Hedgehog/Gli promotes epithelial-mesenchymal transition in lung squamous cell carcinomas
- Dongsheng Yue†1, 2, 3,
- Hui Li†3,
- Juanjuan Che†4,
- Yi Zhang5,
- Hsin-Hui K Tseng3,
- Joy Q Jin3,
- Thomas M Luh3,
- Etienne Giroux-Leprieur3,
- Minli Mo6,
- Qingfeng Zheng3, 7,
- Huaiyin Shi8,
- Hua Zhang1, 2,
- Xishan Hao1, 2Email author,
- Changli Wang1, 2,
- David M Jablons3 and
- Biao He3Email author
© Yue et al.; licensee BioMed Central Ltd. 2014
Received: 21 February 2014
Accepted: 15 April 2014
Published: 24 April 2014
Squamous cell carcinomas (SCC) account for approximately 30% of non-small cell lung cancer. Investigation of the mechanism of invasion and metastasis of lung SCC will be of great help for the development of meaningful targeted therapeutics. This study is intended to understand whether the activation of Hedgehog (Hh) pathway is involved in lung SCC, and whether activated Hh signaling regulates metastasis through epithelial-mesenchymal transition (EMT) in lung SCC.
Two cohorts of patients with lung SCC were studied. Protein expression was examined by immunohistochemistry, Western blot, or immunofluorescence. Protein expression levels in tissue specimens were scored and correlations were analyzed. Vismodegib and a Gli inhibitor were used to inhibit Shh/Gli activity, and recombinant Shh proteins were used to stimulate the Hh pathway in lung SCC cell lines. Cell migration assay was performed in vitro.
Shh/Gli pathway components were aberrantly expressed in lung SCC tissue samples. Gli1 expression was reversely associated with the expression of EMT markers E-Cadherin and β-Catenin in lung SCC specimens. Inhibition of the Shh/Gli pathway suppressed migration and up-regulated E-Cadherin expression in lung SCC cells. Stimulation of the pathway increased migration and down-regulated E-Cadherin expression in lung SCC cells.
Our results suggested that the Shh/Gli pathway may be critical for lung SCC recurrence, metastasis and resistance to chemotherapy. Inhibition of the Shh/Gli pathway activity/function is a potential therapeutic strategy for the treatment of lung SCC patients.
Lung cancer is the leading cause of cancer-related deaths worldwide. 85% of lung cancer is non-small cell lung cancer (NSCLC), with adenocarcinoma (AD) and squamous cell carcinoma (SCC) as the two major pathological subtypes . Lung SCC is closely associated with tobacco smoking, and it accounts 35% of NSCLC, causing an estimated 400,000 deaths per year worldwide . While recent improvements in targeted therapies such as the EGFR tyrosine kinase inhibitors (TKI), bevacizumab and ALK inhibitors have significantly benefited patients with AD, the effectiveness of these treatments are unfortunately disappointing for lung SCC . Lung SCC patients suffer from poor prognosis with significant rates of reoccurrence and metastasis, largely due to the differences in genetic profiles . Recent studies identified potentially actionable genetic abnormalities in lung SCC, such as phosphoinositide 3-kinase (PIK3CA) amplification, fibroblast growth factor receptor 1 (FGFR1) amplification, and discoidin domain receptor 2 (DDR2) mutation. However, significant efforts are still needed to help in the investigation of the biological characteristics of lung SCC in order to decipher and the mechanism underlying the invasion and metastasis of lung SCC.
Epithelial–mesenchymal transition (EMT) was originally characterized during embryonic development. The concept that EMT being a critical event in the invasion, progression and metastasis of epithelial cancers is well established [5, 6]. The molecular basis of EMT involves multiple changes in expression, distribution, and/or function of proteins, i.e. E-cadherin, and the process of EMT is regulated by many molecular events including multiple signaling pathways in various cancers . Furthermore, acquisition of the features of the EMT has been associated with poor prognosis and chemo-resistance, which may allow for recurrence and metastasis to occur after treatment with a standard chemotherapeutic treatment [7–10]. The mechanistic study of EMT regulation could contribute to our understanding of recurrence and metastasis in cancer.
