Regulation of submaxillary gland androgen-regulated protein 3A via estrogen receptor 2 in radioresistant head and neck squamous cell carcinoma cells
© The Author(s). 2017
Received: 13 October 2016
Accepted: 31 January 2017
Published: 6 February 2017
Molecular mechanisms of intrinsic or acquired radioresistance serve as critical barrier for curative therapy of head and neck squamous cell carcinoma (HNSCC) and remain a major obstacle for progression-free and disease-specific survival.
HNSCC cell lines were treated with a protocol of fractionated irradiation (IR, 4× 2Gy) alone or in combination with antagonists of estrogen receptor signaling and viability was determined by a colony-forming assay (CFA). Expression of submaxillary gland androgen-regulated protein 3A (SMR3A) and estrogen receptor 2 (ESR2) were assessed in tumor cells in vitro by RQ-PCR, Western blot analysis and immunofluorescence staining, and by immunohistochemical staining of tissue microarrays containing tumor sections from patients with oropharyngeal squamous cell carcinoma (OPSCC), which were treated by definitive or adjuvant radiotherapy. Subgroups with distinct SMR3A and ESR2 expression patterns were correlated with clinical parameters and survival outcome including multivariable analysis.
Fractionated irradiation (IR) revealed an accumulation of tumor cells with prominent SMR3A expression, which was accompanied by an up-regulation of the estrogen receptor 2 (ESR2). ESR2-dependent regulation of SMR3A was supported by induced expression after stimulation with estradiol (E2), which was impaired by co-treatment with 4-Hydroxytamoxifen (TAM) or Fulvestrant, respectively. Both drugs significantly sensitized FaDu cells to fractionated IR as determined by a CFA and accelerated apoptosis. These data suggest a critical role of ESR2 in radioresistance and that SMR3A might serve as a surrogate marker for active ESR2 signaling. In line with this assumption, ESR2-positive oropharyngeal squamous cell carcinoma (OPSCC) with high SMR3A expression had an unfavorable progression-free and disease-specific survival as compared to those tumors with low SMR3A expression.
In summary, our findings provide compelling experimental evidence that HNSCC with SMR3A and ESR2 co-expression have a higher risk for treatment failure and these patients might benefit from clinically well-established drugs targeting estrogen receptor signaling.
KeywordsESR2 Estrogen Fulvestrant HNSCC OPSCC Radiotherapy SMR3A Tamoxifen
Head and neck cancer is one of the most prevalent human malignancies with an annual incidence of approximately 600,000 new cases worldwide . The majority are head and neck squamous cell carcinoma (HNSCC) originating from the mucosal epithelia of the upper aerodigestive tract. HNSCC is a rather heterogeneous disease and despite aggressive, multimodal treatment of locally advanced tumors a significant proportion of patients develop disease recurrence due to either local or distant failure . Given that many patients with a recurrent HNSCC are no longer amenable to curative therapy, the ensuing morbidity is high and survival is dismal . Consequently, appropriate treatment of HNSCC patients remains a major challenge and there is an urgent demand in a better understanding of molecular principles underlying treatment failure.
In the past, we applied global gene expression profiling on matched samples of primary and recurrent tumors of an orthotopic squamous cell carcinoma model in mice to identify differentially expressed genes . One candidate gene with an increased transcript level in recurrent as compared to primary tumors encoded for the mouse homologue of human submaxillary gland androgen-regulated protein 3A (SMR3A), which belongs to the opiorphin gene family . More recently, SMR3A expression was detected in a subgroup of patients with primary oropharyngeal squamous cell carcinoma (OPSCC) and served as an independent risk factor for unfavorable prognosis . However, the regulation of SMR3A and its putative mode of action in the pathogenesis of HNSCC or in response to treatment have not been addressed, so far.
In the current study, we demonstrate prominent SMR3A expression in tumor cells upon fractionated irradiation (IR) and provide experimental evidence that induced SMR3A expression serves as a surrogate maker for active estrogen receptor 2 (ESR2) signaling in radioresistant tumor cells.
