Cell Biochem Biophys DOI 10.1007/s12013-015-0647-z
ORIGINAL PAPER
SOX9 Overexpression Promotes Glioma Metastasis via Wnt/b-Catenin Signaling Hongwei Liu • Zhixiong Liu • Bing Jiang • Renjun Peng • Zhiming Ma • Jingchen Lu
Ó Springer Science+Business Media New York 2015
Abstract SOX9 gene encodes a transcription factor essential for a central role in the development and differentiation of multiple cell lineages, such as in neurogenesis, neural crest development, etc. Recent study reported that overexpression of SOX9 mRNA is closely associated with poor clinical outcome of patients with malignant gliomas. In the present study, we have explored the regulatory role of SOX9 in glioma metastasis. To investigate the role of SOX9 in glioma metastasis, SOX9 overexpressed in human glioma cell line U251 on cell migration and invasion was evaluated via wound scratch, Transwell assay without or with Matrigel. SOX9-induced changes in EMT process were evaluated by Western blot. Furthermore, the role of bcatenin in the regulatory effect of SOX9 on cell migration and invasion, and EMT process was explored by suppressing b-catenin expression in SOX9-overexpressed U251 cells. SOX9 overexpression in U251 cells resulted in a significant increase in cell migration and invasion. SOX9 overexpression also markedly promoted the EMT process. More importantly, our results revealed that SOX9 stimulated metastasis through activating Wnt/b-catenin signaling. In summary, this study indicated that the promoting effect of SOX9 on glioma metastasis was, at least in part, through Wnt/b-catenin signaling. The findings in this study highlight the effectiveness and therapeutic
H. Liu Z. Liu B. Jiang R. Peng Z. Ma Department of Neurosurgery, Xiangya Hospital, Central South University (CSU), Changsha, China J. Lu (&) Department of Oncology, Xiangya Hospital, Central South University (CSU), Changsha, China e-mail: [email protected]
potential to utilize SOX9 targeted strategies in the treatment of glioma. Keywords SOX9 Metastasis Cell migration Invasion EMT b-Catenin
Introduction Malignant glioma, with the invasive and infiltrative character, can present up to 10 % of tumors in the central nervous system (CNS), and is the leading cause of brain tumor-related death in both developed and developing countries [1, 2]. One of the leading causes of cancer-related death among glioma patients is metastasis, which involves multiple sequential steps including invasion of cancer cells into surrounding tissue, penetrating the walls of lymphatic and/or blood vessels, survival in the circulation system, repenetration through vessels, and growth of a macroscopic secondary tumor in distant organs [3]. Epithelial–mesenchymal transition (EMT), which always happens when tumor cells penetrate the lymphatic and/or blood vessels, renders tumor cells more invasive properties, which facilitate the departure of tumor cells from the epithelial cell community as well as invasion into surrounding tissue or distant organs [4]. SOX9, a member of the SOX (SRY-related high mobility group box) family of transcription factors, plays an essential role in the development and differentiation of multiple cell lineages, such as in neurogenesis, neural crest development, chondrogenesis, male sex gonad or respiratory epithelium development, melanocyte differentiation, and the differentiation of Paneth cells in the gut [5, 6]. Recent researches found that SOX9 is also involved in carcinogenesis in a variety of human cancers. Thomsen
123
Cell Biochem Biophys
et al. found that SOX9 was upregulated and able to suppress growth and tumorigenesis in prostate cancer [7]. SOX9 genes were also found amplified in gastric carcinoma [8]. In addition, SOX9 overexpression was found in breast cancer tissues [9]. Taken together, these studies suggested the role of SOX9 as an oncogene. However, inconsistent findings have been reported on the expression and role of SOX9 in glioma. An early study by Sutter et al. found that the expression levels of SOX9 in primary glioma tissues were similar to or below those in adult brain tissues [10]. However, a later study by Swartling et al. reported the overexpression of SOX9 in glioma cell lines and suppressing SOX9 expression was associated with loss of cell adhesion and inhibition of cell proliferation [11]. Lately, Wang et al. found that a high level of SOX9 mRNA expression was significantly more common in glioma tissues with advanced WHO grade than those with low grade [12]. However, the functional role of SOX9 in the metastasis of glioma remains to be elucidated. In this study, we demonstrated that overexpression of SOX9 in glioma cell line (U251) cells enhanced migration and invasion as well stimulated EMT process. Moreover, we examined the involvement of Wnt/b-catenin signaling pathway in the biological effect of SOX9, which showed that SOX9 may modulate cell migration, invasion, and EMT through Wnt/ b-catenin signaling pathway. Our research revealed that SOX9 functions as a tumor metastasis promoting gene in glioma.
