- Open Access
Interfering with Tumor Hypoxia for Radiotherapy Optimization
Journal of Experimental & Clinical Cancer Research volume 40, Article number: 197 (2021)
Hypoxia in solid tumors is an important predictor of treatment resistance and poor clinical outcome. The significance of hypoxia in the development of resistance to radiotherapy has been recognized for decades and the search for hypoxia-targeting, radiosensitizing agents continues. This review summarizes the main hypoxia-related processes relevant for radiotherapy on the subcellular, cellular and tissue level and discusses the significance of hypoxia in radiation oncology, especially with regard to the current shift towards hypofractionated treatment regimens. Furthermore, we discuss the strategies to interfere with hypoxia for radiotherapy optimization, and we highlight novel insights into the molecular pathways involved in hypoxia that might be utilized to increase the efficacy of radiotherapy.
Solid tumors require the formation of a neovascular network in order to grow and survive [1, 2]. Rapidly proliferating tumor cells create a hypoxic microenvironment that induces tumor angiogenesis. The rapidly growing blood vessels comprising the neovascular network are often aberrant, disorganized and dysfunctional, leading to an inadequate oxygen supply which further increases tumor hypoxia [3, 4]. Oxygen demand, on the other hand, is often deregulated subsequent to tumor-specific metabolic changes. Together, this results in a dynamic, rapidly changing tumor microenvironment characterized by regions of nutrient deprivation and oxygen deficiency [5,6,7]. Tumor hypoxia was recognized as early as in the 1950s, when Gray and Thomlinson demonstrated by mathematical calculations, based on histological sections of human tumors, that regions around necrotic areas display a hypoxic oxygen gradient . Studies in the following decades identified oxygen deficiency to be characteristic for the majority of solid human tumors [9,10,11,12,13,14,15]. In contrast to the early point of view that a radial hypoxia gradient primarily expands from the necrotic tumor core towards the tumor rim, it has since been established that a wide range of fluctuating oxygen tension values (from mild oxygen deficiency to anoxia) are heterogeneously dispersed in the tumor microenvironment. Both hypoxia and necrosis were independently proven to be predictors of poor clinical outcome, independent of the tumor stage, histological grade and lymph node status [16,17,18]. Hypoxia has also been associated with genomic instability , immune suppression [20, 21], the different steps of the metastatic cascade (including invasion, migration, intravasation and extravasation, formation and support of the premetastatic niche) , and an increase in resistance to chemotherapy and radiotherapy [23, 24].
In this review, we focus on the role of hypoxia for the treatment response to radiotherapy and discuss existing and emerging strategies to overcome hypoxia-induced radioresistance.
The Definition of Hypoxia
Hypoxia is generally defined as a reduced partial pressure of oxygen (i.e. pO2 or oxygen tension) below a physiological level. However, attributing a single number to this definition is not so straightforward: physiological pO2 in the human body ranges from ~ 100 mmHg in the lung alveoli to values below 1 mmHg in the mitochondria. This pressure gradient or the “oxygen cascade” ensures the diffusion of oxygen throughout the body, guiding it from the “supplier” (the alveolar gas) to the “sinks” (the cells). Physiologically, all values of pO2 present in this cascade could be considered “normoxic” .
In a recent review , Rey et al. calculated the median oxygen tension in various tissues based on previous publications. The oxygen tension in normal tissues ranged from 30 to 52 mmHg (corresponds to 3.9 to 6.8% oxygen concentration in the gas phase at sea level), while tumoral oxygen tension ranged in between 5.3 to 14 mmHg (0.7 to 1.8%), often with a considerable fraction of cells reaching below 5 mmHg (0.7%) . Although these median values would suggest an overall hypoxic environment in most tumors, the typical tumoral pattern of oxygenation is highly heterogenous and dynamic. Such heterogeneity is a result of the distinct organization, structure and function of the tumor vasculature and ongoing angiogenic processes. In contrast to normal blood vessel networks, the tumor vasculature comprises a chaotic network of aberrant, hierarchically disorganized and often dysfunctional vessels [3, 4]. As a result, two types of hypoxia can be described in the tumor tissue . Chronic or diffusion-limited hypoxia arises from the oxygen diffusion limit of approx. 100 μm and is therefore relevant for cells that do not have access to a blood vessel within this limiting distance. Acute or perfusion-limited hypoxia, on the other hand, is a result of temporary instabilities in the blood flow. Taken together, this illustrates the complexity and heterogeneity of the oxygenation status in solid tumors. Consequently, reducing the definition of hypoxia to a specific oxygen level is not possible without a specific context, which needs to take into account the tissue type and cellular composition, the spatiotemporal heterogeneity of oxygen tension in the tumor and most importantly, the endpoint of interest.
Radiotherapy and Tumor Hypoxia
The first indications that a tissue poorly supplied with oxygen might be more resistant to ionizing radiation (IR) came about as early as in the 1920s [29, 30], culminating in Gray’s seminal paper in 1953 , where he demonstrated on the preclinical level that a decrease in hypoxia, achieved by improving oxygen delivery, results in increased radiosensitivity. These discoveries paved the way to numerous subsequent studies, focusing on elucidating the underlying mechanisms of hypoxia-induced radioresistance, on determining the significance of hypoxia-induced radioresistance on the clinical level, and finally, on developing strategies for radiosensitization.
Direct Interaction of Ionizing Radiation with Oxygen: the Oxygen Fixation Hypothesis
The dependency of cellular responses to IR on the oxygen level has been recognized for almost a century [30, 31]. This dependency is quantitatively described as the oxygen enhancement ratio (OER), defined as the ratio between the radiation dose under hypoxic conditions and the radiation dose under normoxic conditions necessary to achieve the same amount of cell killing (determined in vitro by clonogenic assays) (Fig. 1a). For most cells in culture, the OER between anoxia (i.e. complete oxygen deprivation during irradiation) and air (150 mmHg or 21% oxygen) lies between 2.5 and 3 . In a study measuring the change in OERs over a range of oxygen tension values, Wouters et al.  discovered that the greatest change in radiosensitivity i.e. the highest OER occurs in the range of 0.5–20 mmHg (0.05–2.5%). with an increase above 20 mmHg only showing minor changes in radiosensitivity. Together with the physiological values stated above, it can be concluded that from a radiobiological point of view, most normal tissues under physiological conditions are sufficiently oxygenated. On the other hand, for most tumors, a significant proportion of tumor cells are exposed to hypoxic conditions and consequently are prone to radioresistance.
The mechanism behind the oxygen-driven increase of cellular radiosensitivity is explained by the “oxygen fixation hypothesis” (Fig. 1b) [29, 32]. In short, this hypothesis states that the probability of permanent IR-induced DNA damage is higher in the presence than in the absence of oxygen. Upon impinging on the tissue, photons ionize the atoms of the absorbing material and generate free electrons. These electrons mediate the IR-induced damage, either by directly damaging the macromolecules, or by interacting with water in a process that yields hydroxyl (OH•) and hydrogen (H•) radicals. These short-lived and highly reactive molecules react with and damage the macromolecules. In absence of oxygen, the extent of these reactions is limited due to the radicals’ instability, and the majority of the damage is readily repaired. In presence of oxygen, additional reactive oxygen species (ROS) are formed, e.g. hydrogen peroxides, which increase the overall concentration of DNA damaging agents. Furthermore, free radicals produced in the critical targets (R•) can also react with oxygen to produce first peroxyl radicals (ROO•) and ultimately the more stable and less well repairable ROOH, thereby chemically “fixing” the damage. This phenomenon, also known as the “Sauerstoffeffekt” (i.e. oxygen effect), forms the basis of our understanding of oxygen-mediated changes in radiosensitivity. Notably, the oxygen effect is by definition a purely physicochemical effect and does not depend on the radiobiological processes explained below.
Hypoxia-Induced Processes Relevant for Radiotherapy
In the physiological state, oxygen and nutrient depletion constitute a major source of cellular stress and thereby induce evolutionary conserved adaptive changes that either increase the delivery or decrease the consumption of missing substances [34, 35]. An increase in erythropoiesis and angiogenesis [36, 37], in epithelial permeability  and vascular tone  may all contribute to enhance the delivery of oxygen to the hypoxic tumor area. To attenuate oxygen consumption, hypoxic cells decrease oxidative phosphorylation by switching to anaerobic glycolysis  and reduce the number of mitochondria (i.e. mitophagy) . Furthermore, to facilitate tissue recovery after potential damage induced under such circumstances, hypoxia can induce expression of growth factors and thereby promote cell proliferation and survival . In case these compensatory actions fail and severe hypoxia persists, normal cells undergo cell cycle arrest and eventually cell death [43,44,45].
Many of the aforementioned mechanisms, such as induction of angiogenesis and promotion of cell proliferation, remain active in the tumor. However, unlike in healthy cells, selective pressure of hypoxia-induced cell cycle arrest and apoptosis leads to a selection of more aggressive malignant clones with disruptions in physiological feedback mechanisms that would normally halt uncontrolled cellular growth [42, 46, 47]. As a result, hypoxia-induced adaptive changes effectively promote cancer progression. In addition to the listed physiological mechanisms that cancer cells co-opt, hypoxia directly promotes immortalization , genetic instability [7, 19], immune evasion [20, 21], cancer stem cell survival , invasion and metastasis . All of these mechanisms, but especially the selection of malignant, apoptosis-resistant clones, regulation of angiogenesis and vasculogenesis, metabolic changes and interference with the DNA damage response, determine hypoxia as one of the key factors that mediate resistance to radiotherapy, beyond the oxygen effect. In this review, we discuss the molecular processes behind these radiotherapy resistance-promoting mechanisms. A detailed overview of the complex processes through which hypoxia promotes malignancy is not the scope this review and the reader is referred to other comprehensive literature [6, 26, 50, 51].
Oxygen Sensing Mechanisms: Hypoxia-Inducible Factors and Beyond
Hypoxia-inducible factors (HIFs) constitute a family of transcription factors that act as the main mediators of the cellular response to hypoxia [34, 36, 51,52,53,54,55,56] and were first identified almost three decades ago as the key regulators of hypoxia-induced erythropoietin transcription . HIFs are heterodimers formed by a constitutively expressed β-subunit and an oxygen-sensitive α-subunit. In mammals, three isoforms of the α-subunit differentiate the three paralogs: HIF-1α, HIF-2α and HIF-3α. Upon binding of the HIF-β subunit, these three paralogs compose the three transcription factors HIF-1, HIF-2 and HIF-3, respectively. In this review, we focus on HIF-1 as the most investigated isoform in the context of radiotherapy optimization and only mention the emerging data relating to HIF-2 and radiotherapy. The role of HIF-3 remains elusive and as such, is not covered in this review.
In the presence of oxygen, the α-subunit of HIF-1 is posttranslationally hydroxylated through the interaction with oxygen-sensitive dioxygenases, namely the HIF prolyl-4-hydroxylase domain enzymes (PHDs) [58,59,60]. Hydroxylation allows for binding of the von Hippel-Lindau (VHL) tumor suppressor protein. In this bound state, the α-subunit is targeted for poly-ubiquitination and proteasomal degradation. PHDs require oxygen as a cofactor for activity and are therefore rendered inactive when oxygen is not available. Consequently, in hypoxic conditions HIF-1α and HIF-2α remain stable and bind to the constitutively expressed HIF-β subunit, forming the transcriptionally active heterodimeric HIF-1 and HIF-2 complexes, respectively, which bind hypoxia response elements (HREs) in the regulatory regions of hundreds of target genes [61, 62]. In contrast to this direct binding to the regulatory regions and subsequent gene activation, HIF-1/2 dependent transcriptional modulation can also occur indirectly, through transactivation of genes for long non-coding and micro RNAs [62, 63] as well as DNA and histone modifiers . Through a complex interaction between direct and indirect effects, HIFs modulate the expression of over thousand genes and regulate the majority of the hypoxia-induced adaptive mechanisms (reviewed in e.g. [50, 60, 61, 65]).
The majority of studies over the last decades, that investigated hypoxia-altered gene expression, focused on the HIF-pathway. However, Jumonji-family of histone demethylases has long been speculated to mediate also HIF-independent gene expression regulation . In 2019 Batie et al.  and Chakraborty et al.  clearly demonstrated for the first time direct, HIF-independent oxygen-mediated gene expression modulation by the lysine-specific Jumonji-family demethylases KDM5A and KDM6A. Moreover, cellular phenotype alterations under low pO2 conditions also occur due to numerous adaptive mechanisms other than direct gene expression control. For example, oxygen availability affects mRNA transcription, splicing, stability and translation, protein folding, protein stability and enzymatic activity [32, 50]. Furthermore, metabolic disturbances and other determinants of cellular stress that accompany hypoxia interfere with both HIF-dependent and HIF-independent signaling networks. This includes for example the nutrient-sensing PI3K/AKT/mTOR pathway  and the unfolded protein response (UPR) . Overall, the research into the HIF-independent effects of hypoxia is ongoing, and the potential role of such effects for hypoxia-induced radioresistance remain to be investigated. For a comprehensive overview, the reader is referred to .
Given the role of HIF-1 as a key player for tumor angiogenesis and as part of the cellular responses to cancer treatments, it is not surprising that the status of HIF-1α overexpression correlates with poor prognosis in many cancer types (head and neck, oesophagus, pancreas, stomach, gall bladder, liver, colon, lung, pleura, breast, ovaries, uterus and bladder [71,72,73,74,75,76,77,78,79,80,81,82,83,84]). Similar to HIF-1α, HIF-2α overexpression also correlates with poor prognosis in several cancer types (head and neck, brain, liver, colon, lung, bladder, kidney) [85,86,87,88,89,90,91]. Thus, next to the direct physicochemical role of a low pO2 for radiotherapy resistance (see above), HIF-1 mediates biological processes relevant for hypoxia-related radiotherapy resistance. It is important to note that the transcriptionally active level of HIF-1 starts to exponentially increase already below the oxygen tension of 40 mmHg (6%) [92, 93]. Thus, compared to the physicochemical “oxygen effect” and “radiobiological hypoxia” described above, which occur in the range of 0.5 to 20 mmHg (0.05 to 2.5%), HIF-1-mediated biological effects are exerted at a significant earlier stage of oxygen deprivation.
The Interaction of Radiotherapy and HIF-1
The relationship between HIF-1 and radiotherapy is complex and bilateral. On the one hand, HIF-1 confers radioresistance through various cancer-promoting mechanisms. On the other hand, radiotherapy activates HIF-1 and its downstream targets, both through hypoxia-dependent and -independent mechanisms.
Radiotherapy-Induced HIF-1 Activation
Hypoxia-dependent radiotherapy-induced HIF-1 activation in the tumor occurs primarily through tumor vascular damage and subsequent oxygen deprivation while multiple mechanisms contribute to hypoxia-independent RT-induced HIF-1 activation. The importance of such mechanisms for radiotherapy was undoubtedly demonstrated by Moeller et al. , who identified that HIF-1-induced signaling in irradiated murine tumors follows the pattern of IR-induced reoxygenation and primarily occurs in the tissue with relatively high oxygenation levels. The authors linked this surprising finding to (1) ROS-mediated HIF-1α stabilization and (2) an increase in the translation of HIF-1-regulated transcripts following depolymerization of “stress granules” due to reoxygenation. Several studies suggested that ROS lead to HIF-1α stabilization, probably by reducing the activity of PHD enzymes [95,96,97,98,99]. However, other hypotheses have emerged, for example attributing ROS-mediated regulation of HIF-1α to the ROS-induced activation of the PI3K/AKT/mTOR and Ras/Raf/MEK/ERK pathways, which in turn leads to an increase in HIF-1α expression (see below). This intriguing topic is the focus of several recent reviews (see [100,101,102,103] and references therein). In addition to ROS, heat shock protein 90 (Hsp90) has also been implicated in HIF-1α protein stabilization following irradiation [104, 105].
The PI3K/AKT/mTOR and the Ras/Raf/MEK/ERK (also known as MAPK/ERK) signaling pathways are the major IR-responsive signal transduction cascades linking IR and HIF-1. Mutations in different entities of these pathways are considered key contributors to the hallmarks of cancer, most notably to malignant cell growth, survival, proliferation and metabolism [106, 107]. The HER family of receptor tyrosine kinases (RTKs), which includes the epidermal growth factor receptor (EGFR or HER1), are upstream of both of these pathways. An increase in their phosphorylation and thus activation status in response to IR activate these signaling cascades even in absence of the corresponding growth factor ligands [108, 109]. Furthermore, rapid, IR-induced activation of the Ras/Raf/MEK/ERK pathway has been linked to several other kinases, namely the extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) [108, 110]. Interestingly, an increase in glucose availability has also been proposed as facilitator of IR-induced activation of the PI3K/AKT/mTOR pathway . The activation of the PI3K/AKT/mTOR and Ras/Raf/MEK/ERK pathways in response to IR, in turn, leads to an increase in the expression of HIF-1α [111, 112].
Radiotherapy and the Hypoxic Tumor Microenviroment
Radiotherapy influences the immune cell compartment and the stroma of the tumor microenvironment (TME) [113,114,115]. The topic of the multifaceted role of radiotherapy at the interface of hypoxia-mediated immunosuppression and the anti-tumor immune response is comprehensively reviewed elsewhere [21, 115,116,117,118,119,120,121,122]. Effects of IR on the stroma can be divided into two topics: (1) the interaction with endothelial cells and the tumor vasculature and (2) the interaction with myofibroblasts and radiation fibrosis.