Cell culture and stable clones
Human cell lines (FaDu and Cal27) were purchased from ATCC (http://www.lgcstandards-atcc.org/) and were maintained in Dulbecco’s Modified Eagle’s Medium (Sigma-Aldrich, Germany) supplemented with 10% fetal bovine serum (Invitrogen, Germany), 2 mM L-Glutamine (Invitrogen, Germany) and 50 μg/ml Penicillin-Streptomycin (Invitrogen, Germany) in a humidified atmosphere of 6% CO2 at 37 °C. Authentication of both cell lines was confirmed by the Multiplex Human Cell Line Authentication Test (Multiplexion, Germany, latest update August 11th, 2016). To generate single cell clones, FaDu cells were transfected with either pcDNA3.1 (Invitrogen, USA) or a plasmid encoding a Myc-DDK-tagged ORF of Homo sapiens SMR3A (Origene, USA) using FuGene HD Transfection Reagent (Promega, Germany) according to the manufacturer’s instruction. Following selection with 0.5–0.8 mg/ml G418 (Calbiochem Merck, Germany) for two weeks, single clones were isolated and ectopic SMR3A-Myc/DDK expression was confirmed on transcript and protein level. Two independent mock controls and FaDu-SMR3A clones were selected for further analysis. Parental FaDu and Cal27 cells were treated with the indicated concentrations of estradiol (E2) or 4-Hydroxytamoxifen (TAM) dissolved in ethanol (EtOH) or Fulvestrant dissolved in DMSO. All compounds were purchased from Sigma-Aldrich, Germany.
RNA extraction, cDNA synthesis and real-time quantitative polymerase chain reaction (RQ-PCR) were done as described previously . Primer sequences were selected using Primer-Blast (http://www.ncbi.nlm.nih.gov/tools/primer-blast) and are listed in Additional file 1. To quantify relative SMR3A transcript levels by RQ-PCR analysis cDNA samples were used undiluted. The cycle of threshold (CT) of the gene of interest was standardized to the CT value of the reference gene (LMNB1) using the ∆∆CT method.
Western blot analysis and immunofluorescence staining
Western blot analysis was performed with whole cell lysates as described previously . Membranes were incubated in enhanced chemiluminiscence solution (Thermo Scientific, Germany) and developed with the ImageQuant LAS500 system (GE Healthcare, Germany). Immunofluorescence staining was done as described elsewhere , and pictures were taken with the Fluorescence Microscope BX50F, Olympus XC30 Camera and cellSens Entry imaging software (Olympus, Germany). Antibodies and dilutions that were used for Western blot analysis and immunofluorescence staining are listed in Additional file 2.
20–30,000 cells per well were seeded on glass coverslips in 12-well plates. 48 h after seeding, cells were treated for 2 h with BrdU according to the manufacture’s instruction (BD Pharmingen, Germany). Staining was done as described previously [7, 8] and relative amount of proliferative cells was calculated as the ratio of BrdU-positive versus total cell counts.
Cell migration assays were performed using ibidi Culture Inserts (Ibidi, Germany) according to the instruction of the manufacturer. After removal of the inserts, pictures were taken at the indicated time points using the Keyence BZ-9000 microscope system to calculate the relative gap closure over time.
Colony-formation assay (CFA)
To determine the clonal expansion of tumor cells upon fractionated irradiation, 100, 300 and 1,000 cells were seeded per well in 6-well plates and irradiated on four consecutive days with a daily dose of 2 gray (Gy) using X-RAD 320 (Precision X-Ray, North Branford, CT USA) or kept untreated as controls. Half of the cells were treated daily with 1 μM 4-Hydroxytamoxifen (TAM, Sigma-Adrich, Germany) or every second day with 30 nM Fulvestrant (Sigma-Adrich, Germany). After 10–14 days in culture, clones were stained with crystal violet and total amount of colonies was quantified as described in . The survival fraction was computed according to .