Materials and Methods Cell Lines Human glioma cell line U251 was purchased from China Science Academy, Shanghai, China, and maintained in China Science Academy recommended growth medium supplemented with 10 % fetal bovine serum (Invitrogen Corporation, Carlsbad, CA) at 37 °C in 5 % CO2 humidified atmosphere. Establishment of SOX9-Overexpressing Cell Lines U251 cell line overexpressing SOX9 was established as previously described [13]. Briefly, the amplified full-length cDNA for SOX9 was subcloned into pEGFP-N1 vector (Clontech Laboratories, Inc., Palo Alto, CA). The resulting plasmid was named pEGFP-N1-SOX9. U251 cells were transfected with pEGFP-N1-SOX9 vector to induce excessive SOX9 expression or pEGFP-N1 vector as control. The resulting cell lines were named U251/SOX9 and U251/ vector, respectively. 24 h after transfection, G418 solution was added to cells for selection of stable clones (U251/
123
SOX9 and U251/vector cells). Stable clones were selected and maintained in medium containing G418. Quantitative RT-PCR (qRT-PCR) Total RNA was extracted using the PARISTM Kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s protocol. MultiScribeTM Reverse Transcriptase (Applied Biosystems) was used to synthesize the complementary DNA templates. Real-time RT-PCR was performed in an Applied Biosystems 7500 Detection system using MaximaH SYBR Green/ROX qPCR Master Mix Assays (Fermentas, USA). mRNA expression was determined from the threshold cycle. The relative expression levels were normalized to the expression of human peptidylprolyl isomerase A mRNA [14] and then calculated by the 2[-Delta Delta C(T)] method. The primer sequences for human SOX9 were 50 -TATGACTGGACCCTGGTG-30 (forward); 50 -TGTGGCTTGTTCTTGCTGG-30 (reverse). Western Blot Protein levels were quantified by Bradford assay. Protein (30 lg) from each sample was fractionated by 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). The membranes were blocked in 0.1 % Triton X-100 and 5 % low fat milk powder in phosphate-buffered saline for 1 h at 4 °C and then probed with specific primary antibodies. The membranes were washed thrice with Tris-buffered saline Tween-20 and then incubated in peroxidase-conjugated goat anti-mouse IgG antibody (1:1500, Santa Cruz Biotechnology). Bands were visualized by an enhanced chemiluminescence detection system using medical X-ray films and then quantified by Photoshop (Adobe software). The intensities of the bands of interest were expressed relative to the b-actin intensities from the same sample. The primary antibodies used in this study were as follows: rabbit anti-SOX9 (Santa Cruz Biotechnology, CA, 1:2000), rabbit monoclonal anti-Ecadherin antibody (Cell Signaling Technology, Inc., Beverly, MA, USA, 1:1000), rabbit polyclonal anti-N-cadherin antibody (Boster Biotech Ltd., Wuhan, China, 1:400), rabbit polyclonal anti-vimentin antibody (Bioss Biotech Ltd., Beijing, China, 1:500), rabbit polyclonal anti-fibronectin antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA, 1:2000), mouse monoclonal anti-a-SMA antibody (Boster Biotech Ltd., Wuhan, China, 1:400), rabbit monoclonal anti-b-catenin antibody (Cell Signaling Technology, Inc., Beverly, MA, USA, 1:1000), goat polyclonal anti-Wnt1 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA, 1:1000). The second antibodies used in this study were goat anti-rabbit IgG-HRP, goat anti-
Cell Biochem Biophys
mouse IgG-HRP, and donkey anti-goat IgG-HRP (Beyotime Institute of Biotechnology, Shanghai, China).
cells were fixed with formaldehyde solution. The cells in at least five random microscopic fields (2009) were counted and photographed.
Wound Scratch Assay Suppression of b-Catenin Expression with shRNA Stable transfected cells were seeded into six-well plates and then cultured to 90 % confluence. The confluent cell monolayer was wounded using a sterile 200 lL pipette tip. The suspended cells were washed using normal growth medium. The scratch wound was captured after 24, 36, and 48 h using an OLYMPUS IX81 microscope in three fields of view at 1009 magnification. The area of the open wound was quantified using Photoshop (Adobe). The cellular migratory abilities were represented by the ratios of the decreased open area after 24, 36, 48, or 72 h relative to the open area at 0 h. Three independent assays were performed. Transwell Migration Assay Transwell chambers (polycarbonate filters of 8-m porosity, Corning, NY, USA) were used in this test. The bottom chamber was filled with culture medium containing 20 % FBS. Cells were suspended at a density of 5 9 104 cells/ mL in serum-free medium and 1 9 104 cells were plated in the upper chamber. The cells were removed from the upper chamber by a cotton swab following 24-h incubation. Cells penetrated and attached to the bottom of the filter were fixed with 4 % formaldehyde solution for 20 min before staining with 0.1 % crystal violet for 5 min. The cells were then subjected to imaging under a 209 objective using phase-contrast microscopy (Motic China Group Co., Xiamen, China). The number of cells that finally attached to the bottom dishes was counted under a 2009 objective. Statistical results of cell numbers per each image field were obtained from three independent experiments averaged from five image fields.
shRNA targeted b-catenin was chemically synthesized by Sangon Biotech (Sangon Biotech Co., Ltd., Shanghai, China) according to published sequences [15]. Scramble shRNA which was used as a negative control. shRNA transfection of cells was performed following a standard protocol. Briefly, cells were plated at 5 9 103 cells/well in 6-well culture plate and allowed to reach 70–80 % confluence after 24 h of incubation. The cells were then starved in serum-free culture for 1 h. The transfection mixture containing b-catenin targeted shRNA and Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was incubated for 20 min at room temperature. The cells were then incubated with the above mixture for 5 h at 37 °C in a humidified atmosphere containing 5 % CO2. Subsequently, cells were washed with PBS and maintained in DMEM containing 10 % FBS for 48 h before expression of bcatenin was detected by Western blot analysis. Statistical Analysis Data are expressed as mean ± standard error of the mean. Statistical analysis of differences between groups was performed using analysis of variance (ANOVA) followed by Dunnett’s t test. Values of P \ 0.05 were considered statistically significant.
Results Verification of U251 Cells Stably Overexpressing SOX9
Transwell Invasion Assay 24-well Transwells coated with Matrigel (8-lm pore size; BD Biosciences, San Jose, CA, USA) were used for cell invasion assays. Equal numbers (1 9 105) of untransfected cells as well as cells stably transfected cells were plated onto separate well. Cells were cultured overnight in serum-free medium before trypsinized and resuspended in a density of 2 9 105 cells/ml in DMEM containing 1 % FBS. The cells were then added to the upper chamber with MEM containing 10 % FBS as chemoattractant in the lower chamber. Medium containing 1 % FBS added to the lower chamber was used as control. The Matrigel and the cells remaining in the upper chamber were removed by cotton swabs following 24-h incubation. The cells on the lower surface of the membrane were stained with hematoxylin staining solution after the
A plasmid was constructed by cloning SOX9-coding gene into pEGFP-N1 vector to establish a U251 cell line that can stably express high level of SOX9. The mRNA level of SOX9 was determined by qRT-PCR after stably transfected cells were selected. The SOX9 mRNA level was markedly elevated by more than 3 folds in U251/SOX9 cells compared to U251/Vector cells and parental U251 cells. In addition, 3-fold increase in SOX9 expression was also found in U251/ SOX9 cells, as detected by western blot analysis (Fig. 1b). SOX9 Promotes Cell Migration and Invasion in U251 Cells We tested the cell migratory and invasive abilities through the scratch wound migration assay as well as migration and
123
Cell Biochem Biophys
metastasis of glioma by examining the epithelial marker E-cadherin and mesenchymal markers N-cadherin, vimentin, fibronectin, and a-SMA via Western blots. As shown in Fig. 3, SOX9 overexpression resulted in remarkable downregulation of E-cadherin and upregulation of N-cadherin, vimentin, fibronectin, and a-SMA. Taken together, these results indicated that overexpression of SOX9 promoted the transition from epithelial to mesenchymal phenotype.