In response to IR, endothelial cells mount a stress response, followed by either recovery, or loss of function and cell death. The ultimate fate depends on multiple variables: the total dose, fractionation schedule, and the intrinsic TME properties . The affected endothelial cells and vasculature lose their ability to deliver oxygen and nutrients to the tumor cells, leading either to an adaptive increase in hypoxia-mediated signaling or to indirect radiation-induced cell death (see also “Tumor hypoxia in the age of hypofractionation” below). Vascular endothelial growth factor (VEGF) is at the center of this interaction, as HIF-1 target gene and as essential mediator of tumor (re)vascularization . VEGF activates the endothelial cells and promotes the formation of new blood vessels, thereby facilitating tumor cell survival and radioresistance. Apart from hypoxia-mediated signaling, the damage to the endothelial cells directly facilitates an inflammatory response, mediated in large part by nuclear factor kappa B (NF-κB) [125, 126]. This contributes to a radiation-induced inflammatory state, which in turn activates signaling pathways upstream of HIF-1 (as described above) .
Radiation-induced fibrosis (RIF) is a dose-limiting late side effect of radiotherapy. The development of RIF corresponds to an abnormal wound healing process in response to radiation injury, resulting in a self-propagating fibroproliferative state . The process involves an early inflammatory response to IR-induced DNA damage, followed by endothelial cell dysfunction and hypoxia. Together, this leads to an abnormal activation of fibroblasts (called myofibroblasts in the activated state) and excessive deposition of collagen and other extracellular matrix proteins. Transforming growth factor beta (TGF-β) plays a central role in this process, with ROS-mediated posttranslational activation of TGF-β as one of the main initiating processes of RIF . In addition to the rapid ROS-mediated activation, the production of TFG-β and several other profibrotic proteins such as connective tissue growth factor (CTGF) and platelet-derived growth factor (PDGF) is increased in response to hypoxia [129, 130]. Taken together, this illustrates the complex interaction between radiotherapy, hypoxia and fibrosis in the normal tissue, which is ultimately dose-limiting and thus contributes to radiotherapy treatment failure. Apart from their role in the normal tissue, fibroblast abnormalities, specifically identified in cancer-associated fibroblasts (CAFs) in the TME, may also directly contribute to cancer radioresistance . The role of CAFs in radiotherapy has recently been reviewed elsewhere . In the context of hypoxia, it is important to consider that - similarly to normal tissue - IR-induced, hypoxia-mediated signaling contributes to abnormal activation of CAFs. In turn, irradiated CAFs may facilitate various pro-tumorigenic processes, many of which are already directly activated by hypoxia. These include for example TGF-β-mediated epithelial-to-mesenchymal transition (EMT) , or the paracrine activation of pro-survival pathways such as the PI3K/AKT/mTOR- (through e.g. insulin-like growth factor 1 – IGF-1 ) and the Ras/Raf/MEK/ERK-pathways (through e.g. CXCL1 ). As described above, these pathways can in turn activate HIF-1, thus closing a vicious tumor-promoting circle.
Reprogramming energy metabolism is one of the hallmarks of cancer , which encompasses cancer-associated alterations in metabolic activities, supporting the altered metabolic demands of cancer cells and for tumor growth . The Warburg effect or aerobic glycolysis is central to metabolic reprogramming and comprises a tumor cell-specific shift towards glycolysis and lactate production even under normoxic conditions. Even though aerobic glycolysis to lactate is less efficient than mitochondrial oxidative phosphorylation in terms of ATP generation, a high rate of glucose uptake provides sufficient precursors for anabolic pathways such as the hexosamine pathway, pentose-phosphate pathway (PPP) and one-carbon metabolism. Furthermore the mitochondrial tricarboxylic cycle is still fueled with a few percentages of pyruvate to generate intermediates now used as precursors e.g. for lipid synthesis, thus ultimately also supporting cell growth and proliferation [136,137,138].
As a transcriptional activator of glucose transporters (most importantly, GLUT-1)  and virtually all glycolytic enzymes, including lactate dehydrogenase A (LDH-A) and others, HIF-1 is closely interconnected with this metabolic network . In addition to the upregulation of glycolytic enzymes, HIF-1 supports aerobic glycolysis by suppressing mitochondrial respiration, inhibition of mitochondrial biogenesis and induction of mitochondrial autophagy [41, 140, 141]. Finally, as described above, HIF-1 promotes tumor angiogenesis, which increases the availability of glucose and other nutrients. Overall, HIF-1 activity drives a strong glycolytic flux, which - as part of a positive feedback loop - keeps HIF-1 activity through pyruvate-mediated inhibition of α-ketoglutarate (α-KG) high .. In the absence of the co-factor α-KG, PHDs cannot hydroxylate HIF-1α, thus leading to its stabilization. Similar mechanisms link mutations in the metabolic enzymes isocitrate dehydrogenase 1 and 2 (IDH-1/2), fumarate hydratase (FH) and succinate dehydrogenase (SDH) to a stabilization of HIF-1 even under normoxic conditions: dysfunction of these three enzymes causes accumulation of D-2-hydroxyglutarate (D2HG), fumarate and succinate, respectively, which in turn interfere with PHD activity .
In the context of radiotherapy, glycolysis-induced activation of the PPP is one of the best examples illustrating how HIF-1-mediated metabolic reprogramming may directly confer radioresistance. Activation of PPP results in the regeneration of nicotinamide adenine dinucleotide phosphate (NADPH), which in turn reduces oxidized glutathione to protect cancer cells from ROS [136, 143, 144].
Furthermore, multiple positive feedback loops exist between aerobic glycolysis and radiotherapy-regulated signaling towards HIF-1 activity. The PI3K/AKT/mTOR pathway is one of the key drivers of aerobic glycolysis . Harada et al. observed that IR-induced tumor reoxygenation increases glucose availability, also leading to the activation of the nutrient-sensing PI3K/AKT/mTOR pathway. The subsequent increase of HIF-1α activity further supported glycolysis . However, HIF-1 is known to suppress PI3K/AKT/mTOR signaling in response to hypoxia and thus might also create a negative feedback loop [69, 146, 147]. Thus, in the context of hypoxia and radiotherapy, the role of the PI3K/AKT/mTOR pathway is multifaceted.
These examples only serve to illustrate the network complexity at the interface of tumor metabolism, hypoxia and radiotherapy. Many other signaling pathways, oncogenes and tumor suppressors are also closely connected to the radiotherapy response and are also linked to aerobic glycolysis. Originally identified as the major driver of RT-induced apoptosis in tumor cells, the tumor suppressor protein p53 is also activated under hypoxia in a HIF-dependent manner and thereby selects for a more aggressive treatment resistant tumor phenotype [46, 148,149,150]. More recent data, however, suggest an anti-apoptotic and p53-suppressing role for HIF-1 . Interestingly, p53 also antagonistically regulates glycolysis and oxidative phosphorylation via transcriptional regulation of the downstream genes TP53-induced glycolysis regulator (TIGAR) and assembly of cytochrome c oxidase (SCO2). For further information, the reader is referred to [26, 143, 144, 152,153,154,155].
The Significance of Hypoxia in Radiation Oncology
Detecting Tumor Hypoxia on the Clinical Level
The development of a reliable and routinely usable technique to determine the oxygenation status of human tumors has proven to be challenging. Considering the anatomical position of solid tumors, the range of oxygen tension in human tumors and the spatiotemporal heterogeneity of tumor hypoxia, such a technique would ideally be non-invasive and repeatable, highly sensitive, and with a high spatial and temporal resolution.
Due to technological limitations, early attempts to determine the oxygenation status of human tumor had to rely on indirect measurements. These primarily focused on the vascular component of the tumor, correlating hypoxia to e.g. vascular density based on immunohistochemistry or the perfusion status based on computed tomography (CT), positron emission tomography (PET) or magnetic resonance imaging (MRI) [32, 156]. While informative, such methods were not able to comprehensively capture the complexity of tumor oxygenation, that does not only depend on isolated characteristics of the vascular component.
The introduction of the “Eppendorf” polarographic needle electrode in the 1990s offered for the first time the possibility to measure the oxygenation status of human tumors directly and reproducibly, albeit invasively and with a low spatial resolution . Although these disadvantages precluded its introduction into routine clinical practice, the “Eppendorf” electrode remains historically as one of the major contributors to the recognition of the importance of tumor hypoxia on the clinical level.
In the following years, numerous other techniques for hypoxia detection were developed and refined. In general, the two main approaches include: (1) ex vivo techniques such as immunohistochemistry or gene expression analysis performed on invasively obtained patient material and (2) non-invasive or minimally invasive imaging techniques, which enable in vivo hypoxia detection [32, 50, 158,159,160,161]. Immunohistochemistry is an affordable approach, which allows detection of hypoxic cells with high spatial resolution. Targets visualized by immunohistochemistry include endogenous, hypoxia-induced markers (e.g. HIF-1α, carbonic anhydrase IX - CAIX, HIF-1α, glucose transporters GLUT-1 and GLUT-3, osteopontin – OPN) and exogenous markers that specifically accumulate in hypoxic regions (e.g. 2-nitroimidazole - pimonidazole). As a major advantage, endogenous markers do not require planned intravenous administration of a chemical moiety prior to biopsy, thereby enabling also studies on archival material. On the other hand expression of the majority of endogenous markers is regulated by a multitude of factors other than hypoxia, thereby limiting the specificity of this approach . Furthermore, the pO2 level at which a hypoxia-induced increase in gene expression occurs depends on the endogenous marker of choice but does not necessarily correlate with the pO2 level most relevant to radiation oncology (see above).
The most widely used exogenous markers, 2-nitroimidazoles, bind to their targets specifically at pO2 levels below 10 mmHg . The accumulation of these compounds in respective tumor areas therefore highly correlates with areas of “radiobiological hypoxia”. An additional advantage of exogenous markers is the possibility to label them with probes detectable by non-invasive imaging methods, such as single photon emission computed tomography (SPECT), PET or MRI (see below). Aside from immunohistochemistry, gene expression profiling has recently been established as a promising tool for the ex vivo classification of hypoxic tumors . This molecular biology technique assesses the transcriptional response of the tumor to its microenvironment by using RNA sequencing to detect hypoxia-specific gene upregulation on the whole-tissue as well as on the single-cell level. The selection of relevant genes can be tailored to specific tumors and even individual patients. The resulting “hypoxia gene signature” can then be used to stratify tumors as normoxic or hypoxic, and to guide the therapy in line with personalized hypoxia-oriented treatment approaches.
Imaging techniques are non-invasive or minimally invasive approaches that mostly rely on the administration of exogenous hypoxia-specific compounds bound to probes that can be detected by various imaging modalities. Specialized MRI approaches such as blood oxygen level dependent (BOLD) imaging, however, can indirectly quantify tumor hypoxia without a chemical probe. In addition to their non-invasiveness, imaging techniques are generally sensitive and repeatable. Furthermore, it is possible to obtain multiple measurements over time, thereby capturing at least partially the temporal fluctuation of tumor hypoxia, depending on the temporal resolution of the technique. Despite these advantages, imaging methods still face many limitations, mainly stemming from the spatial and temporal resolution, affordability and availability of such systems. Nevertheless, of the various techniques researched to date, PET is the most widely used in the clinical setting; PET hypoxia tracers such as 18F-fluoromisonidazole (FMISO) and 18F-fluoroazomycin arabinoside (FAZA) have been applied in a number of clinical studies [158, 159].
Recognition of the Role of Tumor Hypoxia in Radiation Oncology
While the evidence from early preclinical studies strongly suggested that tumor hypoxia and consequently its modification could be highly relevant for the radiotherapy response, initial attempts at implementation of oxygen-mimicking drugs in the treatment protocol in the 1970s failed to demonstrate a significant improvement over radiotherapy alone . In addition to this initial lack of positive results, reliable in vivo detection and quantification of hypoxia was a major hurdle at the time (see above). The introduction of the “Eppendorf” electrode, as a robust and reproducible method to measure the oxygenation status of human tumors, paved the way towards the first irrefutable evidence of hypoxia-induced radioresistance. Hoeckel et al.  and Fyles and al . demonstrated the predictive power of tumor oxygenation in the radiation response of cervical cancer, while Nordsmark et al. provided first strong clinical evidence of the importance of tumor hypoxia in the radioresistance of head and neck tumors . Controlled clinical trials employing modification of tumor hypoxia undeniably demonstrated the significance of hypoxia for the treatment response to radiotherapy, in particular for squamous cell carcinoma of the head and neck [168, 169].
The Relevance of Hypoxia in Conventional Radiotherapy
Conventional fractionation has been the mainstay of radiation oncology for decades. By dividing the total radiation dose into 1.8 to 2 Gy fractions given five days per week up to a total dose of 40–70 Gy, conventional fractionation exploits the biological differences between the tumor and the normal tissue, resulting typically in less damage of the normal tissue for the same level of tumor control. This increase in the therapeutic ratio is ultimately the result of a complex interplay between different factors, summarized by Withers as the four R’s of radiotherapy: repair of sublethal cellular damage, repopulation of cells following radiation, redistribution of cells within the cell cycle and reoxygenation of the surviving cells [32, 170, 171]. The phenomenon of reoxygenation occurs after depletion of radiosensitive normoxic cells in response to a single low dose fraction of IR (Fig. 2a). Immediately after IR gradual reoxygenation of the more hypoxic surviving tumor cells occurs within hours or days. With conventional fractionation, this iterative process of reoxygenation is taking place continuously during the course of treatment, thereby partially improving tumor control without an influence on the normoxic normal tissue . Notably, given the proven negative influence of hypoxia on the success of conventional radiotherapy as mentioned above, reoxygenation on its own is evidently insufficient to fully overcome hypoxia-induced radioresistance.
Tumor Hypoxia in the Age of Hypofractionation
In contrast to conventional low dose fractionated regimens, hypofractionation describes a radiation treatment where the total dose is given in a smaller number of larger (> 2 Gy) fractions, resulting in an overall shorter treatment time. When given as a single dose or a small number of fractions, usually with the dose of 8–30 Gy per fraction, the treatment is called stereotactic body radiotherapy (SBRT) . The interest in hypofractionation has been rising steadily along with the technological advances in image guidance and highly conformal dose delivery, which make it possible to deliver a large dose to the tumor, while keeping the dose to the surrounding normal tissues acceptable. Today, hypofractionation is considered routine clinical practice in the treatment of certain disease sites, such as the breast, lung, liver and prostate . Radiobiological implications of hypofractionation, including possible additional biological effects, have been reviewed elsewhere [174, 175]. In short, the success of hypofractionation seems to largely be attributable to an increase in the biologically effective dose to the tumor cells, although vascular damage and antitumor immunity stimulation at high doses per fraction might also play an important role. The question of the relevance of hypoxia for hypofractionation similarly remains open (Fig. 2b). On one hand, hypoxia was suggested to be more detrimental for hypofractionated treatments, primarily due to the loss of reoxygenation and possibly due to vascular damage, leading to oxygen deprivation [174, 176]. On the other hand, high-dose-mediated vascular damage was demonstrated to induce secondary cell death in hypoxic cells, which would normally escape direct IR-mediated death; such an effect might counteract the potential loss of reoxygenation and explain the success of hypofractionation shown in clinical trials . In a recent study investigating for the first time tumor hypoxia in SBRT on the clinical level , Kelada et al. demonstrated that high single doses of radiation given to lung cancer patients as a part of SBRT may induce an increased and persistent state of hypoxia. Overall, the significance of tumor hypoxia in the age of hypofractionation therapy remains elusive, and future clinical investigations are necessary. At the same time the shift from classic fractionated radiotherapy regimens to SBRT resulted in a new search for hypoxia-modifying agents to be applied as part of a combined treatment modality with radiotherapy.
Targeting Tumor Hypoxia
Several strategies have been developed during the last decades to overcome the hurdle of tumor hypoxia for successful radiotherapy. These strategies can be grouped into fundamentally different approaches (Fig. 3). While hypoxic radiosensitizers, hypoxia-activated prodrugs and molecular-targeting agents are preferentially effective in hypoxic parts of the tumor, other approaches aim to increase oxygen availability in the tumor and subsequently to reduce tumor hypoxia and radiation resistance. Finally, radiotherapy-based approaches rely on the concept of personalized medicine to specifically target tumor hypoxia and thereby optimize the treatment planning and delivery on an individual level.
Targeting Hypoxic Cells
The prototype of hypoxic radiosensitizers are the electron-affinic nitroimidazoles, such as misonidazole, etanidazole, pimonidazole and the clinically approved nimorazole, which is given as standard of care to patients for head neck cancer radiotherapy, in particular in Denmark . Originally, already more than 50 years ago, nitrobenzenes followed by the nitroimidazoles were proposed to act as oxygen mimetic agents and to react in hypoxic cells with short-lived, oxidizing, IR-induced free DNA radicals to generate cytotoxic DNA strand breaks, thus creating an adjuvant therapeutic effect . Notably, while oxygen is metabolized already by normoxic cells close to the microvessel, hypoxic radiosensitizers could diffuse further to reach also hypoxic zones within the tumor.