Patient samples and immunohistochemistry
The retrospective study cohort, generation of tissue microarrays, immunohistochemical staining and assessment of the immunoreactivity score for SMR3A was described before . Paraffin-embedded tissue specimens were provided by the tissue bank of the National Center for Tumor Disease (Institute of Pathology, University Hospital Heidelberg) after approval by the local institutional review board (ethic votes: 176/2002 and 206/2005). The study was performed according to the ethical standards of the Declaration of Helsinki. For all tumor samples, clinical and follow-up data were available from the Department of Otolaryngology, Head and Neck Surgery at the University Hospital Heidelberg. Immunohistochemical staining for ESR2 was done using the 3, 3’-diaminobenzidine (DAB) peroxidase substrate kit (Perkin Elmer, Germany) according to the manufacturer’s instruction and the assessment of the immunoreactivity score for ESR2 was according to the procedure described before . Clinical and histopathological features of samples for which informative values for SMR3A and ESR2 were available and which were included in the final analysis are listed in Additional file 3.
Statistical analysis was done using SPSS (version 21) and GraphPad software (http://www.graphpad.com). Differences between patient subgroups were assessed using Chi square test. Disease-specific survival (DSS) was calculated as the time from the date of primary tumor diagnosis to the date of cancer-related death within the follow-up interval (events). Survival times of patients, who were alive or were dead due to causes other than cancer were censored. Progression-free survival (PFS) was calculated from the date of primary tumor diagnosis to the date of the first local recurrence, lymph node or distant metastasis, second primary carcinoma, or date of cancer-related death within the follow-up period (events). Patients without progression (no event) or cancer-unrelated death were censored. The method of Kaplan–Meier was used to estimate survival distributions and differences between subgroups were determined by log-rank tests. To adjust for possible confounders, multivariable Cox proportional hazard regression models were fitted. Models included the covariates gender, age, clinical staging, alcohol and tobacco consumption, HPV status and therapy. Based on availability of complete clinical data sets, 66 cases were included for the analysis of subgroups with ESRhighSMR3Alow versus ESRhighSMR3Ahigh staining patterns and 103 cases were included for the analysis of subgroups with ESRhighSMR3Alow versus all other staining patterns.
In all statistical tests, a p-value of 0.05 or below was considered as statistically significant.
Establishment and analysis of a HNSCC cell line with ectopic SMR3A expression
SMR3A expression was assessed by RQ-PCR analysis with cDNA from human HNSCC cell lines. Transcript levels were close to the detection limit in all cell lines tested, indicating no or a rather low SMR3A expression under normal growth conditions (data not shown). FaDu cells were selected to generate stable clones (FaDu-SMR3A) with ectopic SMR3A expression in order to address its impact on tumor-relevant processes in vitro. Ectopic expression in FaDu-SMR3A clones was confirmed on transcript and protein levels (Additional file 4a-c). However, we did not observe any significant difference in tumor cell proliferation or migration between FaDu-SMR3A clones and mock controls (Additional file 4d-e). In summary, these data suggested that SMR3A has no major impact on tumor cell physiology under normal growth conditions but raised the attractive question, whether it serves as a marker for a distinct subpopulation of tumor cells with higher resistance against well-established treatment options.
SMR3A expression in HNSCC cells upon fractionated irradiation
SMR3A is a downstream target of ESR2 signaling
Inhibition of ESR2 signaling in combination with fractionated IR
Co-expression of ESR2 and SMR3A in tumor samples and correlation with clinical features
Radiotherapy remains a mainstay of local treatment for HNSCC and improvements in intensity-modulated techniques and new protocols of altered fractionation have contributed to reduced mortality and long-term morbidity with a strong impact on the quality of life . However, intrinsic and acquired resistance remains a major obstacle and serves as a critical barrier for curative treatment of cancer patients, including HNSCC . Unraveling molecular principles of radioresistance and subsequent clonal expansion of vital tumor cells is a crucial task to establish prognostic biomarkers for HNSCC patients with a higher risk for treatment failure and to identify new drug targets for more efficient and less toxic therapy.
In this study, we unraveled prominent SMR3A expression in vital tumor cells upon fractionated IR. Although ectopic SMR3A expression had no significant impact on tumor-relevant processes under normal growth conditions in vitro, presented data provide compelling experimental evidence that it might serve as a surrogate marker for a subpopulation of resistant cells as a putative source for tumor relapse after radiotherapy. In line with this assumption, a previous retrospective study unraveled high SMR3A expression as a risk factor for unfavorable progression-free and overall survival in a cohort of OPSCC patients .