Wnt/b-Catenin Signaling is Activated in SOX9 Overexpressed U251 Cells Previous studies have revealed the regulatory role of SOX9 on the transcription of Wnt genes in breast cancer cells [16]. Accordingly, we examined the effect of SOX9 on Wnt/b-catenin signaling pathway by Western blot. Our results showed that protein levels of Wnt1 and b-catenin were dramatically increased in U251/SOX9 cells compared to controls, indicating that overexpression of SOX9 was associated with activation of Wnt/b-catenin signaling (Fig. 4).
Effect of b-Catenin Targeting shRNA on Metastatic Behavior of U251 Induced by SOX9 Overexpression
Fig. 1 Quantification of SOX9 expression in U251 cells after transfection. SOX9 mRNA expression was determined by qRT-PCR (a). SOX9 protein expression was examined via Western blot (b). The values are expressed as the mean ± SD from three independent experiments. Representative images of Western blot from three independent experiments are displayed. **P \ 0.01 versus control
invasion assays to discover whether or not SOX9 can promote the malignancy of glioma in vitro. The results of scratch wound migration assay (Fig. 2a) showed that the U251 cells stably overexpressing SOX9 migrated significantly quicker than the control cells at 24, 36, and 48 h. Transwell experiments reflect cell trans-membrane abilities, including migration and invasion. The migratory and invasive abilities of U251 cells stably overexpressing SOX9 were determined using the Boyden chamber transwell assay. Trans-membrane cell numbers were counted and statistically analyzed. As shown in Fig. 2b, c, SOX9 overexpression significantly enhanced the migratory and invasive abilities of U251 cells compared with the control cells. SOX9 Promotes EMT Process in U251 Cells Next, we examined the effect of SOX9 overexpression on EMT process to further evaluate the role of SOX9 in the
123
In our results before mentioned, we found that SOX9 promoted metastatic behavior and SOX9 overexpression correlated with Wnt/b-catenin signaling activation. Therefore, we established stable U251/SOX9 with suppressed expression of b-catenin to further explore the role of b-catenin in mediating the promoting effect of SOX9 on metastatic behavior. After transfection with bcatenin targeted shRNA, b-catenin expression was effectively decreased (P \ 0.01). Next, the migration and invasion of cells were examined to further investigate the role of b-catenin in mediating the regulatory effect role of SOX9 on cellular function. As shown in Fig. 4b, c, suppressing b-catenin expression resulted in compromised cell migration and invasion ability compared with controls, directly demonstrating that b-catenin acted as essential mediator in SOX9-induced increase in cell migration and invasion. The changes in epithelial marker E-cadherin and mesenchymal markers a-SMA induced by b-catenin silencing were examined by Western blotting. The results revealed that E-cadherin was significantly upregulated while protein levels of N-cadherin, vimentin, fibronectin, and a-SMA were significantly decreased (P \ 0.01) (Fig. 4d), indicating that b-catenin downregulation abolished the promoting effect of SOX9 on EMT.
Cell Biochem Biophys Fig. 2 SOX9 overexpression enhances migration and invasion in U251 cells. Effect of SOX9 overexpression on cell migration was assessed by wound scratch (a) and transwell chamber assay without Matrigel (b). Effect of SOX9 overexpression on cell invasion was detected by transwell assay with Matrigel (c). The values are expressed as the mean ± SD from three independent experiments. Representative images of wound scratch and transwell chamber assay from three independent experiments are displayed. *P \ 0.05 versus control, **P \ 0.01 versus control
Fig. 3 SOX9 overexpression promotes EMT process. Effect of SOX9 overexpression on expression of epithelial cell markers and mesenchymal cell markers (E-cadherin, N-cadherin, vimentin, fibronectin, and a-SMA) were examined by Western blot. The values are expressed as the mean ± SD from three independent experiments. Representative images of Western blot from three independent experiments are displayed. *P \ 0.05 versus control
123
Cell Biochem Biophys
Fig. 4 SOX9 regulates cell migration and invasion through modulating Wnt/b-catenin signaling pathway. SOX9 overexpression correlates with Wnt/b-catenin activation. Activation of Wnt/b-catenin signaling was examined by Western blot (a). Effect of b-catenin knockdown on cell migration was assessed by wound scratch (b) and transwell chamber assay without Matrigel (c). Effect of b-catenin
knockdown on cell invasion was detected by transwell assay with Matrigel (d). Effect of b-catenin knockdown on expression of epithelial cell markers and mesenchymal cell markers (E-cadherin, N-cadherin, vimentin, fibronectin, and a-SMA) were examined by Western blot (e). Representative images of each assay from three independent experiments are displayed. **P \ 0.01 versus control
Discussion
prostate, bladder and pancreas, as well as melanoma, and correlates with poor clinical prognosis [18, 19]. In this study, we investigated the role of SOX9 in the process of metastasis, including cell migration, invasion and EMT, using in vitro glioma model. Our results revealed that SOX9 overexpression increases cell motility, invasiveness, and promoted EMT process. Meanwhile, our findings demonstrated that the regulatory effect of SOX9 on metastasis was, at least in part, mediated through bcatenin. Metastasis consists of multiple sequential steps starting with invasion of cancer cells into surrounding tissues, which requires increased cell motility. A growing body of literature has suggested that SOX9 could mediate mesenchymal features and associated with poor prognosis of cancer patients [12, 20]. In consistent with these findings,
Wnt proteins are a large family of secreted glycoproteins activating the Wnt-pathway. Binding of WNT to Frizzled (Fz) leads to the activation of Disheveled (Dsh), which inhibits the activity of GSK3-b. As a result, dephosphorylation and stabilization of b-catenin occur, enabling it to accumulate within the nucleus, where it interacts with various transcription factors to cause cellular proliferation and differentiation [17]. Several studies revealed that Wnt/ b-catenin signaling pathway plays an essential role in the development and promotion of EMT, and clinical evidences have demonstrated that upregulated b-catenin expressions is significantly associated with invasion and metastasis of carcinomas of the head and neck, esophagus, stomach, colon, liver, lung, breast, female genitalia,