Unfortunately, and despite the clarity of the concept, several reasons might have contributed that these hypoxic radiosensitizers did not find their recognition in the clinical routine. While they indeed enhanced the radiation response of hypoxic tumor cells in vitro and of tumor xenografts in vivo in response to large single doses, hypoxic radiosensitizers did not prove as effective in response to a conventionally fractionated, low dose treatment regimen [164, 178, 179]. Subsequently, these compounds failed to significantly improve radiation therapy as part of classic fractionated low dose treatment regimens with concomitant reoxygenation, rendering them less potent. Furthermore, due to severe toxicities, especially neurotoxicity, many of these compounds could not be applied in sufficiently high doses. For these reasons, there is an ongoing effort required to identify novel drugs and/or strategies that would have lower systemic toxicity but retain hypoxic radiosensitization. One of such novel compounds is RRx-001, a nitric-oxide-generating dinitroazetidine radiosensitizer with direct cytotoxic effects [180, 181]. Based on promising preclinical data, RRx-001 is now being tested as part of a combined treatment modality together with radiotherapy in the treatment of primary brain tumors (NCT02871843) and brain metastases (NCT02215512, ).
Lack of patient selection is an additional reason for the failure of hypoxic radiosensitizers on the clinical level. Using a 15-gene hypoxia classifier to detect a hypoxia gene signature (see above) , Toustrup et al. retrospectively analyzed the beneficial effect of nimorazole in head and neck squamous cell carcinoma (HNSCC) patients treated as part of the Danish Head and Neck Cancer Study (DAHANCA) 5 [184,185,186]. While the original study indeed found a beneficial effect from the addition of nimorazole without pretreatment classification (which eventually led to the introduction of nimorazole into the clinical use), the gene-signature-based classification showed a markedly increased effect of nimorazole in patients whose tumors were classified as more-hypoxic. These findings were confirmed in the IAEA-HypoX study (NCT01507467), where the retrospective analysis of HNSCC patients treated with accelerated radiotherapy in combination with nimorazole demonstrated an increased benefit of nimorazole addition in more-hypoxic tumors . Patient classification based on the hypoxic status of the tumor before treatment administration could therefore uncover previously undetected treatment benefits in the “more-hypoxic” subset of patients. Consistently, prospective stratification based on the hypoxic status of the tumor is now being evaluated as part of two HNSCC clinical studies investigating the addition of nimorazole: gene-signature-based classification is included in the non-inferiority DAHANCA 30 study (NCT02661152) that aims to verify the hypothesis that there is no benefit in nimorazole addition to less-hypoxic patients; FAZA-PET-based classification is being evaluated a part of the DAHANCA 33 study (NCT02976051).
In addition to the ongoing efforts to optimize the use of hypoxic radiosensitizers and to identify novel compounds, it could be of interest to see a renaissance of former drugs, e.g. the hypoxia-activated prodrug tirapazamine (see below), and to probe them as part of hypoxia-stratified, personalized trials with SBRT. Despite the recent success of SBRT, hypoxic tumor entities still represent a major challenge for a hypofractionated treatment regimen with only a few high dose fractions of IR.
Hypoxic radiosensitizers, such as nitroimidazoles, are biochemically reduced under low pO2 conditions to become cellular cytotoxins and are therefore already more toxic to hypoxic than to normoxic cells independent of IR. These findings paved the way for the hypoxia-selective generation of bioreductive prodrugs, which are activated by enzymatic reduction in hypoxic tissues . Five different chemical moieties have been exploited as prodrugs for bioreduction under hypoxic conditions. These include nitro-groups (e.g. nitroimidazoles), quinones (e.g. mitomycin C), aromatic N-oxides (e.g. tirapazamine (TPZ)), aliphatic N-oxides (e.g. AQ4N) and transition metals (e.g. cobalt (III) and copper (II) complexes to release cytotoxic agents ligands). These prodrugs are reduced by concerted mechanisms, which involve either one-electron reductases followed by fragmentation or further reduction of the initially formed prodrug radicals; or two-electron reductions of specific prodrugs. In normoxic conditions, the initial reaction is reversible, while under hypoxic conditions these prodrugs become stable cytotoxins, and act as mono−/di-functional DNA alkylators, intra- and interstrand crosslinkers, and DNA-strandbreakers or additionally poison specific enzymes involved in correct DNA-replication and repair, such as topoisomerase II by TPZ [5, 189].
Even though some of these hypoxia-activated prodrugs (HAPs) were also shown to physico-chemically sensitize for a combined treatment modality with IR (synergistic effect), HAPs rather biologically cooperate with radiotherapy, which is less potent in the hypoxic tumor environment (additive effect). Many of these HAPs have been tested on the preclinical and clinical levels but often with only minimal success in the clinics. Besides differences with regard to classic pharmacokinetic aspects, such as stability and diffusability, the different classes of HAP also vary in their oxygen-dependence for activation, which affects normal tissue toxicity. HAPs that are activated at intermediate rather than very low oxygen tension in the tumor (i.e. at the level of “radiobiological hypoxia” as describe above), such as TPZ and its derivatives, might be most potent to eliminate the relevant fraction of tumor cells at this critical level relevant for radiation resistance. However, such agents run the inherent risk of prodrug activation in normal tissues, which can reach these intermediate oxygen tension values in physiological conditions, thereby resulting in concomitant, limiting side toxicities. Indeed, the initial clinical studies of TPZ in combination with cisplatin and/or radiotherapy failed to demonstrate an additional benefit within the tolerable dose range . However, retrospective analyses later suggested a lack of patient stratification and issues with radiation delivery as potential culprits for this failure [190,191,192]. Three clinical studies are still ongoing, all of them investigating the combination of TPZ and chemo- and immunotherapy in hepatocellular cancer (NCT03259867, NCT02174549, NCT03145558). A combination of TPZ with radiotherapy is currently not being investigated on the clinical level.
Dose-limiting toxicities represent a major hurdle for hypoxia-activated prodrugs. Therefore, a strong interest in the development of novel, less toxic compounds exist. These include new derivatives of TPZ (for example SN30000, a TPZ analogue that demonstrated superior activity in preclinical studies [193, 194]) or compounds with chemical moieties (nitro-compounds and quinones) that are only activated under severe hypoxia.
Activated prodrugs could also induce a bystander effect by the diffusion of the active cytotoxic agents from areas of severe hypoxia to tumor areas with intermediate levels of tumor hypoxia and normoxia. The 2-nitroimidazole conjugated bromoisophosphoramide TH-302 (evofosfamide) and dinitrobenzamide PR-104 represent prototypes of such HAPs [195,196,197]. TH-302 releases upon one-electron reduction and fragmentation a diffusible nitrogen mustard derivative with cytotoxic activity . Although a bystander effect through diffusion was initially thought to substantially contribute to its single-agent activity, recent data suggest the absence of such an effect [199, 200]. Furthermore, despite the promising preclinical data and phase I/II clinical trials [201, 202], TH-302 failed to meet the primary endpoint in the two phase III clinical studies (NCT01440088, NCT01746979). Notably, despite the preclinical evidence of benefit in combination with radiotherapy [195, 203,204,205], all trials except one (NCT02598687 failed to meet the primary endpoint) only combined chemotherapy with TH-302. Currently, no clinical trials with TH-302 are open. Nitrogen mustard-DNA crosslinking-based PR-104 is another novel hypoxia-activated prodrug that failed to fulfill the expectations derived from preclinical data; dose-limiting hematological toxicities prevented the advancement of the drug beyond phase I/II clinical trials [196, 206,207,208,209]. Novel nitrobenzamides based on PR-104 are being developed with CP-506 showing interesting results on the preclinical level . One of the major questions to be resolved is the relevance of scheduling of the combined treatment modality of hypoxia-activated prodrugs in combination with high-dose radiotherapy . Furthermore, adjuvant integration of immune response regulatory agents as part of a combined treatment modality might be a promising approach. Following treatment with HAPs the tumor microenvironment might be less immune suppressive .
Of interest are not only prodrugs that release DNA-oriented cytotoxins but also bioreductive prodrugs which release diffusible inhibitors of non-genotoxic molecular targets. For example, PR-610 releases an irreversible inhibitor of the human EGFR upon bioreductive activation ; the phase I clinical trial NCT016312790 had to be terminated due to unacceptable toxicities. TH-4000 (tarloxotinib) is another prodrug of an EGFR inhibitor that advanced to phase II clinical trials as a single agent. The initial two trials in non-small cell lung cancer (NSCLC) and HNSCC [213, 214] did not meet the interim milestone and were terminated, while the RAIN study (NCT03805841) investigating TH-4000 in NSCLC and other advanced solid tumors is ongoing.
The use of such novel bioreductive prodrugs alone or in combination with IR could increase the local concentration of the active agent in the tumor and could lead to a more personalized and even synergistic treatment approach. Future, intense preclinical and clinical investigations will have to demonstrate under which circumstances a (neo-)adjuvant or a concomitant treatment regimen of these bioreductive cytotoxins with (hypo)fractionated radiotherapy will be most effective.
The identification of key molecular mechanism orchestrating the cellular response to hypoxia uncovered new potential targets for overcoming hypoxia-induced treatment resistance. Targeting the HIF-1 pathway is a particularly interesting approach, as it is a key modulator of the hypoxia-induced phenotype and simultaneously regulates a plethora of resistance-inducing mechanisms (see above). Thus, HIF-1 targeting agents can reverse an aggressive phenotype on their own and at the same time synergize with radiotherapy. The HIF-1 signaling network can theoretically be targeted on multiple levels: by targeting the upstream pathways that regulate HIF-1 expression, by inhibiting HIF-1 protein expression, stability and function and by interference with downstream targets of HIF-1.
The two pathways that are most commonly implicated as upstream regulators of HIF-1 expression are the PI3K/AKT/mTOR and Ras/Raf/MEK/ERK pathways [111, 112]. These are ubiquitous and intensively investigated signaling pathways with pleiotropic effects. Subsequently, interference with these pathways does not specifically inhibit HIF-1, but rather results in a multitude of complex and interdependent effects. As such, the topic of targeting of these pathways in combination with radiotherapy is beyond the scope of this paper. Similarly, HIF-1 modulates the expression of hundreds of genes, and as such inhibition of downstream HIF-1 targets is only partially covered in this review. For a more comprehensive overview, the reader is referred to e.g. [26, 215, 216].
Both rational designed approaches, e.g. with RNA antagonists, and high-throughput screenings, e.g. with chemical libraries, were used to identify HIF-signaling interfering agents and thus, the landscape of HIF-1 inhibitors currently comprises a multitude of compounds with pleiotropic mechanisms. Multiple drugs such as EZN-2968, PX-478, topotecan, bortezomib, vorinostat, ganetespib (STA-9090) and geldanamycin have been reported to (indirectly) target HIF-1 protein expression or stability. EZN-2968 is a HIF-1α mRNA antagonist with evidence of preclinical activity  and encouraging proof-of-concept data from a phase I trial in patients with advanced solid tumors  and awaiting results from two insofar unpublished phase I studies (NCT00466583, NCT02564614). The small molecular compound PX-478 has been reported to act on multiple levels, inhibiting transcription, translation and deubiquitination of HIF-1α [219, 220]. In combination with radiotherapy in prostate cancer  and with gemcitabine in pancreatic cancer , PX-478 showed encouraging results on the preclinical level. The initial findings from the phase I clinical trial in patients with advanced solid tumors (NCT00522652) demonstrated acceptable tolerance in the dose range achieving detectable HIF-1 inhibition . Topotecan is a known chemotherapeutic agent that inhibits topoisomerase I. Surprisingly, a high-throughput screening for HIF-1 inhibitors identified topoisomerase (Topo)-I inhibitors to repress HIF-1 transcriptional activity . A pilot clinical trial confirmed the anti-HIF-1 activity in patients with advanced solid tumors . Topotecan is a part of numerous ongoing clinical trials, however the majority does not investigate its role in the context of hypoxia. Bortezomib is a proteasome inhibitor, clinically used in the treatment of multiple myeloma. Besides its primary mechanism for protein stabilization, recent studies suggest that bortezomib also interferes, presumably as secondary effect, by inhibiting upstream regulators of HIF-1α , repressing HIF-1α translation  or by blocking HIF-1-mediated transcription [228, 229]. The radiosensitization effect of bortezomib in hypoxic cervical cancer cells  and esophageal squamous cancer cells  has been attributed specifically to HIF-1 inhibition, while other studies suggested different mechanism to dominate the increase in radiosensitivity [232,233,234,235]. The histone acetylase inhibitor vorinostat interferes with HIF-1 signaling through translation inhibition, increase in ubiquitination and modulation of nuclear translocation [236, 237]. Preclinical data support a radiosensitizing role of vorinostat through multiple different mechanisms [238,239,240], including in the setting of hypoxia [241, 242]. Ganetespib, geldanamycin and its analogues tanespimycin (17-AAG) and alvespimycin (17-DMAG) all inhibit Hsp90, which in turn leads to enhanced degradation of HIF-1α [105, 243,244,245,246]. Considering the pleiotropic effects of Hsp90 inhibition, these compounds have been extensively tested also in combination with radiotherapy, however not yet in the context of hypoxia.
Inhibitors of HIF-1 transcriptional activity, including for example echinomycin, PX-12 and chetomin, comprise a class of molecular hypoxia-targeting agents with potentially much higher specificity than the compounds mentioned above. Overall, this class of compounds is at an earlier stage of development compared to the aforementioned HIF-1 inhibitors, with only limited testing as part of a combined treatment modality with IR. Echinomycin is a DNA intercalator for which the original clinical trials in the early 1990s failed to demonstrate an anti-tumor effect in an acceptable dose range . Despite these setbacks, preclinical research on echinomycin continued, and in 2005 echinomycin was revealed to act as a potent inhibitor of HIF-1 DNA-binding . In parallel, to overcome toxicities, novel derivatives and formulations of echinomycin are being developed, such as the YK-2000 and liposome-encapsulated echinomycin [247, 249]. There are currently no clinical trials investigating echinomycin or its derivatives. PX-12 is an inhibitor of thioredoxin-1 with multiple proposed mechanisms of cytotoxicity, of which HIF-1 inhibition and ROS generation are of particular interest in the context of hypoxia and radiotherapy [250,251,252,253]. PX-12 has so far only been clinically tested as a single agent, with the lack of an anti-tumor effect leading to the termination of a phase II study . Chetomin is a natural product that inhibits HIF-1 transcriptional activity by blocking the interaction between HIF-1α and its transcriptional coactivator p300 . In vitro data demonstrated a radiosensitization effect in hypoxic glioma, fibrosarcoma and osteosarcoma cells [256,257,258]. In a spontaneous lung cancer model and NSCLC xenografts, chetomin inhibited tumor growth without observable adverse effects .
A more detailed overview of HIF-1-targeting molecular agents, along with insights into emerging compounds can be found in [260, 261]. Overall, the landscape of HIF-1 inhibitors currently comprises a multitude of compounds with pleiotropic mechanisms. Given that HIF-1α proteins are very short-lived with a high turnover, it is not surprising that such compounds show seemingly specific effects on the HIF-1 system because they occur before other cellular effects become apparent. While these HIF-1-directed agents are indeed in various stages of preclinical and even clinical research in the context of hypoxia, it should be emphasized that to date, there are no compounds that could be convincingly considered as specific HIF-1-targeting molecular agents. The main mechanism of action towards increased cell killing in combination with radiotherapy similarly remains to be investigated. HIF-1 targeting could sensitize radioresistant cells to IR-induced cell death or it could simply target the aggressive hypoxia-induced phenotype.
HIF-2 as a target in combination with radiotherapy has been less investigated. Notably, HIF-2α is an emerging target in clear cell renal cell carcinoma, with the compound PT2385 as a first-in-class, highly specific HIF-2α inhibitor currently tested in clinical trials . Several in vitro studies observed a correlation between increased HIF-2α levels with radioresistance and demonstrated an increase in radiosensitivity following HIF-2α inhibition [263,264,265,266,267]. However, the interaction of HIF-2α inhibitors with radiotherapy on the clinical level is currently difficult to predict.
Targeting Tumor Metabolism
As described above, tumor-specific metabolic reprogramming is an important contributor to radioresistance.
Between the different targeting strategies, increasing oxygen availability by targeting oxidative phosphorylation is emerging as a particularly interesting approach, with sufficient tumor and stroma cells still using oxidative phosphorylation as a major source for ATP generation under normoxic conditions [136, 268]. Therefore, compounds reprogramming those cells to anaerobic glycolysis or directly targeting mitochondrial respiration and metabolism sensitize for IR-induced cytotoxic DNA damage by creating (a window of) enhanced tumor oxygenation due to reduced oxygen consumption [269, 270]. A promising candidate belonging to this group of compounds is metformin, one of the most commonly prescribed drugs in the treatment of type II diabetes. The potential role of metformin in cancer development and treatment was first observed in retrospective studies of diabetic cancer patients . Zannella et al. showed that this effect can, at least partially, be attributed to an improved tumor oxygenation and subsequent increase in radiosensitivity . Mechanistically, this occurs through metformin-mediated inhibition of the mitochondrial complex I . Inhibition of complex I, in turn, leads to a redistribution of oxygen and accumulation of α-KG, both of which activate PHDs, and subsequently results in the degradation of HIF-1α. Thus, the contribution of metformin to increased radiosensitivity can occur on two levels: (1) a direct increase in cytotoxic IR-induced ROS through an increase in oxygen availability and (2) an inhibition of HIF-1-mediated prosurvival- and radioresistance-inducing signals . Following these intriguing preclinical observations, metformin also showed promising results in NSCLC patients receiving chemoradiotherapy (NCT02109549, ) and in colorectal cancer patients (NCT03053544). Currently, four clinical trials are investigating metformin as a radiosensitizing, tumor oxygenation-enhancing drug (NCT04170959, NCT03510390, NCT02394652, NCT04275713). The anti-malarial agent atovaquone is an additional example for an “old drug”, that was repurposed in an attempt to exploit its ability to inhibit the mitochondrial complex III . The ability of atovaquone to modify tumor hypoxia and thereby improve the efficacy of radiotherapy was demonstrated in cell lines and tumor xenografts  and is currently being tested in NSCLC patients (NCT0262808). The third example of a repurposed drug is papaverine, a smooth muscle relaxant and complex I inhibitor whose radiosensitizing properties are being tested in a phase I clinical trial in combination with SBRT (, NCT03824327). There are numerous other examples of oxidative phosphorylation inhibitors that have shown impressive results in cell lines and mouse models, but have not yet been able to advance to the clinical level; these include arsenic trioxide [279, 280], nonsteroidal anti-inflammatory drugs  and glucocorticoids . Finally, there is a multitude of promising additional emerging compounds such as BAY-87-2243 and IACS-010759. Both compounds demonstrated antitumor activity as single agents ([283,284,285], NCT03291938, NCT01297530) and in combination with radiotherapy  or radioimmunotherapy .