Activation of Hedgehog (Hh) signaling has been implicated in tumorigenesis and metastasis in various cancer types [11–23]. Hh signaling is orchestrated by two trans-membrane receptors, Patched (Ptch) and Smoothened (Smo). In the canonical Hh pathway, in the absence of the Hh ligand, Ptch inhibits Smo, causing cleavage of Gli to the N-terminal repressor form. Once Hh binds to Ptch, the inhibitory effect on Smo is released, causing active full-length Gli to transport into the nucleus and activate transcription of Hh target genes in a context- and cell-type specific manner. Moreover, several studies have revealed "non-canonical Gli activation" in many cancer cell types by which Gli is activated independent of Hh/Smo regulation [12, 14]. It needs to be elucidated if the canonical Hh pathway or the non-canonical Gli activation is involved in lung SCC, and if Gli activation contributes to the regulation of metastasis.
Studies of EMT regulation by Hh pathway have recently emerged in literature; data, however, is rare and controversial. While Alexaki et al.  and Inaguma et al.  suggested that Gli-factor facilitates cancer cell migration and invasion through E-Cadherin in melanoma and pancreatic cancers, Joost et al.  proposed that inhibition of Gli promoted EMT in pancreatic cancers. Our study intends to extend the research to lung SCC to help us better understand the regulation of EMT by Hh signaling.
We reported the activation of Hh signaling in two cohorts of patient samples, and revealed the reverse association between Gli1 expression and the expression of EMT markers. Inhibition of the Shh/Gli pathway suppressed migration and up-regulated E-Cadherin expression in lung SCC cells. Stimulation of the pathway increased migration and down-regulated E-Cadherin expression in lung SCC cells.
Materials and methods
Tissue specimens of the UCSF cohort were collected from 14 patients who underwent surgical resection for lung SCC at the Thoracic Oncology Program at UCSF. Tissue specimens of the Tianjin cohort were collected from 177 patients who underwent surgical resection for lung SCC at the Tianjin Medical University Cancer Institute and Hospital. Samples were fixed in formalin and embedded in paraffin to make tissue slides. The study with UCSF patient tissues was approved by the Committee on Human Research (CHR approval number: H8714-11647-10) at the University of California, San Francisco (UCSF), and that with Tianjin cohort was approved by the Tianjin Medical University Cancer Institute and Hospital. Written, informed consent was obtained from each patient before specimen collection.
Immunohistochemistry (IHC), immunofluorescence (IF) and Western blot
Immunohistochemistry, immunofluorescence and western blot were performed following standard procedures. Antibodies applied to detect protein expressions in IHC and IF were Gli1 (sc-20687 Santa Cruz Biotechnology, Santa Cruz, CA) at 1:100, Shh (ab 50515 Abcam, Cambridge, MA) at 1:100, Smo (ab 72130 Abcam) at 1:200, Ptch1 (Santa Cruz that Biotechnology,) and E-Cadherin (EMD Millipore) Smo (Sigma, St. Louis, MO) at 1:100, E-cadherin (sc-7870, Santa Cruz Biotechnology) at 1:100, and β-catenin (BD Biosciences, San Jose, California) at 1:400. Antibodies used in Western blot were Gli (C68H3, Abcam) at 1:1000, E-Cad (HECD-1 MED Milliopore, Darmstadt, Germany) at 1:1000 and Actin (A5441, Sigma) at 1:5000. Total protein extraction was performed with M-PER Mammalian Protein Extraction Solution (Thermo Scientific, Waltham, MA), and 40ug of proteins were analyzed in Western blot.