123
Cell Biochem Biophys
our results also demonstrated that SOX9 overexpression increase cell motility and invasiveness in U251 cells. With high migration and invasion capability, U251 cell could easily spread into surrounding tissues and penetrate the walls of lymphatic and/or blood vessels. Hence, the overall effect of SOX9 was to promote metastasis by accelerating the initial steps in the process. EMT is a well-coordinated process during cancer progression, which renders polarity and motility to epithelial cells for tumor invasion and metastasis. During EMT, morphological change was characterized by epithelial polarized cell type changing into the mesenchymal fibroblastoid type [21]. After that, epithelial cells obtained enhanced migratory capabilities due to loss of cell–cell or cell–substrate contacts. The process of EMT is often marked by the decrease of epithelial junction proteins (e.g., E-cadherin, claudins, occludin) as well as downregulation of epithelial differentiation markers including cytokeratins and E-cadherin. In the meanwhile, EMT process is also associated with upregulation of mesenchymal markers, such as vimentin, fibronectin, and N-cadherin. Capaccione et al. reported SOX9 is a negative regulator of E-cadherin, a key protein that is lost during EMT [20]. In consistent with their studies, our results demonstrated that SOX9 overexpression in glioma cells led to molecular changes corresponding to EMT process, providing further evidence of the involvement of SOX9 in the process of EMT. A number of studies have found that nuclear accumulation of b-catenin is also a mark of EMT. Moreover, the nuclear b-catenin accumulation was found in the invasive fronts of primary tumors [22, 23]. In this study, we found increased expression of b-catenin in SOX9 overexpressed glioma cells, suggesting the regulatory role of SOX9 in bcatenin signaling. The study in lung adenocarcinoma cells showed that SOX9 acts downstream of Wnt/b-catenin signaling pathway [20]. On the other hand, Wang et al. suggested that SOX9 is at the center of a positive feedback loop that enhances Wnt/b-catenin signaling by demonstrating SOX9 silencing decreased Wnt/b-catenin activation while SOX9 expression was also increased by Wnt [16]. In this study, we did show that SOX9 played a regulatory role in b-catenin activity. However, we did not address the exact role where SOX9 stands in the whole picture. Elucidation of this question will help us to gain a full understanding of SOX9’s role in cellular functions. In summary we demonstrated that overexpressing SOX9 could promote metastasis of glioma, which could be attributed to the ability of SOX9 to increase cell migration, invasion, and stimulate EMT process. Furthermore, we demonstrated that molecular events involved in SOX9 overexpression-induced phenotype changes were mediated via b-catenin. Our data suggested that targeting SOX9 might provide a potential promising therapeutic strategy to
prevent glioma progression and improve the prognosis of patients with glioma.
References 1. Wei, J., Gabrusiewicz, K., & Heimberger, A. (2013). The controversial role of microglia in malignant gliomas. Clinical and Developmental Immunology, 2013, 285246. 2. Kettenmann, H., et al. (2011). Physiology of microglia. Physiological Reviews, 91(2), 461–553. 3. Steeg, P. S. (2006). Tumor metastasis: Mechanistic insights and clinical challenges. Nature Medicine, 12(8), 895–904. 4. Li, X., et al. (2013). SOX2 promotes tumor metastasis by stimulating epithelial-to-mesenchymal transition via regulation of WNT/beta-catenin signal network. Cancer Letters, 336(2), 379–389. 5. Hersmus, R., et al. (2008). FOXL2 and SOX9 as parameters of female and male gonadal differentiation in patients with various forms of disorders of sex development (DSD). Journal of Pathology, 215(1), 31–38. 6. Jay, P., Berta, P., & Blache, P. (2005). Expression of the carcinoembryonic antigen gene is inhibited by SOX9 in human colon carcinoma cells. Cancer Research, 65(6), 2193–2198. 7. Thomsen, M. K., et al. (2010). SOX9 elevation in the prostate promotes proliferation and cooperates with PTEN loss to drive tumor formation. Cancer Research, 70(3), 979–987. 8. Kimura, M. S., Mutoh, H., & Sugano, K. (2011). SOX9 is expressed in normal stomach, intestinal metaplasia, and gastric carcinoma in humans. Journal of Gastroenterology, 46(11), 1292–1299. 9. Chakravarty, G., et al. (2011). Prognostic significance of cytoplasmic SOX9 in invasive ductal carcinoma and metastatic breast cancer. Experimental Biology and Medicine (Maywood), 236(2), 145–155. 10. Sutter, R., et al. (2010). Cerebellar stem cells act as medulloblastoma-initiating cells in a mouse model and a neural stem cell signature characterizes a subset of human medulloblastomas. Oncogene, 29(12), 1845–1856. 11. Schlierf, B., et al. (2007). Expression of SoxE and SoxD genes in human gliomas. Neuropathology and Applied Neurobiology, 33(6), 621–630. 12. Wang, L., et al. (2012). Oncogenic role of SOX9 expression in human malignant glioma. Medical Oncology, 29(5), 3484– 3490. 13. Shi, G., et al. (2013). Expression and functional role of Sox9 in human epidermal keratinocytes. PLoS One, 8(1), e54355. 14. Jung, M., et al. (2007). In search of suitable reference genes for gene expression studies of human renal cell carcinoma by realtime PCR. BMC Molecular Biology, 8, 47. 15. Zhao, J. H., et al. (2011). Knockdown of beta-catenin through shRNA cause a reversal of EMT and metastatic phenotypes induced by HIF-1 alpha. Cancer Investigation, 29(6), 377–382. 16. Wang, H., et al. (2013). SOX9 regulates low density lipoprotein receptor-related protein 6 (LRP6) and T-cell factor 4 (TCF4) expression and Wnt/beta-catenin activation in breast cancer. Journal of Biological Chemistry, 288(9), 6478–6487. 17. Galera-Ruiz, H., et al. (2012). WNT pathway in laryngeal squamous cell carcinoma and nasopharyngeal carcinoma. Acta Otorhinolaryngologica Italica, 32(2), 122–123. 18. Fu, Y., et al. (2011). Beta-catenin as a potential key target for tumor suppression. International Journal of Cancer, 129(7), 1541–1551.