Apart from the inhibition of mitochondrial functions, other potential targets for radiosensitization in the network of tumor metabolism include for example α-KG as a rate-limiting substrate of the PHD family of enzymes , and a broad spectrum of enzymes involved in glycolysis and the PPP (reviewed in [26, 153, 289]). Overall, current mechanistic insights indeed support the role of numerous metabolic entities in the development of radioresistance, however the effects of interference with these entities as targets in the context of radiotherapy remains to be investigated.
Local application of heat in the range of 39–45 °C, i.e. hyperthermia, is a long-known anti-cancer treatment modality with proven radiosensitizing and cytotoxic effects that preferentially occur in the hypoxic regions (136–140). As part of a combined treatment with chemo- or radiotherapy, hyperthermia is already approved for routine clinical use in several countries. The radiosensitizing effect is thought to occur due to the improvement of oxygen delivery to the tissue [290, 291] and due to interference with the DNA damage response [292,293,294,295,296,297,298]. Cytotoxic effects can be direct and indirect: heat can directly induce necrosis, apoptosis and mitotic catastrophe [299,300,301] and with an applied temperature above 42 °C, heat will indirectly kill by inducing vascular damage . At the same time, however, exposure to elevated temperatures might result in Hsp90-mediated HIF-1α protein stabilization even under normoxic conditions, thereby potentially counteracting the radiosensitizing effect . The details and the relative importance of these effects remain elusive. The optimal temperature, duration and timing of heat exposure still need to be identified. Nevertheless, there is an active interest for hyperthermia in the radiation oncology community, illustrated by the fact that there are currently 16 active clinical trials investigating the combination of hyperthermia and radiotherapy. The addition of hypoxia-targeting compounds to the combination of radiotherapy and hyperthermia might be an interesting future approach, as demonstrated by Sadeghi et al.  who investigated temperature-sensitive liposomes loaded with the hypoxic radiosensitizer pimonidazole.
Targeting the Tumor Vasculature
In contrast to the aforementioned approaches, which primarily target hypoxic tumor cells, inhibitors of angiogenesis (IoAs) aim at cells of the tumor vasculature. Intuitively, IoAs should destroy the tumor vasculature and thus increase tumor hypoxia. Early preclinical data investigating the combination of radiotherapy with IoAs in mouse tumor models offered conflicting results: while there was evidence of an increase in radioresistance following an IoA-mediated increase in hypoxia, there were also reports of a delayed tumor growth when combining the two treatment modalities [304,305,306,307]. Specifically, the fumagalin-derivative TNP-470, which specifically inhibits endothelial cell proliferation, and the endogenous IoA angiostatin, were demonstrated in several animal tumor models to enhance tumor growth delay and to surprisingly and counterintuitively increase tumor oxygenation, when used in combination with IR [305, 306, 308, 309]. In our study using a murine mammary carcinoma allograft model, we found that an IoA-induced increase in tumor hypoxia is nullified when combined with irradiation, most probably due to the decreased demand for oxygen by the drastically reduced number of tumor cells surviving the first few fractions of irradiation . Nevertheless, the existence of contradictory data and the potential to antagonize radiotherapy contributed for a long time to the reservations to combine IoAs with radiotherapy in the clinics.
In 2001, Jain coined the (now widely accepted) term “normalization” of the tumor vasculature to explain the paradoxical findings of IoA-induced potentiation of the effects of chemotherapy and radiotherapy [311,312,313]. Tumor hypoxia is caused by a dysregulated tumor vasculature and an overproduction of angiogenic growth factors. Anti-angiogenic strategies were demonstrated to destruct immature microvessels, to stabilize leaky vessels and to remodel the dysfunctional tumor vasculature to a normal phenotype with increased tumor blood flow and oxygen delivery. This process could contribute to the increased treatment response originally observed and now aimed for by the combined treatment modality of IR with IoAs. A major obstacle though is that normalization of the tumor vasculature is a transient process only occurring within a short time window and highly dependent on correct dosing of the respective inhibitor and on the individual tumor environment. Nevertheless, this line of thinking resulted in preclinical studies, that successfully demonstrated an enhanced radiation response when tumors were irradiated during a transient increase of tumor oxygenation [314,315,316]. These studies were performed on tumor xenografts and orthotopic tumor models treated with different classes of anti-angiogenic agents, such as the VEGF-directed antibody bevacizumab, the VEGF-receptor directed antibody DC101, the anti-angiogenic peptide anginex or multiple VEGF-RTK-inhibitors [309, 317,318,319,320,321]. These preclinical findings motivated a large number of clinical trials, the majority of which investigated the combination of bevacizumab with radiotherapy, but also the broad-sprectrum IoA endostatin, RTK-inhibitors sorafenib, sunitinib, vandetanib and semaxanib; and thalidomide (extensively reviewed in [314, 322]). Overall, the results of these studies so far have failed to show a substantial benefit of the addition of IoAs to a radiotherapy treatment regimen; but do not exclude the feasibility of such an approach, with patient selection and optimization of dosing and scheduling remaining the key challenge. Interestingly, not only VEGF but also other pro-angiogenic factors such as placental growth factor (PlGF) are secreted in response to irradiation and in a dose- and time-dependent manner. Our own recent studies showed a strong paracrine vasculature-protective role of PlGF as part of a p53-regulated IR-induced resistance mechanism and suggest PlGF as a promising target for a combined treatment modality with radiotherapy . Furthermore, not only endothelial cell-directed compounds, but also tumor cell signaling-directed agents may contribute to a window of tumor vasculature normalization, e.g. by the reduction of VEGF-secretion, leading to reduced radiation resistance [319, 324, 325].
Overall, pharmacological-induced normalization of the tumor vasculature with concomitant reoxygenation is an interesting concept. However, a fractionated treatment regimen of IR with concomitant IR-induced reoxygenation might even supersede this effect. We currently cannot predict which tumor entities and phenotypes will respond to anti-angiogenic compounds accordingly, not even on the preclinical experimental level. Even more challenges exist in the clinics so that a defined window of tumor vasculature normalization could be identified on the individual patient situation, e.g. using hypoxia PET imaging, to be exploited for SBRT. Conflicting data on the outcome of different treatment schedulings exist when using IoAs in combination with IR. The studies suggest that it is not a neo-adjuvant but rather a concomitant and even more so an adjuvant treatment regimen that results in an enhanced tumor response to irradiation in combination with several investigated anti-angiogenic compounds [308, 309, 326,327,328,329]. As such, preclinical scheduling experiments suggest additional mechanisms to contribute to radiosensitization by anti-angiogenic agents, besides normalization of the tumor vasculature. IR affects the tumor vasculature in multiple ways, including the switch to other forms of angiogenesis [330, 331]. But irradiation also induces endothelial cell apoptosis, and the apoptotic response on the level of the tumor vasculature correlates with the tumor response to single high doses of IR (>15Gy) . IoAs also induce endothelial cell apoptosis and thus might enhance the fragility of the tumor vasculature also to low dose fractions of irradiation and thereby increase the efficacy of radiotherapy [333,334,335].
Increasing Oxygen Delivery
Tumor hypoxia can be regarded as a shifted balance between demand and supply of oxygen relative to the normal tissue. Several earlier strategies were developed to increase the oxygen transport capacity of the blood and perfusion of the tumor with more oxygen. Hyperbaric oxygen (HBO) therapy  and blood transfusions in anemic patients [184, 337] failed to achieve a significant improvement in the outcome, which together with the complexity of the procedure and patient compliance led to a loss of interest into these approaches. An increase of the hemoglobin concentration could also be achieved by erythropoietin injections, but clinical studies revealed increased radiation resistance upon erythropoietin, which can be linked to radiation protective erythropoietin-induced signaling in certain tumor cells [337,338,339]. The approach with the most clinical success is the combination of carbogen (95% normobaric oxygen + 5% carbon dioxide) to deal with diffusion-limited chronic hypoxia and nicotinamide to overcome acute hypoxia. Two clinical trials, BCON in bladder cancer [340, 341] and ARCON in HNSCC [342,343,344] reported improvements in the outcome.
Other approaches to modify oxygen delivery include allosteric hemoglobin modifiers (e.g. RSR13, that reached phase III trials, but failed to improve overall survival ), agents that improve the diffusion of oxygen (e.g. trans sodium crocetinate – TSC , currently investigated in phase III clinical trials in glioblastoma (NCT03393000), and oxygen transport agents based on hemoglobin and fluorocarbons (e.g. NVX-108 [347, 348], currently tested in phase II clinical trials in glioblastoma (NCT03862430)). The allosteric effector of hemoglobin myo-inositol trispyrophosphate (ITPP) was also shown to locally increase the pO2 in hypoxic tumors. Even though mixed results exists on the potency of ITPP, which is primarily due to pharmacokinetic aspects, we and others demonstrated its chemo- and radiosensitization capacity in both immunocompromised and immunocompetent tumor models [349,350,351,352], For a more comprehensive review on the topic, see .
Optimizing Radiotherapy Treatment Planning and Delivery: Dose Painting
Recent developments in the field of medical physics made advanced, highly conformal techniques of dose delivery, such as intensity-modulated radiotherapy (IMRT) and volumetric arc therapy (VMAT), a routine part of clinical practice . Together with the development of real-time in vivo hypoxia-detecting methods such as FMISO- or FAZA-PET (see above), the concept of “dose painting”, which includes first identification of hypoxic regions and then a targeted increase in the dose to those regions, was established . Although promising, such PET-guided hypoxia dose painting faces inherent problems derived from the spatiotemporal heterogeneity of tumor hypoxia and the resolution limit of PET . The large multicenter trial RTEP5 (NCT01576796), in which patients with NSCLC received a targeted dose increase in hypoxic regions identified by FMISO-PET, initially failed to demonstrate an improvement in the tumor control by dose painting . However, long-term follow-up showed an increase in the overall survival in the patients who received the radiotherapy boost . Currently, the NCT02352792 trial is recruiting HNSCC patients and will investigate FMISO-PET-based dose escalation, with an interim analysis demonstrating the feasibility of the approach . MRI-based identification of hypoperfused volumes is another strategy to define hypoxic areas in the tumor; such an approach is used in an ongoing clinical trial that investigates targeted dose escalation in HNSCC (NCT02031250). In contrast to the conventional dose-boosting strategies described above, Tubin et al. have taken an innovative approach to partially irradiate only hypoxic segments of bulky tumors, with a short course of hypofractionated radiotherapy [359, 360]. Based on the promising preclinical data, suggesting the occurrence of bystander and abscopal responses , and the success from the initial clinical experience [359, 360], this approach is now being tested in a phase I clinical trial (NCT04168320).
A century of preclinical and clinical research on tumor hypoxia and its impact on ionizing radiation-induced cell killing have consolidated that a reduced pO2-level in the tumor represents a major hurdle for successful radiotherapy. The concept of fractionated low dose radiotherapy with concomitant iterative reoxygenation in between fractions and its clinical application for many tumor entities remain the most powerful but still insufficient approach to overcome hypoxia-related radiation resistance. Lack of successful pharmaceutical interventions and its integration into combined treatment modalities is not only due to the complexity of tumor hypoxia on its own but is also linked to the physics- and imaging-oriented discipline of radiation oncology and its strong focus on technical innovations.
Molecular, cellular and (patho-)physiological processes all contribute to a differential pO2 in the tumor in its complex dynamics during tumor growth and in response to treatment. Furthermore, invasive and non-invasive methodologies probe tumor hypoxia at differential spatiotemporal resolutions, and only complementary approaches characterize the hypoxia status of an individual tumor sufficiently. It is not surprising that only a few pharmaceutical approaches have reliably proven to decrease tumor hypoxia, rendering personalized strategies even more difficult. At the same time, radiobiological research during the last decades demonstrated that different treatment regimens influence blood perfusion and oxygen diffusion in a differential way. However, we only start to understand these processes on the mechanistic level to optimally increase the efficacy of radiotherapy in combination with clinically relevant hypoxia-modifying agents.
Radiotherapy is nowadays often combined with immuno-modulatory approaches resulting in improved locoregional control and overall survival for some tumor entities [116, 118,119,120]. However, tumor hypoxia not only represents a hurdle for successful radiotherapy but also creates an immunosuppressive environment [21, 117, 121]. As such, the development of potent tumor hypoxia-modifying agents and its integration into current novel treatment strategies in the field of radiotherapy holds great promise to further improve tumor control, the therapeutic window and successful radiotherapy.
Availability of Data and Materials
Data sharing is not applicable to this article as no datasets were generated or analysed during the current study.
adenosine-monophosphate-activated protein kinase
- BOLD MRI:
blood oxygen level dependent magnetic resonance imaging
connective tissue growth factor
Danish Head and Neck Cancer Study
epidermal growth factor receptor
fumarase or fumarate hydratase
head and neck squamous cell carcinoma
hypoxia response element
insulin-like growth factor
inhibitor of angiogenesis
magnetic resonance imaging
nicotinamide adenine dinucleotide phosphate
- NF- κB:
nuclear factor kappa B
non-small cell lung cancer
oxygen enhancement ratio
platelet-derived growth factor
positron emission tomography
prolyl hydroxylase domain
placental growth factor
pentose phosphate pathway
reactive oxygen species
receptor tyrosine kinase
stereotactic body radiotherapy
single photo emission computed tomography
tricarboxylic acid cycle
transforming growth factor β
unfolded protein response
vascular endothelial growth factor
volumetric arc radiotherapy
Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 2000;100(1):57–70. https://doi.org/10.1016/S0092-8674(00)81683-9.
Hanahan D, Weinberg RA. Hallmarks of cancer: The next generation. Cell. 2011;144(5):646–74. https://doi.org/10.1016/j.cell.2011.02.013.
Nagy JA, Chang S-H, Shih S-C, Dvorak AM, Dvorak HF. Heterogeneity of the tumor vasculature. Semin Thromb Hemost. 2010;36(03):321–31. https://doi.org/10.1055/s-0030-1253454.
Baluk P, Hashizume H, McDonald DM. Cellular abnormalities of blood vessels as targets in cancer. Curr Opin Genet Dev. 2005;15(1):102–11. https://doi.org/10.1016/j.gde.2004.12.005.
Wilson WR, Hay MP. Targeting hypoxia in cancer therapy. Nat Rev Cancer. 2011;11(6):393–410. https://doi.org/10.1038/nrc3064.
Harris AL. Hypoxia — a key regulatory factor in tumour growth. Nat Rev Cancer. 2002;2(1):38–47. https://doi.org/10.1038/nrc704.
Bhandari V, Hoey C, Liu LY, Lalonde E, Ray J, Livingstone J, et al. Molecular landmarks of tumor hypoxia across cancer types. Nat Genet. 2019;51(2):308–18. https://doi.org/10.1038/s41588-018-0318-2.
Thomlinson RH, Gray LH. The histological structure of some human lung cancers and the possible implications for radiotherapy. Br J Cancer. 1955;9(4):539–49. https://doi.org/10.1038/bjc.1955.55.
Vaupel P, Kelleher DK, Höckel M. Oxygenation status of malignant tumors: Pathogenesis of hypoxia and significance for tumor therapy. Semin Oncol. 2001;28(2 Suppl 8):29–35. https://doi.org/10.1016/S0093-7754(01)90210-6.
Vaupel P, Thews O, Hoeckel M. Treatment resistance of solid tumors. Med Oncol. 2001;18(4):243–59. https://doi.org/10.1385/MO:18:4:243.
Nordsmark M, Alsner J, Keller J, Nielsen OS, Jensen OM, Horsman MR, et al. Hypoxia in human soft tissue sarcomas: Adverse impact on survival and no association with p53 mutations. Br J Cancer. 2001;84(8):1070–5. https://doi.org/10.1054/bjoc.2001.1728.
Nordsmark M, Bentzen SM, Rudat V, Brizel D, Lartigau E, Stadler P, et al. Prognostic value of tumor oxygenation in 397 head and neck tumors after primary radiation therapy. An international multi-center study. Radiother Oncol. 2005;77(1):18–24. https://doi.org/10.1016/j.radonc.2005.06.038.
Brown JM. Tumor hypoxia in cancer therapy. Methods Enzymol. 2007;435:295–321. https://doi.org/10.1016/S0076-6879(07)35015-5.
Dhani N, Fyles A, Hedley D, Milosevic M. The clinical significance of hypoxia in human cancers. Semin Nucl Med. 2015;45(2):110–21. https://doi.org/10.1053/j.semnuclmed.2014.11.002.
Bhandari V, Li CH, Bristow RG, Boutros PC. Divergent mutational processes distinguish hypoxic and normoxic tumours. Nat Commun. 2020;11(1):737. https://doi.org/10.1038/s41467-019-14052-x.