Cell culture, drug treatment and migration assay
Human lung SCC cell lines H2170, H1703, H1869 and SK-MES-1 were purchased from the Cell Culture Core Facility at Harvard University (Boston, MA, USA). The cell lines were cultured in RPMI 1640 (Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) and antibiotics. Cells were seeded one day before treatment with Gli-I and vismodegib (Selleck) at different concentrations and Shh recombinant proteins (eBioscience) for 30 hours, with vehicle (DMSO) as controls. Cells were subjected to the following analyses of immunofluorescence and migration assay. In migration assays, four wounds were made in each condition, and cell migration was presented by the average of distance differences between 30 hr and 0 hr. All experiments have been conducted for more than three times, and representative results were included in the text.
Kappa test was used to evaluate the association between the expressions of Hh pathway components and EMT markers, and between Gli1 and recurrence/metastasis. IHC scores of 1–3 were grouped as positive “+” , and 0 was grouped as negative “-” for dichotomized analysis. Non-parametric Kendall’s tau-b statistics was used to determine the correlation between IHC staining of Hh components. Two-sided student’s t-test was performed for migration assays. A p value <0.05 was indicated as *, 0.01 as **, and 0.001 as *** in corresponding figures. Data analysis was performed using SPSS 17.0 software.
Results and discussion
Aberrant activation of the Shh pathway in lung SCC
Characteristics of the lung SCC patients (Tianjin cohort)
Next we analyzed the association between expressions of key components in the Shh pathway. Kendall’s tau-b correlation tests yielded significant correlations between every two factors (p = 0.000), while Kappa’s test suggested strong positive association between SHh and Gli1(p = 0.000) (Figure 1C), suggesting the canonical Shh pathway is activated in lung SCC. These data are consistent with previous reports that the upstream Shh signaling has correlations with downstream targets in NSCLC [29, 30]. Taken together, our results suggest that aberrant activation of the Shh pathway plays an important role in lung SCC.
Gli expression reversely correlates with EMT markers
Shh/Gli signaling promotes cell migration by down-regulating E-Cadherin expression
To further understand the role of Shh/Gli in EMT regulation in lung SCC, we manipulated the Shh/Gli signaling pathway in lung SCC cell lines to examine its impact on cell migration and E-Cadherin expression. To inhibit the Shh/Gli activity, we applied two small molecule compounds: Vismodegib and a novel Gli inhibitor. Vismodegib (also known as GDC-0449) is a Smo inhibitor recently approved by the U.S. Food and Drug Administration to treat adult patients with basal cell carcinoma [32–35]. Multiple clinical trials are evaluating the use of vismodegib in other types of cancer, in addition to other candidate drugs that targets Hh signaling [32, 36]. The novel Gli inhibitor (Gli-I) developed by our lab specifically inhibits Gli1 and Gli2 transcriptional activity . To stimulate the pathway, we applied recombinant Shh proteins.
Our study provides evidence for aberrant activation of Shh/Gli pathway and a strong association between expressions of Gli proteins and EMT markers in human lung SCC, as well as the implication of activated Shh/Gli pathway in cell migration and EMT process. Our findings suggest that the Shh/Gli pathway may be a critical component in lung SCC recurrence, metastasis and resistance to chemotherapy. Inhibition of the Shh/Gli pathway activity/function is a potential therapeutic strategy for the treatment of lung SCC patients.
This work was supported by NIH/NCI R01CA125030, and the Eileen D. Ludwig Endowed for Thoracic Oncology Research (to B He); The Bonnie J. Addario Lung Cancer Foundation, the Kazan, McClain, Abrams, Fernandez, Lyons, Greenwood, Harley & Oberman Foundation, the Ziegelmam Family Foundation, and the Barbara Isackson Lung Cancer Research Fund (to DM Jablons); Tianjin Municipal Science and Technology Commission (12JCYBJC17800) and the Key Program for Anti-cancer Research of Tianjin Municipal Science and Technology Commission (12ZCDZSY15400) (to CL Wang).
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