123
Cell Biochem Biophys 19. Valenta, T., Hausmann, G., & Basler, K. (2012). The many faces and functions of beta-catenin. EMBO Journal, 31(12), 2714–2736. 20. Capaccione, K. M., et al. (2014). Sox9 mediates Notch1-induced mesenchymal features in lung adenocarcinoma. Oncotarget, 5(11), 3636–3650. 21. Yang, J., & Weinberg, R. A. (2008). Epithelial–mesenchymal transition: At the crossroads of development and tumor metastasis. Developmental Cell, 14(6), 818–829.
123
22. Polette, M., et al. (2007). Beta-catenin and ZO-1: Shuttle molecules involved in tumor invasion-associated epithelial–mesenchymal transition processes. Cells Tissues Organs, 185(1–3), 61–65. 23. Brabletz, T., et al. (2001). Variable beta-catenin expression in colorectal cancers indicates tumor progression driven by the tumor environment. Proceedings of the National Academy Sciences USA, 98(18), 10356–10361.
ORIGINAL PAPER
SOX9 Overexpression Promotes Glioma Metastasis via Wnt/b-Catenin Signaling Hongwei Liu • Zhixiong Liu • Bing Jiang • Renjun Peng • Zhiming Ma • Jingchen Lu
Ó Springer Science+Business Media New York 2015
Abstract SOX9 gene encodes a transcription factor essential for a central role in the development and differentiation of multiple cell lineages, such as in neurogenesis, neural crest development, etc. Recent study reported that overexpression of SOX9 mRNA is closely associated with poor clinical outcome of patients with malignant gliomas. In the present study, we have explored the regulatory role of SOX9 in glioma metastasis. To investigate the role of SOX9 in glioma metastasis, SOX9 overexpressed in human glioma cell line U251 on cell migration and invasion was evaluated via wound scratch, Transwell assay without or with Matrigel. SOX9-induced changes in EMT process were evaluated by Western blot. Furthermore, the role of bcatenin in the regulatory effect of SOX9 on cell migration and invasion, and EMT process was explored by suppressing b-catenin expression in SOX9-overexpressed U251 cells. SOX9 overexpression in U251 cells resulted in a significant increase in cell migration and invasion. SOX9 overexpression also markedly promoted the EMT process. More importantly, our results revealed that SOX9 stimulated metastasis through activating Wnt/b-catenin signaling. In summary, this study indicated that the promoting effect of SOX9 on glioma metastasis was, at least in part, through Wnt/b-catenin signaling. The findings in this study highlight the effectiveness and therapeutic
H. Liu Z. Liu B. Jiang R. Peng Z. Ma Department of Neurosurgery, Xiangya Hospital, Central South University (CSU), Changsha, China J. Lu (&) Department of Oncology, Xiangya Hospital, Central South University (CSU), Changsha, China e-mail: [email protected]
potential to utilize SOX9 targeted strategies in the treatment of glioma. Keywords SOX9 Metastasis Cell migration Invasion EMT b-Catenin
Introduction Malignant glioma, with the invasive and infiltrative character, can present up to 10 % of tumors in the central nervous system (CNS), and is the leading cause of brain tumor-related death in both developed and developing countries [1, 2]. One of the leading causes of cancer-related death among glioma patients is metastasis, which involves multiple sequential steps including invasion of cancer cells into surrounding tissue, penetrating the walls of lymphatic and/or blood vessels, survival in the circulation system, repenetration through vessels, and growth of a macroscopic secondary tumor in distant organs [3]. Epithelial–mesenchymal transition (EMT), which always happens when tumor cells penetrate the lymphatic and/or blood vessels, renders tumor cells more invasive properties, which facilitate the departure of tumor cells from the epithelial cell community as well as invasion into surrounding tissue or distant organs [4]. SOX9, a member of the SOX (SRY-related high mobility group box) family of transcription factors, plays an essential role in the development and differentiation of multiple cell lineages, such as in neurogenesis, neural crest development, chondrogenesis, male sex gonad or respiratory epithelium development, melanocyte differentiation, and the differentiation of Paneth cells in the gut [5, 6]. Recent researches found that SOX9 is also involved in carcinogenesis in a variety of human cancers. Thomsen
123
Cell Biochem Biophys
et al. found that SOX9 was upregulated and able to suppress growth and tumorigenesis in prostate cancer [7]. SOX9 genes were also found amplified in gastric carcinoma [8]. In addition, SOX9 overexpression was found in breast cancer tissues [9]. Taken together, these studies suggested the role of SOX9 as an oncogene. However, inconsistent findings have been reported on the expression and role of SOX9 in glioma. An early study by Sutter et al. found that the expression levels of SOX9 in primary glioma tissues were similar to or below those in adult brain tissues [10]. However, a later study by Swartling et al. reported the overexpression of SOX9 in glioma cell lines and suppressing SOX9 expression was associated with loss of cell adhesion and inhibition of cell proliferation [11]. Lately, Wang et al. found that a high level of SOX9 mRNA expression was significantly more common in glioma tissues with advanced WHO grade than those with low grade [12]. However, the functional role of SOX9 in the metastasis of glioma remains to be elucidated. In this study, we demonstrated that overexpression of SOX9 in glioma cell line (U251) cells enhanced migration and invasion as well stimulated EMT process. Moreover, we examined the involvement of Wnt/b-catenin signaling pathway in the biological effect of SOX9, which showed that SOX9 may modulate cell migration, invasion, and EMT through Wnt/ b-catenin signaling pathway. Our research revealed that SOX9 functions as a tumor metastasis promoting gene in glioma.