Vaupel P. Prognostic potential of the pretherapeutic tumor oxygenation status. Adv Exp Med Biol. 2009;645:241–6. https://doi.org/10.1007/978-0-387-85998-9_36.
Höckel M, Knoop C, Schlenger K, Vorndran B, Bauβnann E, Mitze M, et al. Intratumoral pO2 predicts survival in advanced cancer of the uterine cervix. Radiother Oncol. 1993;26(1):45–50. https://doi.org/10.1016/0167-8140(93)90025-4.
Richards CH, Mohammed Z, Qayyum T, Horgan PG, McMillan DC. The prognostic value of histological tumor necrosis in solid organ malignant disease: a systematic review. Future Oncol. 2011;7(10):1223–35. https://doi.org/10.2217/fon.11.99.
Bristow RG, Hill RP. Hypoxia and metabolism. Hypoxia, DNA repair and genetic instability. Nat Rev Cancer. 2008;8(3):180–92. https://doi.org/10.1038/nrc2344.
Facciabene A, Peng X, Hagemann IS, Balint K, Barchetti A, Wang L-P, et al. Tumour hypoxia promotes tolerance and angiogenesis via CCL28 and T reg cells. Nature. 2011;475(7355):226–30. https://doi.org/10.1038/nature10169.
Noman MZ, Hasmim M, Messai Y, Terry S, Kieda C, Janji B, et al. Hypoxia: a key player in antitumor immune response. A Review in the theme: Cellular responses to hypoxia. Am J Physiol-Cell Physiol. 2015;309(9):C569–79. https://doi.org/10.1152/ajpcell.00207.2015.
Rankin EB, Giaccia AJ. Hypoxic control of metastasis. Science. 2016;352(6282):175–80. https://doi.org/10.1126/science.aaf4405.
Grau C, Overgaard J. Effect of etoposide, carmustine, vincristine, 5-fluorouracil, or methotrexate on radiobiologically oxic and hypoxic cells in a C3H mouse mammary carcinoma in situ. Cancer Chemother Pharmacol. 1992;30(4):277–80. https://doi.org/10.1007/BF00686295.
Dewhirst MW, Cao Y, Moeller B. Cycling hypoxia and free radicals regulate angiogenesis and radiotherapy response. Nat Rev Cancer. 2008;8(6):425–37. https://doi.org/10.1038/nrc2397.
Rey S, Schito L, Koritzinsky M, Wouters BG. Molecular targeting of hypoxia in radiotherapy. Adv Drug Deliv Rev. 2017;109:45–62. https://doi.org/10.1016/j.addr.2016.10.002.
Mortensen LS, Buus S, Nordsmark M, Bentzen L, Munk OL, Keiding S, et al. Identifying hypoxia in human tumors: A correlation study between 18F-FMISO PET and the Eppendorf oxygen-sensitive electrode. Acta Oncol Stockh Swed. 2010;49(7):934–40. https://doi.org/10.3109/0284186X.2010.516274.
Bayer C, Shi K, Astner ST, Maftei C-A, Vaupel P. Acute versus chronic hypoxia: Why a simplified classification is simply not enough. Int J Radiat Oncol Biol Phys. 2011;80(4):965–8. https://doi.org/10.1016/j.ijrobp.2011.02.049.
Hall EJ, Giaccia AJ. Radiobiology for the radiologist. 7th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2012.
Crabtree HG, Cramer W, Murray JA. The action of radium on cancer cells. II.—Some factors determining the susceptibility of cancer cells to radium. Proc R Soc Lond Ser B Contain Pap Biol Character. 1933;113(782):238–50. https://doi.org/10.1098/rspb.1933.0044.
Gray LH, Conger AD, Ebert M, Hornsey S, Scott OCA. The concentration of oxygen dissolved in tissues at the time of irradiation as a factor in radiotherapy. Br J Radiol. 1953;26(312):638–48. https://doi.org/10.1259/0007-1285-26-312-638.
Joiner MC, van der Kogel AJ, van der Kogel AJ. Basic clinical radiobiology. Boca Raton, FL: CRC Press; 2018. https://doi.org/10.1201/9780429490606.
Wouters BG, Brown JM. Cells at intermediate oxygen levels can be more important than the “hypoxic fraction” in determining tumor response to fractionated radiotherapy. Radiat Res. 1997;147(5):541–50. https://doi.org/10.2307/3579620.
Wenger RH. Cellular adaptation to hypoxia: O2-sensing protein hydroxylases, hypoxia-inducible transcription factors, and O2-regulated gene expression. FASEB J. 2002;16(10):1151–62. https://doi.org/10.1096/fj.01-0944rev.
Lee P, Chandel NS, Simon MC. Cellular adaptation to hypoxia through hypoxia inducible factors and beyond. Nat Rev Mol Cell Biol. 2020;21(5):268–83. https://doi.org/10.1038/s41580-020-0227-y.
Semenza GL, Wang GL. A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation. Mol Cell Biol. 1992;12(12):5447–54. https://doi.org/10.1128/MCB.12.12.5447.
Shweiki D, Itin A, Soffer D, Keshet E. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature. 1992;359(6398):843–5. https://doi.org/10.1038/359843a0.
Synnestvedt K, Furuta GT, Comerford KM, Louis N, Karhausen J, Eltzschig HK, et al. Ecto-5′-nucleotidase (CD73) regulation by hypoxia-inducible factor-1 mediates permeability changes in intestinal epithelia. J Clin Invest. 2002;110(7):993–1002. https://doi.org/10.1172/JCI15337.
Bodi I, Bishopric NH, Discher DJ, Wu X, Webster KA. Cell-specificity and signaling pathway of endothelin-1 gene regulation by hypoxia. Cardiovasc Res. 1995;30(6):975–84. https://doi.org/10.1016/S0008-6363(95)00164-6.
Semenza GL, Roth PH, Fang HM, Wang GL. Transcriptional regulation of genes encoding glycolytic enzymes by hypoxia-inducible factor 1. J Biol Chem. 1994;269(38):23757–63. https://doi.org/10.1016/S0021-9258(17)31580-6.
Semenza GL. Oxygen-dependent regulation of mitochondrial respiration by hypoxia-inducible factor 1. Biochem J. 2007;405(1):1–9. https://doi.org/10.1042/BJ20070389.
Vaupel P. Tumor microenvironmental physiology and its implications for radiation oncology. Semin Radiat Oncol. 2004;14(3):198–206. https://doi.org/10.1016/j.semradonc.2004.04.008.
Goda N, Ryan HE, Khadivi B, McNulty W, Rickert RC, Johnson RS. Hypoxia-inducible factor 1alpha is essential for cell cycle arrest during hypoxia. Mol Cell Biol. 2003;23(1):359–69. https://doi.org/10.1128/mcb.23.1.359-369.2003.
Hackenbeck T, Knaup KX, Schietke R, Schödel J, Willam C, Wu X, et al. HIF-1 or HIF-2 induction is sufficient to achieve cell cycle arrest in NIH3T3 mouse fibroblasts independent from hypoxia. Cell Cycle. 2009;8(9):1386–95. https://doi.org/10.4161/cc.8.9.8306.
Carmeliet P, Dor Y, Herbert J-M, Fukumura D, Brusselmans K, Dewerchin M, et al. Role of HIF-1α in hypoxia-mediated apoptosis, cell proliferation and tumour angiogenesis. Nature. 1998;394(6692):485–90. https://doi.org/10.1038/28867.
Graeber TG, Osmanian C, Jacks T, Housman DE, Koch CJ, Lowe SW, et al. Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumours. Nature. 1996;379(6560):88–91. https://doi.org/10.1038/379088a0.
Vaupel P. Hypoxia and aggressive tumor phenotype: implications for therapy and prognosis. Oncologist. 2008;13(S3):21–6. https://doi.org/10.1634/theoncologist.13-S3-21.
Seimiya H, Tanji M, Oh-hara T, Tomida A, Naasani I, Tsuruo T. Hypoxia up-regulates telomerase activity via mitogen-activated protein kinase signaling in human solid tumor cells. Biochem Biophys Res Commun. 1999;260(2):365–70. https://doi.org/10.1006/bbrc.1999.0910.
Keith B, Simon MC. Hypoxia-inducible factors, stem cells, and cancer. Cell. 2007;129(3):465–72. https://doi.org/10.1016/j.cell.2007.04.019.
Luo W, Wang Y. Hypoxia mediates tumor malignancy and therapy resistance. Adv Exp Med Biol. 2019;1136:1–18. https://doi.org/10.1007/978-3-030-12734-3_1.
Sørensen BS, Horsman MR. Tumor hypoxia: Impact on radiation therapy and molecular pathways. Front Oncol. 2020;10:562. https://doi.org/10.3389/fonc.2020.00562.
Wang GL, Semenza GL. General involvement of hypoxia-inducible factor 1 in transcriptional response to hypoxia. Proc Natl Acad Sci. 1993;90(9):4304–8. https://doi.org/10.1073/pnas.90.9.4304.
Wang GL, Semenza GL. Characterization of hypoxia-inducible factor 1 and regulation of DNA binding activity by hypoxia. J Biol Chem. 1993;268(29):21513–8. https://doi.org/10.1016/S0021-9258(20)80571-7.
Wang GL, Jiang BH, Rue EA, Semenza GL. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci. 1995;92(12):5510–4. https://doi.org/10.1073/pnas.92.12.5510.
Iyer NV, Kotch LE, Agani F, Leung SW, Laughner E, Wenger RH, et al. Cellular and developmental control of O2 homeostasis by hypoxia-inducible factor 1α. Genes Dev. 1998;12(2):149–62. https://doi.org/10.1101/gad.12.2.149.
Wenger RH, Stiehl DP, Camenisch G. Integration of oxygen signaling at the consensus HRE. Sci STKE. 2005;2005:re12. https://doi.org/10.1126/stke.3062005re12.
Wang GL, Semenza GL. Molecular basis of hypoxia-induced erythropoietin expression. Curr Opin Hematol. 1996;3(2):156–62. https://doi.org/10.1097/00062752-199603020-00009.
Ivan M, Kondo K, Yang H, Kim W, Valiando J, Ohh M, et al. HIFα targeted for VHL-mediated destruction by proline hydroxylation: Implications for O2 sensing. Science. 2001;292(5516):464–8. https://doi.org/10.1126/science.1059817.
Jaakkola P, Mole DR, Tian Y-M, Wilson MI, Gielbert J, Gaskell SJ, et al. Targeting of HIF-α to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science. 2001;292(5516):468–72. https://doi.org/10.1126/science.1059796.
Kaelin WG, Ratcliffe PJ. Oxygen sensing by metazoans: The central role of the HIF hydroxylase pathway. Mol Cell. 2008;30(4):393–402. https://doi.org/10.1016/j.molcel.2008.04.009.
Semenza GL. Hypoxia-inducible factors in physiology and medicine. Cell. 2012;148(3):399–408. https://doi.org/10.1016/j.cell.2012.01.021.
Mole DR, Blancher C, Copley RR, Pollard PJ, Gleadle JM, Ragoussis J, et al. Genome-wide association of hypoxia-inducible factor (HIF)-1α and HIF-2α DNA binding with expression profiling of hypoxia-inducible transcripts. J Biol Chem. 2009;284(25):16767–75. https://doi.org/10.1074/jbc.M901790200.
Camps C, Saini HK, Mole DR, Choudhry H, Reczko M, Guerra-Assunção JA, et al. Integrated analysis of microRNA and mRNA expression and association with HIF binding reveals the complexity of microRNA expression regulation under hypoxia. Mol Cancer. 2014;13(1):28. https://doi.org/10.1186/1476-4598-13-28.
Xia X, Lemieux ME, Li W, Carroll JS, Brown M, Liu XS, et al. Integrative analysis of HIF binding and transactivation reveals its role in maintaining histone methylation homeostasis. Proc Natl Acad Sci. 2009;106(11):4260–5. https://doi.org/10.1073/pnas.0810067106.
Huang Y, Lin D, Taniguchi CM. Hypoxia inducible factor (HIF) in the tumor microenvironment: Friend or foe? Sci China Life Sci. 2017;60(10):1114–24. https://doi.org/10.1007/s11427-017-9178-y.
Melvin A, Rocha S. Chromatin as an oxygen sensor and active player in the hypoxia response. Cell Signal. 2012;24(1):35–43. https://doi.org/10.1016/j.cellsig.2011.08.019.
Batie M, Frost J, Frost M, Wilson JW, Schofield P, Rocha S. Hypoxia induces rapid changes to histone methylation and reprograms chromatin. Science. 2019;363(6432):1222–6. https://doi.org/10.1126/science.aau5870.
Chakraborty AA, Laukka T, Myllykoski M, Ringel AE, Booker MA, Tolstorukov MY, et al. Histone demethylase KDM6A directly senses oxygen to control chromatin and cell fate. Science. 2019;363(6432):1217–22. https://doi.org/10.1126/science.aaw1026.
Liu L, Cash TP, Jones RG, Keith B, Thompson CB, Simon MC. Hypoxia-induced energy stress regulates mRNA translation and cell growth. Mol Cell. 2006;21(4):521–31. https://doi.org/10.1016/j.molcel.2006.01.010.
Hetz C, Saxena S. ER stress and the unfolded protein response in neurodegeneration. Nat Rev Neurol. 2017;13(8):477–91. https://doi.org/10.1038/nrneurol.2017.99.
Kurokawa T, Miyamoto M, Kato K, Cho Y, Kawarada Y, Hida Y, et al. Overexpression of hypoxia-inducible-factor 1 α (HIF-1 α ) in oesophageal squamous cell carcinoma correlates with lymph node metastasis and pathologic stage. Br J Cancer. 2003;89(6):1042–7. https://doi.org/10.1038/sj.bjc.6601186.
Baba Y, Nosho K, Shima K, Irahara N, Chan AT, Meyerhardt JA, et al. HIF1A overexpression is associated with poor prognosis in a cohort of 731 colorectal cancers. Am J Pathol. 2010;176(5):2292–301. https://doi.org/10.2353/ajpath.2010.090972.
Miyake K, Yoshizumi T, Imura S, Sugimoto K, Batmunkh E, Kanemura H, et al. Expression of hypoxia-inducible factor-1α, histone deacetylase 1, and metastasis-associated protein 1 in pancreatic carcinoma: Correlation with poor prognosis with possible regulation. Pancreas. 2008;36(3):e1–9. https://doi.org/10.1097/MPA.0b013e31815f2c2a.
Birner P, Schindl M, Obermair A, Breitenecker G, Oberhuber G. Expression of hypoxia-inducible factor 1α in epithelial ovarian tumors: Its impact on prognosis and on response to chemotherapy. Clin Cancer Res. 2001;7(6):1661–8.
Yan M, Rayoo M, Takano EA, Thorne H, Fox SB. BRCA1 tumours correlate with a HIF-1 α phenotype and have a poor prognosis through modulation of hydroxylase enzyme profile expression. Br J Cancer. 2009;101(7):1168–74. https://doi.org/10.1038/sj.bjc.6605287.
Seeber LM, Horrée N, van der Groep P, van der Wall E, Verheijen RH, van Diest PJ. Necrosis related HIF-1α expression predicts prognosis in patients with endometrioid endometrial carcinoma. BMC Cancer. 2010;10(1):307. https://doi.org/10.1186/1471-2407-10-307.
Batmunkh E, Shimada M, Morine Y, Imura S, Kanemura H, Arakawa Y, et al. Expression of hypoxia-inducible factor-1 alpha (HIF-1α) in patients with the gallbladder carcinoma. Int J Clin Oncol. 2010;15(1):59–64. https://doi.org/10.1007/s10147-009-0011-7.
Chen L, Shi Y, Yuan J, Han Y, Qin R, Wu Q, et al. HIF-1 alpha overexpression correlates with poor overall survival and disease-free survival in gastric cancer patients post-gastrectomy. PLoS One. 2014;9(3):e90678. https://doi.org/10.1371/journal.pone.0090678.
Huang M, Chen Q, Xiao J, Yao T, Bian L, Liu C, et al. Overexpression of hypoxia-inducible factor-1α is a predictor of poor prognosis in cervical cancer: a clinicopathologic study and a meta-analysis. Int J Gynecol Cancer Off J Int Gynecol Cancer Soc. 2014;24(6):1054–64. https://doi.org/10.1097/IGC.0000000000000162.
Wang Q, Hu D, Rui Y, Jiang A, Liu Z, Huang L. Prognosis value of HIF-1α expression in patients with non-small cell lung cancer. Gene. 2014;541(2):69–74. https://doi.org/10.1016/j.gene.2014.03.025.
Zheng Y, Ni Y, Huang X, Wang Z, Han W. Overexpression of HIF-1α indicates a poor prognosis in tongue carcinoma and may be associated with tumour metastasis. Oncol Lett. 2013;5(4):1285–9. https://doi.org/10.3892/ol.2013.1185.
Theodoropoulos VE, Lazaris AC, Sofras F, Gerzelis I, Tsoukala V, Ghikonti I, et al. Hypoxia-inducible factor 1α expression correlates with angiogenesis and unfavorable prognosis in bladder cancer. Eur Urol. 2004;46(2):200–8. https://doi.org/10.1016/j.eururo.2004.04.008.
Klabatsa A, Sheaff MT, Steele JPC, Evans MT, Rudd RM, Fennell DA. Expression and prognostic significance of hypoxia-inducible factor 1α (HIF-1α) in malignant pleural mesothelioma (MPM). Lung Cancer. 2006;51(1):53–9. https://doi.org/10.1016/j.lungcan.2005.07.010.