Studies in rodents revealed a positive regulation of opiorphin family members by hormone signaling [11–13], and our findings indicate that ESR2 signaling not only induces SMR3A expression but also plays a critical role in resistance to treatment. This assumption is further supported by recent reports demonstrating that the presence of ESR2 modulates response to several therapeutic agents in breast and lung cancer cells [16–18]. So far, only a limited number of studies focused on estrogen receptor signaling in the pathogenesis or prognosis of HNSCC. Already in 1988, Somers and colleagues demonstrated that estrogen treatment potentiated growth of laryngeal tumors in a xenograft model in vivo . More recently, a functional crosstalk between estrogen and EGF receptor signaling was reported, which might contribute to neoplastic transformation and disease progression of HNSCC . Conflicting data were published with regard to the expression pattern of ESR1 and ESR2 in HNSCC and their correlation with clinical outcome [20–23]. However, in line with our data two studies found a frequent expression of ESR2 but not ESR1 in HNSCC cell lines and tumor tissues [21, 22], suggesting a more prominent role of ESR2-related signaling.
In contrast to ESR1, ESR2 is usually described as a tumor suppressor in estrogen-sensitive malignancies . However, we detected a positive staining for ESR2 expression as a common event in OPSCC, while ESR1-positive tumor cells were a rather rare event (data not shown). In line with our hypothesis that SMR3A serves as a surrogate marker for active ESR2 signaling and predicts treatment resistance, the combined expression of both proteins was associated with an unfavorable clinical outcome concerning progression-free and disease-specific survival after definitive or adjuvant radiotherapy. Although multivariable Cox proportional hazard regression models supported the assumption that ESR2 and SMR3A staining patterns are associated with clinical outcome, our retrospective study is limited by the low amount of patients for distinct subgroups. A major challenge for the future will be the analysis of ESR2 and SMR3A but also other downstream targets in larger cohort studies, including specimens from HNSCC of other locations.
It is worth noting that OPSCC without detectable ESR2 expression were also correlated with a poor survival, indicating that the prognostic value of ESR2 expression is context dependent and that ESR2-negative and positive tumors represent two distinct subgroups of HNSCC which most likely differ in their cellular and molecular traits. In the past, the prognostic value of ESR2 expression in other human malignancies, including breast, ovarian, bladder, prostate and lung cancer, was controversially discussed further supporting a strong context dependency [17, 25–33]. Nevertheless, a negative staining in a tumor biopsy taken at the time point of diagnosis or during primary surgery does not exclude induction and/or expansion of ESR2-positive tumor cells during fractionated radiotherapy. Innovative tools, such as irradiation of ex vivo cultures derived from vital tumor tissue [34, 35], might be appropriate pre-clinical models to address this issue and could help to select individual patients for new treatment options.
In summary, our data suggest that HNSC’C with a combined ESR2 and SMR3A expression are at a higher risk for treatment failure upon radiotherapy, but might benefit from treatment with TAM or Fulvestrant, two clinically well-established inhibitors targeting estrogen receptor signaling . Indeed, both drugs significantly sensitized tumor cells to fractionated IR and an accelerated sensitivity to chemotherapy after administration of Fulvestrant was demonstrated recently for of estrogen receptor positive breast and lung cancer cells [37, 38]. However, additional cohort studies and preclinical models are required to confirm the clinical relevance of our findings and as a proof-of-concept for the efficacy of TAM or Fulvestrant in combination with radiotherapy for HNSCC patients.
Estrogen receptor 1
Estrogen receptor 2
Head and neck squamous cell carcinoma
Oropharyngeal squamous cell carcinoma
Submaxillary gland androgen-regulated protein 3A
We gratefully acknowledge the excellent technical support by Leoni Erdinger, Ines Kaden, Nataly Henfling, Antje Schuhmann and Ingeborg Vogt. We thank the tissue bank of the National Center for Tumor Disease (Institute of Pathology, University Hospital Heidelberg) for providing tumor specimens of OPSCC patients. We acknowledge the financial support of the China Scholarship Council for a PhD fellowship to CR, and the German Research Foundation and Ruprecht-Karls-Universität Heidelberg within the funding program Open Access Publishing.
This work was in part supported by the German Research Foundation (HE 5760/3-1 to JH) and the German Cancer Aid (70112000 to JH).