Materials and Methods Cell Lines Human glioma cell line U251 was purchased from China Science Academy, Shanghai, China, and maintained in China Science Academy recommended growth medium supplemented with 10 % fetal bovine serum (Invitrogen Corporation, Carlsbad, CA) at 37 °C in 5 % CO2 humidified atmosphere. Establishment of SOX9-Overexpressing Cell Lines U251 cell line overexpressing SOX9 was established as previously described [13]. Briefly, the amplified full-length cDNA for SOX9 was subcloned into pEGFP-N1 vector (Clontech Laboratories, Inc., Palo Alto, CA). The resulting plasmid was named pEGFP-N1-SOX9. U251 cells were transfected with pEGFP-N1-SOX9 vector to induce excessive SOX9 expression or pEGFP-N1 vector as control. The resulting cell lines were named U251/SOX9 and U251/ vector, respectively. 24 h after transfection, G418 solution was added to cells for selection of stable clones (U251/
123
SOX9 and U251/vector cells). Stable clones were selected and maintained in medium containing G418. Quantitative RT-PCR (qRT-PCR) Total RNA was extracted using the PARISTM Kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s protocol. MultiScribeTM Reverse Transcriptase (Applied Biosystems) was used to synthesize the complementary DNA templates. Real-time RT-PCR was performed in an Applied Biosystems 7500 Detection system using MaximaH SYBR Green/ROX qPCR Master Mix Assays (Fermentas, USA). mRNA expression was determined from the threshold cycle. The relative expression levels were normalized to the expression of human peptidylprolyl isomerase A mRNA [14] and then calculated by the 2[-Delta Delta C(T)] method. The primer sequences for human SOX9 were 50 -TATGACTGGACCCTGGTG-30 (forward); 50 -TGTGGCTTGTTCTTGCTGG-30 (reverse). Western Blot Protein levels were quantified by Bradford assay. Protein (30 lg) from each sample was fractionated by 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). The membranes were blocked in 0.1 % Triton X-100 and 5 % low fat milk powder in phosphate-buffered saline for 1 h at 4 °C and then probed with specific primary antibodies. The membranes were washed thrice with Tris-buffered saline Tween-20 and then incubated in peroxidase-conjugated goat anti-mouse IgG antibody (1:1500, Santa Cruz Biotechnology). Bands were visualized by an enhanced chemiluminescence detection system using medical X-ray films and then quantified by Photoshop (Adobe software). The intensities of the bands of interest were expressed relative to the b-actin intensities from the same sample. The primary antibodies used in this study were as follows: rabbit anti-SOX9 (Santa Cruz Biotechnology, CA, 1:2000), rabbit monoclonal anti-Ecadherin antibody (Cell Signaling Technology, Inc., Beverly, MA, USA, 1:1000), rabbit polyclonal anti-N-cadherin antibody (Boster Biotech Ltd., Wuhan, China, 1:400), rabbit polyclonal anti-vimentin antibody (Bioss Biotech Ltd., Beijing, China, 1:500), rabbit polyclonal anti-fibronectin antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA, 1:2000), mouse monoclonal anti-a-SMA antibody (Boster Biotech Ltd., Wuhan, China, 1:400), rabbit monoclonal anti-b-catenin antibody (Cell Signaling Technology, Inc., Beverly, MA, USA, 1:1000), goat polyclonal anti-Wnt1 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA, 1:1000). The second antibodies used in this study were goat anti-rabbit IgG-HRP, goat anti-
Cell Biochem Biophys
mouse IgG-HRP, and donkey anti-goat IgG-HRP (Beyotime Institute of Biotechnology, Shanghai, China).
cells were fixed with formaldehyde solution. The cells in at least five random microscopic fields (2009) were counted and photographed.
Wound Scratch Assay Suppression of b-Catenin Expression with shRNA Stable transfected cells were seeded into six-well plates and then cultured to 90 % confluence. The confluent cell monolayer was wounded using a sterile 200 lL pipette tip. The suspended cells were washed using normal growth medium. The scratch wound was captured after 24, 36, and 48 h using an OLYMPUS IX81 microscope in three fields of view at 1009 magnification. The area of the open wound was quantified using Photoshop (Adobe). The cellular migratory abilities were represented by the ratios of the decreased open area after 24, 36, 48, or 72 h relative to the open area at 0 h. Three independent assays were performed. Transwell Migration Assay Transwell chambers (polycarbonate filters of 8-m porosity, Corning, NY, USA) were used in this test. The bottom chamber was filled with culture medium containing 20 % FBS. Cells were suspended at a density of 5 9 104 cells/ mL in serum-free medium and 1 9 104 cells were plated in the upper chamber. The cells were removed from the upper chamber by a cotton swab following 24-h incubation. Cells penetrated and attached to the bottom of the filter were fixed with 4 % formaldehyde solution for 20 min before staining with 0.1 % crystal violet for 5 min. The cells were then subjected to imaging under a 209 objective using phase-contrast microscopy (Motic China Group Co., Xiamen, China). The number of cells that finally attached to the bottom dishes was counted under a 2009 objective. Statistical results of cell numbers per each image field were obtained from three independent experiments averaged from five image fields.
shRNA targeted b-catenin was chemically synthesized by Sangon Biotech (Sangon Biotech Co., Ltd., Shanghai, China) according to published sequences [15]. Scramble shRNA which was used as a negative control. shRNA transfection of cells was performed following a standard protocol. Briefly, cells were plated at 5 9 103 cells/well in 6-well culture plate and allowed to reach 70–80 % confluence after 24 h of incubation. The cells were then starved in serum-free culture for 1 h. The transfection mixture containing b-catenin targeted shRNA and Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was incubated for 20 min at room temperature. The cells were then incubated with the above mixture for 5 h at 37 °C in a humidified atmosphere containing 5 % CO2. Subsequently, cells were washed with PBS and maintained in DMEM containing 10 % FBS for 48 h before expression of bcatenin was detected by Western blot analysis. Statistical Analysis Data are expressed as mean ± standard error of the mean. Statistical analysis of differences between groups was performed using analysis of variance (ANOVA) followed by Dunnett’s t test. Values of P \ 0.05 were considered statistically significant.