Unruh A, Ressel A, Mohamed HG, Johnson RS, Nadrowitz R, Richter E, et al. The hypoxia-inducible factor-1 α is a negative factor for tumor therapy. Oncogene. 2003;22(21):3213–20. https://doi.org/10.1038/sj.onc.1206385.
Gao Z-J, Wang Y, Yuan W, Yuan J, Yuan K. HIF-2α not HIF-1α overexpression confers poor prognosis in non–small cell lung cancer. Tumor Biol. 2017;39(6):1010428317709637. https://doi.org/10.1177/1010428317709637.
Han S, Huang T, Li W, Liu S, Yang W, Shi Q, et al. Association between hypoxia-inducible factor-2α (HIF-2α) expression and colorectal cancer and its prognostic role: a systematic analysis. Cell Physiol Biochem Int J Exp Cell Physiol Biochem Pharmacol. 2018;48(2):516–27. https://doi.org/10.1159/000491806.
Scrideli CA, Carlotti CG, Mata JF, Neder L, Machado HR, Oba-Sinjo SM, et al. Prognostic significance of co-overexpression of the EGFR/IGFBP-2/HIF-2A genes in astrocytomas. J Neuro-Oncol. 2007;83(3):233–9. https://doi.org/10.1007/s11060-007-9328-0.
Bangoura G, Liu Z-S, Qian Q, Jiang C-Q, Yang G-F, Jing S. Prognostic significance of HIF-2α/EPAS1 expression in hepatocellular carcinoma. World J Gastroenterol WJG. 2007;13(23):3176–82. https://doi.org/10.3748/wjg.v13.i23.3176.
Lim E, Kuo C-C, Tu H-F, Yang C-C. The prognosis outcome of oral squamous cell carcinoma using HIF-2α. J Chin Med Assoc. 2017;80(10):651–6. https://doi.org/10.1016/j.jcma.2017.06.005.
Ioachim E, Michael M, Salmas M, Michael MM, Stavropoulos NE, Malamou-Mitsi V. Hypoxia-inducible factors HIF-1α and HIF-2α expression in bladder cancer and their associations with other angiogenesis-related proteins. Urol Int. 2006;77(3):255–63. https://doi.org/10.1159/000094819.
Martínez-Sáez O, Gajate Borau P, Alonso-Gordoa T, Molina-Cerrillo J, Grande E. Targeting HIF-2 α in clear cell renal cell carcinoma: A promising therapeutic strategy. Crit Rev Oncol Hematol. 2017;111:117–23. https://doi.org/10.1016/j.critrevonc.2017.01.013.
Jiang BH, Semenza GL, Bauer C, Marti HH. Hypoxia-inducible factor 1 levels vary exponentially over a physiologically relevant range of O2 tension. Am J Physiol-Cell Physiol. 1996;271(4):C1172–80. https://doi.org/10.1152/ajpcell.1996.271.4.C1172.
Yu AY, Frid MG, Shimoda LA, Wiener CM, Stenmark K, Semenza GL. Temporal, spatial, and oxygen-regulated expression of hypoxia-inducible factor-1 in the lung. Am J Physiol Lung Cell Mol Physiol. 1998;275(4):L818–26. https://doi.org/10.1152/ajplung.1998.275.4.L818.
Moeller BJ, Cao Y, Li CY, Dewhirst MW. Radiation activates HIF-1 to regulate vascular radiosensitivity in tumors: Role of reoxygenation, free radicals, and stress granules. Cancer Cell. 2004;5(5):429–41. https://doi.org/10.1016/S1535-6108(04)00115-1.
Chandel NS, McClintock DS, Feliciano CE, Wood TM, Melendez JA, Rodriguez AM, et al. Reactive oxygen species generated at mitochondrial complex III stabilize hypoxia-inducible factor-1α during hypoxia. J Biol Chem. 2000;275(33):25130–8. https://doi.org/10.1074/jbc.M001914200.
Gerald D, Berra E, Frapart YM, Chan DA, Giaccia AJ, Mansuy D, et al. JunD reduces tumor angiogenesis by protecting cells from oxidative stress. Cell. 2004;118(6):781–94. https://doi.org/10.1016/j.cell.2004.08.025.
Bell E, Klimova T, Eisenbart J, Morase C, Murphy M, Budinger S, et al. The Qo site of the mitochondrial complex III is required for the transduction of hypoxic signaling via reactive oxygen species production. J Cell Biol. 2007;177(6):1029–936. https://doi.org/10.1083/jcb.200609074.
Klimova T, Chandel NS. Mitochondrial complex III regulates hypoxic activation of HIF. Cell Death Differ. 2008;15(4):660–6. https://doi.org/10.1038/sj.cdd.4402307.
Sasabe E, Yang Z, Ohno S, Yamamoto T. Reactive oxygen species produced by the knockdown of manganese-superoxide dismutase up-regulate hypoxia-inducible factor-1alpha expression in oral squamous cell carcinoma cells. Free Radic Biol Med. 2010;48(10):1321–9. https://doi.org/10.1016/j.freeradbiomed.2010.02.013.
Movafagh S, Crook S, Vo K. Regulation of hypoxia-inducible factor-1a by reactive oxygen species : New developments in an old debate. J Cell Biochem. 2015;116(5):696–703. https://doi.org/10.1002/jcb.25074.
Waypa GB, Smith KA, Schumacker PT. O2 sensing, mitochondria and ROS signaling: The fog is lifting. Mol Asp Med. 2016;47–48:76–89. https://doi.org/10.1016/j.mam.2016.01.002.
Smith KA, Waypa GB, Schumacker PT. Redox signaling during hypoxia in mammalian cells. Redox Biol. 2017;13:228–34. https://doi.org/10.1016/j.redox.2017.05.020.
Chen R, Lai UH, Zhu L, Singh A, Ahmed M, Forsyth NR. Reactive oxygen species formation in the brain at different oxygen levels: The role of hypoxia inducible factors. Front Cell Dev Biol. 2018;6 https://doi.org/10.3389/fcell.2018.00132.
Kim W-Y, Oh SH, Woo J-K, Hong WK, Lee H-Y. Targeting heat shock protein 90 overrides the resistance of lung cancer cells by blocking radiation-induced stabilization of hypoxia-inducible factor-1alpha. Cancer Res. 2009;69(4):1624–32. https://doi.org/10.1158/0008-5472.CAN-08-0505.
Katschinski DM, Le L, Heinrich D, Wagner KF, Hofer T, Schindler SG, et al. Heat induction of the unphosphorylated form of hypoxia-inducible factor-1alpha is dependent on heat shock protein-90 activity. J Biol Chem. 2002;277(11):9262–7. https://doi.org/10.1074/jbc.M110377200.
Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144(5):646–74. https://doi.org/10.1016/j.cell.2011.02.013.
Asati V, Kar Mahapatra D, Kumar BS. PI3K/Akt/mTOR and Ras/Raf/MEK/ERK signaling pathways inhibitors as anticancer agents: Structural and pharmacological perspectives. Eur J Med Chem. 2016;109:314–41. https://doi.org/10.1016/j.ejmech.2016.01.012.
Hein AL, Ouellette MM, Yan Y. Radiation-induced signaling pathways that promote cancer cell survival (Review). Int J Oncol. 2014;45(5):1813–9. https://doi.org/10.3892/ijo.2014.2614.
Wee P, Wang Z. Epidermal growth factor receptor cell proliferation signaling pathways. Cancers. 2017;9(5):52. https://doi.org/10.3390/cancers9050052.
Munshi A, Ramesh R. Mitogen-activated protein kinases and their role in radiation response. Genes Cancer. 2013;4(9–10):401–8. https://doi.org/10.1177/1947601913485414.
Harada H, Itasaka S, Kizaka-Kondoh S, Shibuya K, Morinibu A, Shinomiya K, et al. The Akt/mTOR pathway assures the synthesis of HIF-1α protein in a glucose- and reoxygenation-dependent manner in irradiated tumors. J Biol Chem. 2009;284(8):5332–42. https://doi.org/10.1074/jbc.M806653200.
Fukuda R, Hirota K, Fan F, Jung YD, Ellis LM, Semenza GL. Insulin-like growth factor 1 induces hypoxia-inducible factor 1-mediated vascular endothelial growth factor expression, which is dependent on MAP kinase and phosphatidylinositol 3-kinase aignaling in colon cancer cells. J Biol Chem. 2002;277(41):38205–11. https://doi.org/10.1074/jbc.M203781200.
Barker HE, Paget JTE, Khan AA, Harrington KJ. The Tumour microenvironment after radiotherapy: Mechanisms of resistance and recurrence. Nat Rev Cancer. 2015;15(7):409–25. https://doi.org/10.1038/nrc3958.
Barcellos-Hoff MH. Remodeling the irradiated tumor microenvironment: The fifth R of radiobiology? In: Tofilon PJ, Camphausen K, editors. Increasing the therapeutic ratio of radiotherapy. Cham: Springer International Publishing; 2017. p. 135–49. https://doi.org/10.1007/978-3-319-40854-5_6.
Jarosz-Biej M, Smolarczyk R, Cichoń T, Kułach N. Tumor microenvironment as a “game changer” in cancer radiotherapy. Int J Mol Sci. 2019;20(13):3212. https://doi.org/10.3390/ijms20133212.
Herrera FG, Bourhis J, Coukos G. Radiotherapy combination opportunities leveraging immunity for the next oncology practice. CA Cancer J Clin. 2017;67(1):65–85. https://doi.org/10.3322/caac.21358.
Wennerberg E, Lhuillier C, Vanpouille-Box C, Pilones KA, García-Martínez E, Rudqvist N-P, et al. Barriers to radiation-induced in situ tumor vaccination. Front Immunol. 2017;8:229. https://doi.org/10.3389/fimmu.2017.00229.
Grassberger C, Ellsworth SG, Wilks MQ, Keane FK, Loeffler JS. Assessing the interactions between radiotherapy and antitumour immunity. Nat Rev Clin Oncol. 2019;16(12):729–45. https://doi.org/10.1038/s41571-019-0238-9.
Deutsch E, Chargari C, Galluzzi L, Kroemer G. Optimising efficacy and reducing toxicity of anticancer radioimmunotherapy. Lancet Oncol. 2019;20(8):e452–63. https://doi.org/10.1016/S1470-2045(19)30171-8.
Pitroda SP, Chmura SJ, Weichselbaum RR. Integration of radiotherapy and immunotherapy for treatment of oligometastases. Lancet Oncol. 2019;20(8):e434–42. https://doi.org/10.1016/S1470-2045(19)30157-3.
Li Y, Patel SP, Roszik J, Qin Y. Hypoxia-driven immunosuppressive metabolites in the tumor microenvironment: New approaches for combinational immunotherapy. Front Immunol. 2018;9:1591. https://doi.org/10.3389/fimmu.2018.01591.
Wang B, Zhao Q, Zhang Y, Liu Z, Zheng Z, Liu S, et al. Targeting hypoxia in the tumor microenvironment: a potential strategy to improve cancer immunotherapy. J Exp Clin Cancer Res. 2021;40(1):24. https://doi.org/10.1186/s13046-020-01820-7.
Park HJ, Griffin RJ, Hui S, Levitt SH, Song CW. Radiation-induced vascular damage in tumors: Implications of vascular damage in ablative hypofractionated radiotherapy (SBRT and SRS). Radiat Res. 2012;177(3):311–27. https://doi.org/10.1667/RR2773.1.
Carmeliet P. VEGF as a key mediator of angiogenesis in cancer. Oncology. 2005;69(3):4–10. https://doi.org/10.1159/000088478.
Hellweg CE. The Nuclear Factor κB pathway: A link to the immune system in the radiation response. Cancer Lett. 2015;368(2):275–89. https://doi.org/10.1016/j.canlet.2015.02.019.
Baselet B, Sonveaux P, Baatout S, Aerts A. Pathological effects of ionizing radiation: endothelial activation and dysfunction. Cell Mol Life Sci. 2019;76(4):699–728. https://doi.org/10.1007/s00018-018-2956-z.
Gandhi S, Chandna S. Radiation-induced inflammatory cascade and its reverberating crosstalks as potential cause of post-radiotherapy second malignancies. Cancer Metastasis Rev. 2017;36(2):375–93. https://doi.org/10.1007/s10555-017-9669-x.
Yarnold J, Vozenin Brotons M-C. Pathogenetic mechanisms in radiation fibrosis. Radiother Oncol. 2010;97(1):149–61. https://doi.org/10.1016/j.radonc.2010.09.002.
Distler JHW, Jüngel A, Pileckyte M, Zwerina J, Michel BA, Gay RE, et al. Hypoxia-induced increase in the production of extracellular matrix proteins in systemic sclerosis. Arthritis Rheum. 2007;56(12):4203–15. https://doi.org/10.1002/art.23074.
Darby I, Hewitson T. Hypoxia in tissue repair and fibrosis. Cell Tissue Res. 2016;365(3):553–62. https://doi.org/10.1007/s00441-016-2461-3.
Barbazán J, Matic VD. Cancer associated fibroblasts: is the force the path to the dark side? Curr Opin Cell Biol. 2019;56:71–9. https://doi.org/10.1016/j.ceb.2018.09.002.
Wang Z, Tang Y, Tan Y, Wei Q, Yu W. Cancer-associated fibroblastas in radiotherapy: challenges and new opportunities. Cell Commun Signal. 2019;17(1):47. https://doi.org/10.1186/s12964-019-0362-2.
Tam SY, Wu VWC, Law HKW. Hypoxia-induced epithelial-mesenchymal transition in cancers: HIF-1α and beyond. Front Oncol. 2020;10:486. https://doi.org/10.3389/fonc.2020.00486.
Tommelein J, De Vlieghere E, Verset L, Melsens E, Leenders J, Descamps B, et al. Radiotherapy-activated cancer-associated fibroblasts promote tumor progression through paracrine IGF1R activation. Cancer Res. 2018;78(3):659–70. https://doi.org/10.1158/0008-5472.CAN-17-0524.
Zhang H, Yue J, Jiang Z, Zhou R, Xie R, Xu Y, et al. CAF-secreted CXCL1 conferred radioresistance by regulating DNA damage response in a ROS-dependent manner in esophageal squamous cell carcinoma. Cell Death Dis. 2017;8(5):e2790. https://doi.org/10.1038/cddis.2017.180.
DeBerardinis RJ, Chandel NS. Fundamentals of cancer metabolism. Sci Adv. 2016;2(5):e1600200. https://doi.org/10.1126/sciadv.1600200.
Heiden MGV, Cantley LC, Thompson CB. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science. 2009;324(5930):1029–33. https://doi.org/10.1126/science.1160809.
Frezza C. Metabolism and cancer: the future is now. Br J Cancer. 2020;122(2):133–5. https://doi.org/10.1038/s41416-019-0667-3.
Ebert BL, Firth JD, Ratcliffe PJ. Hypoxia and mitochondrial inhibitors regulate expression of glucose transporter-1 via distinct cis-acting sequences (*). J Biol Chem. 1995;270(49):29083–9. https://doi.org/10.1074/jbc.270.49.29083.
Zhang H, Gao P, Fukuda R, Kumar G, Krishnamachary B, Zeller KI, et al. HIF-1 inhibits mitochondrial biogenesis and cellular respiration in VHL-deficient renal cell carcinoma by repression of C-MYC activity. Cancer Cell. 2007;11(5):407–20. https://doi.org/10.1016/j.ccr.2007.04.001.
Semenza GL. Mitochondrial autophagy: Life and breath of the cell. Autophagy. 2008;4(4):534–6. https://doi.org/10.4161/auto.5956.
Nagao A, Kobayashi M, Koyasu S, Chow CCT, Harada H. HIF-1-dependent reprogramming of glucose metabolic pathway of cancer cells and its therapeutic significance. Int J Mol Sci. 2019;20(2):238. https://doi.org/10.3390/ijms20020238.
Meijer TWH, Kaanders JHAM, Span PN, Bussink J. Targeting hypoxia, HIF-1, and tumor glucose metabolism to Improve radiotherapy efficacy. Clin Cancer Res. 2012;18(20):5585–94. https://doi.org/10.1158/1078-0432.CCR-12-0858.
Courtnay R, Ngo DC, Malik N, Ververis K, Tortorella SM, Karagiannis TC. Cancer metabolism and the Warburg effect: the role of HIF-1 and PI3K. Mol Biol Rep. 2015;42(4):841–51. https://doi.org/10.1007/s11033-015-3858-x.
DeYoung MP, Horak P, Sofer A, Sgroi D, Ellisen LW. Hypoxia regulates TSC1/2–mTOR signaling and tumor suppression through REDD1-mediated 14–3–3 shuttling. Genes Dev. 2008;22(2):239–51. https://doi.org/10.1101/gad.1617608.
Brugarolas J, Lei K, Hurley RL, Manning BD, Reiling JH, Hafen E, et al. Regulation of mTOR function in response to hypoxia by REDD1 and the TSC1/TSC2 tumor suppressor complex. Genes Dev. 2004;18(23):2893–904. https://doi.org/10.1101/gad.1256804.
Chen D, Li M, Luo J, Gu W. Direct Interactions between HIF-1α and Mdm2 Modulate p53 Function*. J Biol Chem. 2003;278(16):13595–8. https://doi.org/10.1074/jbc.C200694200.