Availability of data and materials
The datasets supporting the conclusions of this article are included within the article and its additional files.
JG and CR performed experiments, data acquisition and statistical analysis; CF, KZ and PKP provided and evaluated patient samples, clinical and pathological data; KJW provided technical support and access to equipment; JH devised the study and was responsible for its conceptual design; JG and JH wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
Human tissue specimens were collected and provided by the tissue bank of the National Center for Tumor Disease (Institute of Pathology, University Hospital Heidelberg) after approval by the local institutional review board of the Medical Faculty Heidelberg (ethic votes: 176/2002 and 206/2005). The study was performed according to the ethical standards of the Declaration of Helsinki.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Ferlay J, Soerjomataram I, Ervik M, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray F: GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 11 [Internet]. Lyon, France: International Agency for Research on Cancer Available from: http://globocan.iarc.fr/. Accessed 10 June 2016.
- Leemans CR, Braakhuis BJ, Brakenhoff RH. The molecular biology of head and neck cancer. Nat Rev Cancer. 2011;11:9–22.View ArticlePubMedGoogle Scholar
- Sacco AG, Cohen EE. Current treatment options for recurrent or metastatic head and neck squamous cell carcinoma. J Clin Oncol. 2015;33:3305–13.View ArticlePubMedGoogle Scholar
- Acuna Sanhueza GA, Faller L, George B, Koffler J, Misetic V, Flechtenmacher C, Dyckhoff G, Plinkert PP, Angel P, Simon C, Hess J. Opposing function of MYBBP1A in proliferation and migration of head and neck squamous cell carcinoma cells. BMC Cancer. 2012;12:72.View ArticlePubMedPubMed CentralGoogle Scholar
- Wisner A, Dufour E, Messaoudi M, Nejdi A, Marcel A, Ungeheuer MN, Rougeot C. Human Opiorphin, a natural antinociceptive modulator of opioid-dependent pathways. Proc Natl Acad Sci U S A. 2006;103:17979–84.View ArticlePubMedPubMed CentralGoogle Scholar
- Koffler J, Holzinger D, Sanhueza GA, Flechtenmacher C, Zaoui K, Lahrmann B, Grabe N, Plinkert PK, Hess J. Submaxillary gland androgen-regulated protein 3A expression is an unfavorable risk factor for the survival of oropharyngeal squamous cell carcinoma patients after surgery. Eur Arch Otorhinolaryngol. 2013;270:1493–500.View ArticlePubMedGoogle Scholar
- Schrader CH, Kolb M, Zaoui K, Flechtenmacher C, Grabe N, Weber KJ, Hielscher T, Plinkert PK, Hess J. Kallikrein-related peptidase six regulates epithelial-to-mesenchymal transition and serves as prognostic biomarker for head and neck squamous cell carcinoma patients. Mol Cancer. 2015;14:107.View ArticlePubMedPubMed CentralGoogle Scholar
- Bayo P, Jou A, Stenzinger A, Shao C, Gross M, Jensen A, Grabe N, Mende CH, Rados PV, Debus J, et al. Loss of SOX2 expression induces cell motility via vimentin up-regulation and is an unfavorable risk factor for survival of head and neck squamous cell carcinoma. Mol Oncol. 2015;9:1704–19.View ArticlePubMedGoogle Scholar
- Niyazi M, Niyazi I, Belka C. Counting colonies of clonogenic assays by using densitometric software. Radiat Oncol. 2007;2:4.View ArticlePubMedPubMed CentralGoogle Scholar
- Franken NA, Rodermond HM, Stap J, Haveman J, van Bree C. Clonogenic assay of cells in vitro. Nat Protoc. 2006;1:2315–9.View ArticlePubMedGoogle Scholar