Results Verification of U251 Cells Stably Overexpressing SOX9
Transwell Invasion Assay 24-well Transwells coated with Matrigel (8-lm pore size; BD Biosciences, San Jose, CA, USA) were used for cell invasion assays. Equal numbers (1 9 105) of untransfected cells as well as cells stably transfected cells were plated onto separate well. Cells were cultured overnight in serum-free medium before trypsinized and resuspended in a density of 2 9 105 cells/ml in DMEM containing 1 % FBS. The cells were then added to the upper chamber with MEM containing 10 % FBS as chemoattractant in the lower chamber. Medium containing 1 % FBS added to the lower chamber was used as control. The Matrigel and the cells remaining in the upper chamber were removed by cotton swabs following 24-h incubation. The cells on the lower surface of the membrane were stained with hematoxylin staining solution after the
A plasmid was constructed by cloning SOX9-coding gene into pEGFP-N1 vector to establish a U251 cell line that can stably express high level of SOX9. The mRNA level of SOX9 was determined by qRT-PCR after stably transfected cells were selected. The SOX9 mRNA level was markedly elevated by more than 3 folds in U251/SOX9 cells compared to U251/Vector cells and parental U251 cells. In addition, 3-fold increase in SOX9 expression was also found in U251/ SOX9 cells, as detected by western blot analysis (Fig. 1b). SOX9 Promotes Cell Migration and Invasion in U251 Cells We tested the cell migratory and invasive abilities through the scratch wound migration assay as well as migration and
123
Cell Biochem Biophys
metastasis of glioma by examining the epithelial marker E-cadherin and mesenchymal markers N-cadherin, vimentin, fibronectin, and a-SMA via Western blots. As shown in Fig. 3, SOX9 overexpression resulted in remarkable downregulation of E-cadherin and upregulation of N-cadherin, vimentin, fibronectin, and a-SMA. Taken together, these results indicated that overexpression of SOX9 promoted the transition from epithelial to mesenchymal phenotype.
Wnt/b-Catenin Signaling is Activated in SOX9 Overexpressed U251 Cells Previous studies have revealed the regulatory role of SOX9 on the transcription of Wnt genes in breast cancer cells [16]. Accordingly, we examined the effect of SOX9 on Wnt/b-catenin signaling pathway by Western blot. Our results showed that protein levels of Wnt1 and b-catenin were dramatically increased in U251/SOX9 cells compared to controls, indicating that overexpression of SOX9 was associated with activation of Wnt/b-catenin signaling (Fig. 4).
Effect of b-Catenin Targeting shRNA on Metastatic Behavior of U251 Induced by SOX9 Overexpression
Fig. 1 Quantification of SOX9 expression in U251 cells after transfection. SOX9 mRNA expression was determined by qRT-PCR (a). SOX9 protein expression was examined via Western blot (b). The values are expressed as the mean ± SD from three independent experiments. Representative images of Western blot from three independent experiments are displayed. **P \ 0.01 versus control
invasion assays to discover whether or not SOX9 can promote the malignancy of glioma in vitro. The results of scratch wound migration assay (Fig. 2a) showed that the U251 cells stably overexpressing SOX9 migrated significantly quicker than the control cells at 24, 36, and 48 h. Transwell experiments reflect cell trans-membrane abilities, including migration and invasion. The migratory and invasive abilities of U251 cells stably overexpressing SOX9 were determined using the Boyden chamber transwell assay. Trans-membrane cell numbers were counted and statistically analyzed. As shown in Fig. 2b, c, SOX9 overexpression significantly enhanced the migratory and invasive abilities of U251 cells compared with the control cells. SOX9 Promotes EMT Process in U251 Cells Next, we examined the effect of SOX9 overexpression on EMT process to further evaluate the role of SOX9 in the
123
In our results before mentioned, we found that SOX9 promoted metastatic behavior and SOX9 overexpression correlated with Wnt/b-catenin signaling activation. Therefore, we established stable U251/SOX9 with suppressed expression of b-catenin to further explore the role of b-catenin in mediating the promoting effect of SOX9 on metastatic behavior. After transfection with bcatenin targeted shRNA, b-catenin expression was effectively decreased (P \ 0.01). Next, the migration and invasion of cells were examined to further investigate the role of b-catenin in mediating the regulatory effect role of SOX9 on cellular function. As shown in Fig. 4b, c, suppressing b-catenin expression resulted in compromised cell migration and invasion ability compared with controls, directly demonstrating that b-catenin acted as essential mediator in SOX9-induced increase in cell migration and invasion. The changes in epithelial marker E-cadherin and mesenchymal markers a-SMA induced by b-catenin silencing were examined by Western blotting. The results revealed that E-cadherin was significantly upregulated while protein levels of N-cadherin, vimentin, fibronectin, and a-SMA were significantly decreased (P \ 0.01) (Fig. 4d), indicating that b-catenin downregulation abolished the promoting effect of SOX9 on EMT.