Moeller BJ, Dreher MR, Rabbani ZN, Schroeder T, Cao Y, Li CY, et al. Pleiotropic effects of HIF-1 blockade on tumor radiosensitivity. Cancer Cell. 2005;8(2):99–110. https://doi.org/10.1016/j.ccr.2005.06.016.
Moeller BJ, Dewhirst MW. HIF-1 and tumour radiosensitivity. Br J Cancer. 2006;95(1):1–5. https://doi.org/10.1038/sj.bjc.6603201.
Rohwer N, Cramer T. Hypoxia-mediated drug resistance: Novel insights on the functional interaction of HIFs and cell death pathways. Drug Resist Updat. 2011;14(3):191–201. https://doi.org/10.1016/j.drup.2011.03.001.
Huang R, Zhou P-K. HIF-1 signaling: A key orchestrator of cancer radioresistance. Radiat Med Prot. 2020;1(1):7–14. https://doi.org/10.1016/j.radmp.2020.01.006.
Tang L, Wei F, Wu Y, He Y, Shi L, Xiong F, et al. Role of metabolism in cancer cell radioresistance and radiosensitization methods. J Exp Clin Cancer Res CR. 2018;37(1):87. https://doi.org/10.1186/s13046-018-0758-7.
Madan E, Gogna R, Bhatt M, Pati U, Kuppusamy P, Mahdi AA. Regulation of glucose metabolism by p53: emerging new roles for the tumor suppressor. Oncotarget. 2011;2(12):948–57. https://doi.org/10.18632/oncotarget.389.
Green DR, Chipuk JE. p53 and Metabolism: Inside the TIGAR. Cell. 2006;126(1):30–2. https://doi.org/10.1016/j.cell.2006.06.032.
Vaupel P, Kallinowski F, Okunieff P. Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: a review. Cancer Res. 1989;49(23):6449–65.
Vaupel P, Schlenger K, Knoop C, Höckel M. Oxygenation of human tumors: evaluation of tissue oxygen distribution in breast cancers by computerized O2 tension measurements. Cancer Res. 1991;51(12):3316–22.
Horsman MR, Mortensen LS, Petersen JB, Busk M, Overgaard J. Imaging hypoxia to improve radiotherapy outcome. Nat Rev Clin Oncol. 2012;9(12):674–87. https://doi.org/10.1038/nrclinonc.2012.171.
Stieb S, Eleftheriou A, Warnock G, Guckenberger M, Riesterer O. Longitudinal PET imaging of tumor hypoxia during the course of radiotherapy. Eur J Nucl Med Mol Imaging. 2018;45(12):2201–17. https://doi.org/10.1007/s00259-018-4116-y.
Rickard AG, Palmer GM, Dewhirst MW. Clinical and pre-clinical methods for quantifying tumor hypoxia. In: Gilkes DM, editor. Hypoxia cancer metastasis. Cham: Springer International Publishing; 2019. p. 19–41. https://doi.org/10.1007/978-3-030-12734-3_2.
Bussink J, Kaanders JHAM, van der Kogel AJ. Tumor hypoxia at the micro-regional level: clinical relevance and predictive value of exogenous and endogenous hypoxic cell markers. Radiother Oncol. 2003;67(1):3–15. https://doi.org/10.1016/S0167-8140(03)00011-2.
Mayer A, Höckel M, Vaupel P. Endogenous hypoxia markers: Case not proven! In: Kang KA, Harrison DK, Bruley DF, editors. Oxygen transport to tissue XXIX, vol. 614. Boston, MA: Springer; 2008.
Harris BHL, Barberis A, West CML, Buffa FM. Gene expression signatures as biomarkers of tumour xypoxia. Clin Oncol. 2015;27(10):547–60. https://doi.org/10.1016/j.clon.2015.07.004.
Brown JM. Clinical trials of radiosensitizers: What should we expect? Int J Radiat Oncol. 1984;10(3):425–9. https://doi.org/10.1016/0360-3016(84)90063-4.
Höckel M, Vorndran B, Schlenger K, Baußmann E, Knapstein PG. Tumor oxygenation: A new predictive parameter in locally advanced cancer of the uterine cervix. Gynecol Oncol. 1993;51(2):141–9. https://doi.org/10.1006/gyno.1993.1262.
Fyles AW, Milosevic M, Wong R, Kavanagh M-C, Pintilie M, Sun A, et al. Oxygenation predicts radiation response and survival in patients with cervix cancer. Radiother Oncol. 1998;48(2):149–56. https://doi.org/10.1016/S0167-8140(98)00044-9.
Nordsmark M, Overgaard M, Overgaard J. Pretreatment oxygenation predicts radiation response in advanced squamous cell carcinoma of the head and neck. Radiother Oncol. 1996;41(1):31–9. https://doi.org/10.1016/S0167-8140(96)91811-3.
Overgaard J. Hypoxic radiosensitization: adored and ignored. J Clin Oncol. 2007;25(26):4066–74. https://doi.org/10.1200/JCO.2007.12.7878.
Overgaard J. Hypoxic modification of radiotherapy in squamous cell carcinoma of the head and neck--a systematic review and meta-analysis. Radiother Oncol. 2011;100(1):22–32. https://doi.org/10.1016/j.radonc.2011.03.004.
Brown JM, Wilson WR. Exploiting tumour hypoxia in cancer treatment. Nat Rev Cancer. 2004;4(6):437–47. https://doi.org/10.1038/nrc1367.
Withers HR, Lett JT, Adler H. The four R’s of radiotherapy. Adv Radiat Biol. 1975;5:241–71. https://doi.org/10.1016/B978-0-12-035405-4.50012-8.
Potters L, Kavanagh B, Galvin JM, Hevezi JM, Janjan NA, Larson DA, et al. American Society for Therapeutic Radiology and Oncology (ASTRO) and American College of Radiology (ACR) practice guideline for the performance of stereotactic body radiation therapy. Int J Radiat Oncol Biol Phys. 2010;76:326–32. https://doi.org/10.1016/j.ijrobp.2009.09.042.
Lo SS, Fakiris AJ, Chang EL, Mayr NA, Wang JZ, Papiez L, et al. Stereotactic body radiation therapy: a novel treatment modality. Nat Rev Clin Oncol. 2010;7(1):44–54. https://doi.org/10.1038/nrclinonc.2009.188.
Brown JM, Carlson DJ, Brenner DJ. The tumor radiobiology of SRS and SBRT: Are more than the 5 R’s involved? Int J Radiat Oncol Biol Phys. 2014;88(2):254–62. https://doi.org/10.1016/j.ijrobp.2013.07.022.
Song CW, Glatstein E, Marks LB, Emami B, Grimm J, Sperduto PW, et al. Biological principles of stereotactic body radiation therapy (SBRT) and stereotactic radiation surgery (SRS): Indirect cell death. Int J Radiat Oncol. 2019;110(1):21–34. https://doi.org/10.1016/j.ijrobp.2019.02.047.
Carlson DJ, Keall PJ, Loo BW, Chen ZJ, Brown JM. Hypofractionation results in reduced tumor cell kill compared to conventional fractionation for tumors with regions of hypoxia. Int J Radiat Oncol. 2011;79(4):1188–95. https://doi.org/10.1016/j.ijrobp.2010.10.007.
Adams GE, Cooke MS. Electron-affinic sensitization. Int J Radiat Biol Relat Stud Phys Chem Med. 1969;15(5):457–71. https://doi.org/10.1080/09553006914550741.
Denekamp J, Fowler JF, McNally NJ. Hypoxic cell sensitizers: early or late in fractionated therapy? Br J Cancer. 1978;37(5):858–60. https://doi.org/10.1038/bjc.1978.125.
Overgaard J. Clinical evaluation of nitroimidazoles as modifiers of hypoxia in solid tumors. Oncol Res. 1994;6(10-11):509–18.
Das DS, Tian Z, Ray A, Ravillah D, Song Y, Richardson PG, et al. Anti-myeloma activity of a novel free radical inducer Rrx-001. Blood. 2014;124(21):4712. https://doi.org/10.1182/blood.V124.21.4712.4712.
Oronsky B, Scicinski J, Ning S, Peehl D, Oronsky A, Cabrales P, et al. RRx-001, A novel dinitroazetidine radiosensitizer. Investig New Drugs. 2016;34(3):371–7. https://doi.org/10.1007/s10637-016-0326-y.
Kim MM, Parmar HA, Schipper M, Devasia T, Aryal MP, Kesari S, et al. BRAINSTORM: A multi-institutional phase 1/2 Study of RRx-001 in combination with whole brain radiation therapy for patients with brain metastases. Int J Radiat Oncol. 2020;107(3):478–86. https://doi.org/10.1016/j.ijrobp.2020.02.639.
Toustrup K, Sørensen BS, Nordsmark M, Busk M, Wiuf C, Alsner J, et al. Development of a hypoxia gene expression classifier with predictive impact for hypoxic modification of radiotherapy in head and neck cancer. Cancer Res. 2011;71(17):5923–31. https://doi.org/10.1158/0008-5472.CAN-11-1182.
Overgaard J, Sand Hansen H, Overgaard M, Bastholt L, Berthelsen A, Specht L, et al. A randomized double-blind phase III study of nimorazole as a hypoxic radiosensitizer of primary radiotherapy in supraglottic larynx and pharynx carcinoma. Results of the Danish Head and Neck Cancer Study (DAHANCA) Protocol 5-85. Radiother Oncol. 1998;46(2):135–46. https://doi.org/10.1016/S0167-8140(97)00220-X.
Toustrup K, Sørensen BS, Alsner J, Overgaard J. Hypoxia gene expression signatures as prognostic and predictive markers in head and neck radiotherapy. Semin Radiat Oncol. 2012;22(2):119–27. https://doi.org/10.1016/j.semradonc.2011.12.006.
Toustrup K, Sørensen BS, Lassen P, Wiuf C, Alsner J, Overgaard J. Gene expression classifier predicts for hypoxic modification of radiotherapy with nimorazole in squamous cell carcinomas of the head and neck. Radiother Oncol. 2012;102(1):122–9. https://doi.org/10.1016/j.radonc.2011.09.010.
Hassan Metwally MA, Ali R, Kuddu M, Shouman T, Strojan P, Iqbal K, et al. IAEA-HypoX. A randomized multicenter study of the hypoxic radiosensitizer nimorazole concomitant with accelerated radiotherapy in head and neck squamous cell carcinoma. Radiother Oncol. 2015;116(1):15–20. https://doi.org/10.1016/j.radonc.2015.04.005.
Mohindra JK, Rauth AM. Increased cell killing by metronidazole and nitrofurazone of hypoxic compared to aerobic mammalian cells. Cancer Res. 1976;36(3):930–6.
Ahn G-O, Brown M. Targeting tumors with hypoxia-activated cytotoxins. Front Biosci J Virtual Libr. 2007;12(8-12):3483–501. https://doi.org/10.2741/2329.
Mistry IN, Thomas M, Calder EDD, Conway SJ, Hammond EM. Clinical advances of hypoxia-activated prodrugs in combination with radiation therapy. Int J Radiat Oncol. 2017;98(5):1183–96. https://doi.org/10.1016/j.ijrobp.2017.03.024.
Le Q-T, Taira A, Budenz S, Dorie MJ, Goffinet DR, Fee WE, et al. Mature results from a randomized Phase II trial of cisplatin plus 5-fluorouracil and radiotherapy with or without tirapazamine in patients with resectable stage IV head and neck squamous cell carcinomas. Cancer. 2006;106(9):1940–9. https://doi.org/10.1002/cncr.21785.
Peters LJ, O’Sullivan B, Giralt J, Fitzgerald TJ, Trotti A, Bernier J, et al. Critical impact of radiotherapy protocol compliance and quality in the treatment of advanced head and neck cancer: results from TROG 02.02. J Clin Oncol. 2010;28(18):2996–3001. https://doi.org/10.1200/JCO.2009.27.4498.
Hay M, Hicks K, Wilson W. Discovery of the hypoxia-activated prodrug SN30000. In: Comprehensive medicinal chemistry III. 3rd ed. Amsterdam: Elsevier; 2017. p. 58–94.
Mao X, McManaway S, Jaiswal JK, Hong CR, Wilson WR, Hicks KO. Schedule-dependent potentiation of chemotherapy drugs by the hypoxia-activated prodrug SN30000. Cancer Biol Ther. 2019;20(9):1258–69. https://doi.org/10.1080/15384047.2019.1617570.
Liu Q, Sun JD, Wang J, Ahluwalia D, Baker AF, Cranmer LD, et al. TH-302, a hypoxia-activated prodrug with broad in vivo preclinical combination therapy efficacy: optimization of dosing regimens and schedules. Cancer Chemother Pharmacol. 2012;69(6):1487–98. https://doi.org/10.1007/s00280-012-1852-8.
McKeage MJ, Jameson MB, Ramanathan RK, Rajendran J, Gu Y, Wilson WR, et al. PR-104 a bioreductive pre-prodrug combined with gemcitabine or docetaxel in a phase Ib study of patients with advanced solid tumours. BMC Cancer. 2012;12(1):496. https://doi.org/10.1186/1471-2407-12-496.
Sun JD, Liu Q, Wang J, Ahluwalia D, Ferraro D, Wang Y, et al. Selective tumor hypoxia targeting by hypoxia-activated prodrug TH-302 inhibits tumor growth in preclinical models of cancer. Clin Cancer Res. 2012;18(3):758–70. https://doi.org/10.1158/1078-0432.CCR-11-1980.
Meng F, Evans JW, Bhupathi D, Banica M, Lan L, Lorente G, et al. Molecular and cellular pharmacology of the hypoxia-activated prodrug TH-302. Mol Cancer Ther. 2012;11(3):740–51. https://doi.org/10.1158/1535-7163.MCT-11-0634.
Hong CR, Dickson BD, Jaiswal JK, Pruijn FB, Hunter FW, Hay MP, et al. Cellular pharmacology of evofosfamide (TH-302): A critical re-evaluation of its bystander effects. Biochem Pharmacol. 2018;156:265–80. https://doi.org/10.1016/j.bcp.2018.08.027.
Hong CR, Wilson WR, Hicks KO. An intratumor pharmacokinetic/pharmacodynamic model for the hypoxia-activated prodrug evofosfamide (TH-302): Monotherapy activity is not dependent on a bystander effect. Neoplasia. 2019;21(2):159–71. https://doi.org/10.1016/j.neo.2018.11.009.
Chawla SP, Cranmer LD, Van Tine BA, Reed DR, Okuno SH, Butrynski JE, et al. Phase II study of the safety and antitumor activity of the hypoxia-activated prodrug TH-302 in combination with doxorubicin in patients with advanced soft tissue sarcoma. J Clin Oncol. 2014;32(29):3299–306. https://doi.org/10.1200/JCO.2013.54.3660.
Borad MJ, Reddy SG, Bahary N, Uronis HE, Sigal D, Cohn AL, et al. Randomized phase II trial of gemcitabine plus TH-302 versus gemcitabine in patients with advanced pancreatic cancer. J Clin Oncol. 2015;33(13):1475–81. https://doi.org/10.1200/JCO.2014.55.7504.
Nytko KJ, Grgic I, Bender S, Ott J, Guckenberger M, Riesterer O, et al. The hypoxia-activated prodrug evofosfamide in combination with multiple regimens of radiotherapy. Oncotarget. 2017;8(14):23702–12. https://doi.org/10.18632/oncotarget.15784.
Peeters SGJA, Zegers CML, Biemans R, Lieuwes NG, van Stiphout RGPM, Yaromina A, et al. TH-302 in combination with radiotherapy enhances the therapeutic outcome and is associated with pretreatment [18F]HX4 hypoxia PET imaging. Clin Cancer Res. 2015;21(13):2984–92. https://doi.org/10.1158/1078-0432.CCR-15-0018.
Hajj C, Russell J, Hart CP, Goodman KA, Lowery MA, Haimovitz-Friedman A, et al. A combination of radiation and the hypoxia-activated prodrug evofosfamide (TH-302) is efficacious against a human orthotopic pancreatic tumor model. Transl Oncol. 2017;10(5):760–5. https://doi.org/10.1016/j.tranon.2017.06.010.
Jameson MB, Rischin D, Pegram M, Gutheil J, Patterson AV, Denny WA, et al. A phase I trial of PR-104, a nitrogen mustard prodrug activated by both hypoxia and aldo-keto reductase 1C3, in patients with solid tumors. Cancer Chemother Pharmacol. 2010;65(4):791–801. https://doi.org/10.1007/s00280-009-1188-1.
Konopleva M, Thall PF, Yi CA, Borthakur G, Coveler A, Bueso-Ramos C, et al. Phase I/II study of the hypoxia-activated prodrug PR104 in refractory/relapsed acute myeloid leukemia and acute lymphoblastic leukemia. Haematologica. 2015;100(7):927–34. https://doi.org/10.3324/haematol.2014.118455.
Abou-Alfa GK, Chan SL, Lin C-C, Chiorean EG, Holcombe RF, Mulcahy MF, et al. PR-104 plus sorafenib in patients with advanced hepatocellular carcinoma. Cancer Chemother Pharmacol. 2011;68(2):539–45. https://doi.org/10.1007/s00280-011-1671-3.
McKeage MJ, Gu Y, Wilson WR, Hill A, Amies K, Melink TJ, et al. A phase I trial of PR-104, a pre-prodrug of the bioreductive prodrug PR-104A, given weekly to solid tumour patients. BMC Cancer. 2011;11(1):432. https://doi.org/10.1186/1471-2407-11-432.