- Rosinski-Chupin I, Tronik D, Rougeon F. High level of accumulation of a mRNA coding for a precursor-like protein in the submaxillary gland of male rats. Proc Natl Acad Sci U S A. 1988;85:8553–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Senorale-Pose M, Jacqueson A, Rougeon F, Rosinski-Chupin I. Acinar cells are target cells for androgens in mouse submandibular glands. J Histochem Cytochem. 1998;46:669–78.View ArticlePubMedGoogle Scholar
- Chua RG, Calenda G, Zhang X, Siragusa J, Tong Y, Tar M, Aydin M, DiSanto ME, Melman A, Davies KP. Testosterone regulates erectile function and Vcsa1 expression in the corpora of rats. Mol Cell Endocrinol. 2009;303:67–73.View ArticlePubMedPubMed CentralGoogle Scholar
- Gregoire V, Langendijk JA, Nuyts S. Advances in radiotherapy for head and neck cancer. J Clin Oncol. 2015;33:3277–84.View ArticlePubMedGoogle Scholar
- Barker HE, Paget JT, Khan AA, Harrington KJ. The tumour microenvironment after radiotherapy: mechanisms of resistance and recurrence. Nat Rev Cancer. 2015;15:409–25.View ArticlePubMedPubMed CentralGoogle Scholar
- Pons DG, Torrens-Mas M, Nadal-Serrano M, Sastre-Serra J, Roca P, Oliver J. The presence of estrogen receptor beta modulates the response of breast cancer cells to therapeutic agents. Int J Biochem Cell Biol. 2015;66:85–94.View ArticlePubMedGoogle Scholar
- Wang Z, Li Z, Ding X, Shen Z, Liu Z, An T, Duan J, Zhong J, Wu M, Zhao J, et al. ERbeta localization influenced outcomes of EGFR-TKI treatment in NSCLC patients with EGFR mutations. Sci Rep. 2015;5:11392.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhang G, Yanamala N, Lathrop KL, Zhang L, Klein-Seetharaman J, Srinivas H. Ligand-independent antiapoptotic function of estrogen receptor-beta in lung cancer cells. Mol Endocrinol. 2010;24:1737–47.View ArticlePubMedPubMed CentralGoogle Scholar
- Somers KD, Koenig M, Schechter GL. Growth of head and neck squamous cell carcinoma in nude mice: potentiation of laryngeal carcinoma by 17 beta-estradiol. J Natl Cancer Inst. 1988;80:688–91.View ArticlePubMedGoogle Scholar
- Egloff AM, Rothstein ME, Seethala R, Siegfried JM, Grandis JR, Stabile LP. Cross-talk between estrogen receptor and epidermal growth factor receptor in head and neck squamous cell carcinoma. Clin Cancer Res. 2009;15:6529–40.View ArticlePubMedPubMed CentralGoogle Scholar
- Doll C, Arsenic R, Lage H, Johrens K, Hartwig S, Nelson K, Raguse JD. Expression of estrogen receptors in oscc in relation to histopathological grade. Anticancer Res. 2015;35:5867–72.PubMedGoogle Scholar
- Shatalova EG, Klein-Szanto AJ, Devarajan K, Cukierman E, Clapper ML. Estrogen and cytochrome P450 1B1 contribute to both early- and late-stage head and neck carcinogenesis. Cancer Prev Res (Phila). 2011;4:107–15.View ArticleGoogle Scholar
- Lukits J, Remenar E, Raso E, Ladanyi A, Kasler M, Timar J. Molecular identification, expression and prognostic role of estrogen- and progesterone receptors in head and neck cancer. Int J Oncol. 2007;30:155–60.PubMedGoogle Scholar
- Dey P, Barros RP, Warner M, Strom A, Gustafsson JA. Insight into the mechanisms of action of estrogen receptor beta in the breast, prostate, colon, and CNS. J Mol Endocrinol. 2013;51:T61–74.View ArticlePubMedGoogle Scholar
- Tan W, Li Q, Chen K, Su F, Song E, Gong C. Estrogen receptor beta as a prognostic factor in breast cancer patients: a systematic review and meta-analysis. Oncotarget. 2016;7:10373–85.PubMedPubMed CentralGoogle Scholar