Thiolloy S, Deschoemaeker S, Ongenae N, Gilissen J, Dubois L, Yaromina A, et al. Abstract 4959: CP-506, a next generation hypoxia-activated prodrug, as promising novel anti-cancer therapeutic. Cancer Res. 2018;78:4959. https://doi.org/10.1158/1538-7445.AM2018-4959.
Jayaprakash P, Ai M, Liu A, Budhani P, Bartkowiak T, Sheng J, et al. Targeted hypoxia reduction restores T cell infiltration and sensitizes prostate cancer to immunotherapy. J Clin Invest. 2018;128(11):5137–49. https://doi.org/10.1172/JCI96268.
Patterson AV, Jaiswal J, Carlin K, Abbattista MR, Guise CP, Silva S, et al. Abstract B278: PR610: A novel hypoxia-selective tyrosine kinase inhibitor in phase I clinical trial. Mol Cancer Ther. 2013;12:B278. https://doi.org/10.1158/1535-7163.TARG-13-B278.
Liu SV, Aggarwal C, Brzezniak C, Doebele RC, Gerber DE, Gitlitz B, et al. Phase 2 study of tarloxotinib bromide (TRLX) in patients (pts) with EGFR-Mutant, T790M-Negative NSCLC progressing on an EGFR TKI. J Clin Oncol. 2016;34(15_suppl):TPS9100. https://doi.org/10.1200/JCO.2016.34.15_suppl.TPS9100.
Rischin D, Boyer MJ, Brzezniak CE, Colevas AD, Doebele RC, Gilbert J, et al. A phase 2 study of tarloxotinib bromide (TRLX) in patients (Pts) with recurrent or metastatic (R/M) squamous cell carcinoma of the head and neck (SCCHN) or skin (SCCS). J Clin Oncol. 2016;34(15_suppl):TPS6105. https://doi.org/10.1200/JCO.2016.34.15_suppl.TPS6105.
Hua H, Kong Q, Zhang H, Wang J, Luo T, Jiang Y. Targeting mTOR for cancer therapy. J Hematol Oncol. 2019;12(1):71. https://doi.org/10.1186/s13045-019-0754-1.
Drosten M, Barbacid M. Targeting the MAPK pathway in KRAS-driven tumors. Cancer Cell. 2020;37(4):543–50. https://doi.org/10.1016/j.ccell.2020.03.013.
Greenberger LM, Horak ID, Filpula D, Sapra P, Westergaard M, Frydenlund HF, et al. A RNA antagonist of hypoxia-inducible factor-1α, EZN-2968, inhibits tumor cell growth. Mol Cancer Ther. 2008;7(11):3598–608. https://doi.org/10.1158/1535-7163.MCT-08-0510.
Jeong W, Rapisarda A, Park SR, Kinders RJ, Chen A, Melillo G, et al. Pilot trial of EZN-2968, an antisense oligonucleotide inhibitor of hypoxia-inducible factor-1 alpha (HIF-1α), in patients with refractory solid tumors. Cancer Chemother Pharmacol. 2014;2(2):343–8. https://doi.org/10.1007/s00280-013-2362-z.
Welsh S, Williams R, Kirkpatrick L, Paine-Murrieta G, Powis G. Antitumor activity and pharmacodynamic properties of PX-478, an inhibitor of hypoxia-inducible factor-1α. Mol Cancer Ther. 2004;3(3):233–44.
Koh MY, Spivak-Kroizman T, Venturini S, Welsh S, Williams RR, Kirkpatrick DL, et al. Molecular mechanisms for the activity of PX-478, an antitumor inhibitor of the hypoxia-inducible factor-1α. Mol Cancer Ther. 2008;7(1):90–100. https://doi.org/10.1158/1535-7163.MCT-07-0463.
Palayoor ST, Mitchell JB, Cerna D, DeGraff W, John-Aryankalayil M, Coleman CN. PX-478, an inhibitor of hypoxia-inducible factor-1α, enhances radiosensitivity of prostate carcinoma cells. Int J Cancer. 2008;123(10):2430–7. https://doi.org/10.1002/ijc.23807.
Zhao T, Ren H, Jia L, Chen J, Xin W, Yan F, et al. Inhibition of HIF-1α by PX-478 enhances the anti-tumor effect of gemcitabine by inducing immunogenic cell death in pancreatic ductal adenocarcinoma. Oncotarget. 2014;6(4):2250–62. https://doi.org/10.18632/oncotarget.2948.
Tibes R, Falchook GS, Von Hoff DD, Weiss GJ, Iyengar T, Kurzrock R, et al. Results from a phase I, dose-escalation study of PX-478, an orally available inhibitor of HIF-1α. J Clin Oncol. 2010;28(15_suppl):3076. https://doi.org/10.1200/jco.2010.28.15_suppl.3076.
Rapisarda A, Uranchimeg B, Scudiero DA, Selby M, Sausville EA, Shoemaker RH, et al. Identification of small molecule inhibitors of hypoxia-inducible factor 1 transcriptional activation pathway. Cancer Res. 2002;62(15):4316–24.
Kummar S, Raffeld M, Juwara L, Horneffer Y, Strassberger A, Allen D, et al. Multihistology, target-driven pilot trial of oral topotecan as an inhibitor of hypoxia-inducible factor-1α in advanced solid tumors. Clin Cancer Res. 2011;17(15):5123–31. https://doi.org/10.1158/1078-0432.CCR-11-0682.
Befani CD, Vlachostergios PJ, Hatzidaki E, Patrikidou A, Bonanou S, Simos G, et al. Bortezomib represses HIF-1α protein expression and nuclear accumulation by inhibiting both PI3K/Akt/TOR and MAPK pathways in prostate cancer cells. J Mol Med. 2012;90(1):45–54. https://doi.org/10.1007/s00109-011-0805-8.
Zhu K, Chan W, Heymach J, Wilkinson M, McConkey DJ. Control of HIF-1α expression by eIF2α phosphorylation–mediated translational repression. Cancer Res. 2009;69(5):1836–43. https://doi.org/10.1158/0008-5472.CAN-08-4103.
Kaluz S, Kaluzová M, Stanbridge EJ. Proteasomal inhibition attenuates transcriptional activity of hypoxia-inducible factor 1 (HIF-1) via specific effect on the HIF-1α C-terminal activation domain. Mol Cell Biol. 2006;26(15):5895–907. https://doi.org/10.1128/MCB.00552-06.
Shin DH, Chun Y-S, Lee DS, Huang LE, Park J-W. Bortezomib inhibits tumor adaptation to hypoxia by stimulating the FIH-mediated repression of hypoxia-inducible factor-1. Blood. 2008;111(6):3131–6. https://doi.org/10.1182/blood-2007-11-120576.
Cui H, Qin Q, Yang M, Zhang H, Liu Z, Yang Y, et al. Bortezomib enhances the radiosensitivity of hypoxic cervical cancer cells by inhibiting HIF-1α expression. Int J Clin Exp Pathol. 2015;8(8):9032–41.
Wang D, Qin Q, Jiang Q-J, Wang D-F. Bortezomib sensitizes esophageal squamous cancer cells to radiotherapy by suppressing the expression of HIF-1α and apoptosis proteins. J X-Ray Sci Technol. 2016;24(4):639–46. https://doi.org/10.3233/XST-160571.
Goktas S, Baran Y, Ural AU, Yazici S, Aydur E, Basal S, et al. Proteasome inhibitor bortezomib increases radiation sensitivity in androgen independent human prostate cancer cells. Urology. 2010;75(4):793–8. https://doi.org/10.1016/j.urology.2009.07.1215.
Chiu H-W, Lin S-W, Lin L-C, Hsu Y-H, Lin Y-F, Ho S-Y, et al. Synergistic antitumor effects of radiation and proteasome inhibitor treatment in pancreatic cancer through the induction of autophagy and the downregulation of TRAF6. Cancer Lett. 2015;365(2):229–39. https://doi.org/10.1016/j.canlet.2015.05.025.
Wu Y-H, Wu W-S, Lin L-C, Liu C-S, Ho S-Y, Wang B-J, et al. Bortezomib enhances radiosensitivity in oral cancer through inducing autophagy-mediated TRAF6 oncoprotein degradation. J Exp Clin Cancer Res CR. 2018;37(1):91. https://doi.org/10.1186/s13046-018-0760-0.
Forster C, Schilling D, Wank M, Schmid TE, Combs SE. EP-2323: Effects of the proteasome inhibitor bortezomib on radiosensitivity of glioblastoma cells. Radiother Oncol. 2018;127:S1281–2. https://doi.org/10.1016/S0167-8140(18)32632-X.
Hutt DM, Roth DM, Vignaud H, Cullin C, Bouchecareilh M. The histone deacetylase inhibitor, Vorinostat, represses hypoxia inducible factor 1 alpha expression through translational inhibition. PLoS One. 2014;9(8):e106224. https://doi.org/10.1371/journal.pone.0106224.
Zhang C, Yang C, Feldman MJ, Wang H, Pang Y, Maggio DM, et al. Vorinostat suppresses hypoxia signaling by modulating nuclear translocation of hypoxia inducible factor 1 alpha. Oncotarget. 2017;8(34):56110–25. https://doi.org/10.18632/oncotarget.18125.
Baschnagel A, Russo A, Burgan WE, Carter D, Beam K, Palmieri D, et al. Vorinostat enhances the radiosensitivity of a breast cancer brain metastatic cell line grown in vitro and as intracranial xenografts. Mol Cancer Ther. 2009;8(6):1589–95. https://doi.org/10.1158/1535-7163.MCT-09-0038.
Munshi A, Tanaka T, Hobbs ML, Tucker SL, Richon VM, Meyn RE. Vorinostat, a histone deacetylase inhibitor, enhances the response of human tumor cells to ionizing radiation through prolongation of γ-H2AX foci. Mol Cancer Ther. 2006;5(8):1967–74. https://doi.org/10.1158/1535-7163.MCT-06-0022.
Diss E, Nalabothula N, Nguyen D, Chang E, Kwok Y, Carrier F. Vorinostat promotes hyper-radiosensitivity in wild type p53 human glioblastoma cells. J Clin Oncol Res. 2014;2:1–9.
Saelen MG, Ree AH, Kristian A, Fleten KG, Furre T, Hektoen HH, et al. Radiosensitization by the histone deacetylase inhibitor vorinostat under hypoxia and with capecitabine in experimental colorectal carcinoma. Radiat Oncol. 2012;7(1):165. https://doi.org/10.1186/1748-717X-7-165.
Jonsson M, Ragnum HB, Julin CH, Yeramian A, Clancy T, Frikstad K-AM, et al. Hypoxia-independent gene expression signature associated with radiosensitisation of prostate cancer cell lines by histone deacetylase inhibition. Br J Cancer. 2016;115(8):929–39. https://doi.org/10.1038/bjc.2016.278.
Jhaveri K, Modi S. Ganetespib: research and clinical development. OncoTargets Ther. 2015;8:1849–58. https://doi.org/10.2147/OTT.S65804.
Isaacs JS, Jung Y-J, Mimnaugh EG, Martinez A, Cuttitta F, Neckers LM. Hsp90 regulates a von Hippel Lindau-independent hypoxia-inducible factor-1 alpha-degradative pathway. J Biol Chem. 2002;277(33):29936–44. https://doi.org/10.1074/jbc.M204733200.
Mabjeesh NJ, Post DE, Willard MT, Kaur B, Van Meir EG, Simons JW, et al. Geldanamycin induces degradation of hypoxia-inducible factor 1α protein via the proteosome pathway in prostate cancer cells. Cancer Res. 2002;62(9):2478–82.
Hollingshead M, Alley M, Burger AM, Borgel S, Pacula-Cox C, Fiebig H-H, et al. In vivo antitumor efficacy of 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride), a water-soluble geldanamycin derivative. Cancer Chemother Pharmacol. 2005;56(2):115–25. https://doi.org/10.1007/s00280-004-0939-2.
Park Y-S, Shin W-S, Kim C-S, Ahn CM, Qi X-F, Kim S-K. Molecular and cellular toxicological profiling of DNA bis-intercalator, quinoxaline compounds: echinomycin as the versatile lead. Mol Cell Toxicol. 2018;14(1):9–18. https://doi.org/10.1007/s13273-018-0002-8.
Kong D, Park EJ, Stephen AG, Calvani M, Cardellina JH, Monks A, et al. Echinomycin, a small-molecule inhibitor of hypoxia-inducible factor-1 DNA-binding activity. Cancer Res. 2005;65(19):9047–55. https://doi.org/10.1158/0008-5472.CAN-05-1235.
Wang Y, Liu Y, Bailey C, Zhang H, He M, Sun D, et al. Therapeutic targeting of TP53 -mutated acute myeloid leukemia by inhibiting HIF-1α with echinomycin. Oncogene. 2020;39(14):3015–27. https://doi.org/10.1038/s41388-020-1201-z.
Baker AF, Dragovich T, Tate WR, Ramanathan RK, Roe D, Hsu C-H, et al. The antitumor thioredoxin-1 inhibitor PX-12 (1-methylpropyl 2-imidazolyl disulfide) decreases thioredoxin-1 and VEGF levels in cancer patient plasma. J Lab Clin Med. 2006;147(2):83–90. https://doi.org/10.1016/j.lab.2005.09.001.
Kim YH, Coon A, Baker AF, Powis G. Antitumor agent PX-12 inhibits HIF-1α protein levels through an Nrf2/PMF-1-mediated increase in spermidine/spermine acetyl transferase. Cancer Chemother Pharmacol. 2011;68(2):405–13. https://doi.org/10.1007/s00280-010-1500-0.
You BR, Shin HR, Park WH. PX-12 inhibits the growth of A549 lung cancer cells via G2/M phase arrest and ROS-dependent apoptosis. Int J Oncol. 2014;44(1):301–8. https://doi.org/10.3892/ijo.2013.2152.
You BR, Shin HR, Han BR, Park WH. PX-12 induces apoptosis in Calu-6 cells in an oxidative stress-dependent manner. Tumor Biol. 2015;36(3):2087–95. https://doi.org/10.1007/s13277-014-2816-x.
Ramanathan RK, Abbruzzese J, Dragovich T, Kirkpatrick L, Guillen JM, Baker AF, et al. A randomized phase II study of PX-12, an inhibitor of thioredoxin in patients with advanced cancer of the pancreas following progression after a gemcitabine-containing combination. Cancer Chemother Pharmacol. 2011;67(3):503–9. https://doi.org/10.1007/s00280-010-1343-8.
Kung AL, Zabludoff SD, France DS, Freedman SJ, Tanner EA, Vieira A, et al. Small molecule blockade of transcriptional coactivation of the hypoxia-inducible factor pathway. Cancer Cell. 2004;6(1):33–43. https://doi.org/10.1016/j.ccr.2004.06.009.
Jin Z, Aixi Y, Baiwen Q, Zonghuan L, Xiang H. Inhibition of hypoxia-inducible factor-1 alpha radiosensitized MG-63 human osteosarcoma cells in vitro. Tumori J. 2015;101(5):578–84. https://doi.org/10.5301/tj.5000243.
Staab A, Loeffler J, Said HM, Diehlmann D, Katzer A, Beyer M, et al. Effects of HIF-1 inhibition by chetomin on hypoxia-related transcription and radiosensitivity in HT 1080 human fibrosarcoma cells. BMC Cancer. 2007;7(1):213. https://doi.org/10.1186/1471-2407-7-213.
Kessler J, Hahnel A, Wichmann H, Rot S, Kappler M, Bache M, et al. HIF-1α inhibition by siRNA or chetomin in human malignant glioma cells: effects on hypoxic radioresistance and monitoring via CA9 expression. BMC Cancer. 2010;10(1):605. https://doi.org/10.1186/1471-2407-10-605.
Min S, Wang X, Du Q, Gong H, Yang Y, Wang T, et al. Chetomin, a Hsp90/HIF1α pathway inhibitor, effectively targets lung cancer stem cells and non-stem cells. Cancer Biol Ther. 2020;21(8):1–11. https://doi.org/10.1080/15384047.2020.1763147.
DiGiacomo JW, Gilkes DM. Therapeutic strategies to block the hypoxic response. In: Gilkes DM, editor. Hypoxia cancer metastasis. Cham: Springer International Publishing; 2019. p. 141–57. https://doi.org/10.1007/978-3-030-12734-3_10.
Li Z, You Q, Zhang X. Small-molecule modulators of the hypoxia-inducible factor pathway: Development and therapeutic applications. J Med Chem. 2019;62(12):5725–49. https://doi.org/10.1021/acs.jmedchem.8b01596.
Courtney KD, Ma Y, de Leon AD, Christie A, Xie Z, Woolford L, et al. HIF-2 complex dissociation, target inhibition, and acquired resistance with PT2385, a first-in-class HIF-2 inhibitor, in patients with clear cell renal cell carcinoma. Clin Cancer Res. 2020;26(4):793–803. https://doi.org/10.1158/1078-0432.CCR-19-1459.
Bhatt RS, Landis DM, Zimmer M, Torregrossa J, Chen S, Sukhatme VP, et al. Hypoxia-inducible factor-2α: effect on radiation sensitivity and differential regulation by an mTOR inhibitor. BJU Int. 2008;102(3):358–63. https://doi.org/10.1111/j.1464-410X.2008.07558.x.
Bertout JA, Majmundar AJ, Gordan JD, Lam JC, Ditsworth D, Keith B, et al. HIF2α inhibition promotes p53 pathway activity, tumor cell death, and radiation responses. Proc Natl Acad Sci. 2009;106(34):14391–6. https://doi.org/10.1073/pnas.0907357106.