- Honma N, Horii R, Iwase T, Saji S, Younes M, Takubo K, Matsuura M, Ito Y, Akiyama F, Sakamoto G. Clinical importance of estrogen receptor-beta evaluation in breast cancer patients treated with adjuvant tamoxifen therapy. J Clin Oncol. 2008;26:3727–34.View ArticlePubMedGoogle Scholar
- Hopp TA, Weiss HL, Parra IS, Cui Y, Osborne CK, Fuqua SA. Low levels of estrogen receptor beta protein predict resistance to tamoxifen therapy in breast cancer. Clin Cancer Res. 2004;10:7490–9.View ArticlePubMedGoogle Scholar
- van Kruchten M, van der Marel P, de Munck L, Hollema H, Arts H, Timmer-Bosscha H, de Vries E, Hospers G, Reyners A. Hormone receptors as a marker of poor survival in epithelial ovarian cancer. Gynecol Oncol. 2015;138:634–9.View ArticlePubMedGoogle Scholar
- Kauffman EC, Robinson BD, Downes M, Marcinkiewicz K, Vourganti S, Scherr DS, Gudas LJ, Mongan NP. Estrogen receptor-beta expression and pharmacological targeting in bladder cancer. Oncol Rep. 2013;30:131–8.PubMedPubMed CentralGoogle Scholar
- Zellweger T, Sturm S, Rey S, Zlobec I, Gsponer JR, Rentsch CA, Terracciano LM, Bachmann A, Bubendorf L, Ruiz C. Estrogen receptor beta expression and androgen receptor phosphorylation correlate with a poor clinical outcome in hormone-naive prostate cancer and are elevated in castration-resistant disease. Endocr Relat Cancer. 2013;20:403–13.View ArticlePubMedGoogle Scholar
- Nakajima Y, Akaogi K, Suzuki T, Osakabe A, Yamaguchi C, Sunahara N, Ishida J, Kako K, Ogawa S, Fujimura T, et al. Estrogen regulates tumor growth through a nonclassical pathway that includes the transcription factors ERbeta and KLF5. Sci Signal. 2011;4:ra22.View ArticlePubMedGoogle Scholar
- Stabile LP, Dacic S, Land SR, Lenzner DE, Dhir R, Acquafondata M, Landreneau RJ, Grandis JR, Siegfried JM. Combined analysis of estrogen receptor beta-1 and progesterone receptor expression identifies lung cancer patients with poor outcome. Clin Cancer Res. 2011;17:154–64.View ArticlePubMedGoogle Scholar
- Ma L, Zhan P, Liu Y, Zhou Z, Zhu Q, Miu Y, Wang X, Jin J, Li Q, Lv T, Song Y. Prognostic value of the expression of estrogen receptor beta in patients with non-small cell lung cancer: a meta-analysis. Transl Lung Cancer Res. 2016;5:202–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Affolter A, Muller MF, Sommer K, Stenzinger A, Zaoui K, Lorenz K, Wolf T, Sharma S, Wolf J, Perner S, et al. Targeting irradiation-induced mitogen-activated protein kinase activation in vitro and in an ex vivo model for human head and neck cancer. Head Neck. 2016;38 Suppl 1:E2049–2061.View ArticlePubMedGoogle Scholar
- Freudlsperger C, Horn D, Weissfuss S, Weichert W, Weber KJ, Saure D, Sharma S, Dyckhoff G, Grabe N, Plinkert P, et al. Phosphorylation of AKT(Ser473) serves as an independent prognostic marker for radiosensitivity in advanced head and neck squamous cell carcinoma. Int J Cancer. 2015;136:2775–85.View ArticlePubMedGoogle Scholar
- Osborne CK, Wakeling A, Nicholson RI. Fulvestrant: an oestrogen receptor antagonist with a novel mechanism of action. Br J Cancer. 2004;90 Suppl 1:S2–6.View ArticlePubMedPubMed CentralGoogle Scholar
- Wang J, Yang Q, Haffty BG, Li X, Moran MS. Fulvestrant radiosensitizes human estrogen receptor-positive breast cancer cells. Biochem Biophys Res Commun. 2013;431:146–51.View ArticlePubMedGoogle Scholar
- Hamilton DH, Matthews Griner L, Keller JM, Hu X, Southall N, Marugan J, David JM, Ferrer M, Palena C. Targeting estrogen receptor signaling with fulvestrant enhances immune and chemotherapy-mediated cytotoxicity of human lung cancer. Clin Cancer Res. 2016;22:6204–16.View ArticlePubMedGoogle Scholar