Cell Biochem Biophys Fig. 2 SOX9 overexpression enhances migration and invasion in U251 cells. Effect of SOX9 overexpression on cell migration was assessed by wound scratch (a) and transwell chamber assay without Matrigel (b). Effect of SOX9 overexpression on cell invasion was detected by transwell assay with Matrigel (c). The values are expressed as the mean ± SD from three independent experiments. Representative images of wound scratch and transwell chamber assay from three independent experiments are displayed. *P \ 0.05 versus control, **P \ 0.01 versus control
Fig. 3 SOX9 overexpression promotes EMT process. Effect of SOX9 overexpression on expression of epithelial cell markers and mesenchymal cell markers (E-cadherin, N-cadherin, vimentin, fibronectin, and a-SMA) were examined by Western blot. The values are expressed as the mean ± SD from three independent experiments. Representative images of Western blot from three independent experiments are displayed. *P \ 0.05 versus control
123
Cell Biochem Biophys
Fig. 4 SOX9 regulates cell migration and invasion through modulating Wnt/b-catenin signaling pathway. SOX9 overexpression correlates with Wnt/b-catenin activation. Activation of Wnt/b-catenin signaling was examined by Western blot (a). Effect of b-catenin knockdown on cell migration was assessed by wound scratch (b) and transwell chamber assay without Matrigel (c). Effect of b-catenin
knockdown on cell invasion was detected by transwell assay with Matrigel (d). Effect of b-catenin knockdown on expression of epithelial cell markers and mesenchymal cell markers (E-cadherin, N-cadherin, vimentin, fibronectin, and a-SMA) were examined by Western blot (e). Representative images of each assay from three independent experiments are displayed. **P \ 0.01 versus control
Discussion
prostate, bladder and pancreas, as well as melanoma, and correlates with poor clinical prognosis [18, 19]. In this study, we investigated the role of SOX9 in the process of metastasis, including cell migration, invasion and EMT, using in vitro glioma model. Our results revealed that SOX9 overexpression increases cell motility, invasiveness, and promoted EMT process. Meanwhile, our findings demonstrated that the regulatory effect of SOX9 on metastasis was, at least in part, mediated through bcatenin. Metastasis consists of multiple sequential steps starting with invasion of cancer cells into surrounding tissues, which requires increased cell motility. A growing body of literature has suggested that SOX9 could mediate mesenchymal features and associated with poor prognosis of cancer patients [12, 20]. In consistent with these findings,
Wnt proteins are a large family of secreted glycoproteins activating the Wnt-pathway. Binding of WNT to Frizzled (Fz) leads to the activation of Disheveled (Dsh), which inhibits the activity of GSK3-b. As a result, dephosphorylation and stabilization of b-catenin occur, enabling it to accumulate within the nucleus, where it interacts with various transcription factors to cause cellular proliferation and differentiation [17]. Several studies revealed that Wnt/ b-catenin signaling pathway plays an essential role in the development and promotion of EMT, and clinical evidences have demonstrated that upregulated b-catenin expressions is significantly associated with invasion and metastasis of carcinomas of the head and neck, esophagus, stomach, colon, liver, lung, breast, female genitalia,
123
Cell Biochem Biophys
our results also demonstrated that SOX9 overexpression increase cell motility and invasiveness in U251 cells. With high migration and invasion capability, U251 cell could easily spread into surrounding tissues and penetrate the walls of lymphatic and/or blood vessels. Hence, the overall effect of SOX9 was to promote metastasis by accelerating the initial steps in the process. EMT is a well-coordinated process during cancer progression, which renders polarity and motility to epithelial cells for tumor invasion and metastasis. During EMT, morphological change was characterized by epithelial polarized cell type changing into the mesenchymal fibroblastoid type [21]. After that, epithelial cells obtained enhanced migratory capabilities due to loss of cell–cell or cell–substrate contacts. The process of EMT is often marked by the decrease of epithelial junction proteins (e.g., E-cadherin, claudins, occludin) as well as downregulation of epithelial differentiation markers including cytokeratins and E-cadherin. In the meanwhile, EMT process is also associated with upregulation of mesenchymal markers, such as vimentin, fibronectin, and N-cadherin. Capaccione et al. reported SOX9 is a negative regulator of E-cadherin, a key protein that is lost during EMT [20]. In consistent with their studies, our results demonstrated that SOX9 overexpression in glioma cells led to molecular changes corresponding to EMT process, providing further evidence of the involvement of SOX9 in the process of EMT. A number of studies have found that nuclear accumulation of b-catenin is also a mark of EMT. Moreover, the nuclear b-catenin accumulation was found in the invasive fronts of primary tumors [22, 23]. In this study, we found increased expression of b-catenin in SOX9 overexpressed glioma cells, suggesting the regulatory role of SOX9 in bcatenin signaling. The study in lung adenocarcinoma cells showed that SOX9 acts downstream of Wnt/b-catenin signaling pathway [20]. On the other hand, Wang et al. suggested that SOX9 is at the center of a positive feedback loop that enhances Wnt/b-catenin signaling by demonstrating SOX9 silencing decreased Wnt/b-catenin activation while SOX9 expression was also increased by Wnt [16]. In this study, we did show that SOX9 played a regulatory role in b-catenin activity. However, we did not address the exact role where SOX9 stands in the whole picture. Elucidation of this question will help us to gain a full understanding of SOX9’s role in cellular functions. In summary we demonstrated that overexpressing SOX9 could promote metastasis of glioma, which could be attributed to the ability of SOX9 to increase cell migration, invasion, and stimulate EMT process. Furthermore, we demonstrated that molecular events involved in SOX9 overexpression-induced phenotype changes were mediated via b-catenin. Our data suggested that targeting SOX9 might provide a potential promising therapeutic strategy to
prevent glioma progression and improve the prognosis of patients with glioma.
References 1. Wei, J., Gabrusiewicz, K., & Heimberger, A. (2013). The controversial role of microglia in malignant gliomas. Clinical and Developmental Immunology, 2013, 285246. 2. Kettenmann, H., et al. (2011). Physiology of microglia. Physiological Reviews, 91(2), 461–553. 3. Steeg, P. S. (2006). Tumor metastasis: Mechanistic insights and clinical challenges. Nature Medicine, 12(8), 895–904. 4. Li, X., et al. (2013). SOX2 promotes tumor metastasis by stimulating epithelial-to-mesenchymal transition via regulation of WNT/beta-catenin signal network. Cancer Letters, 336(2), 379–389. 5. Hersmus, R., et al. (2008). FOXL2 and SOX9 as parameters of female and male gonadal differentiation in patients with various forms of disorders of sex development (DSD). Journal of Pathology, 215(1), 31–38. 6. Jay, P., Berta, P., & Blache, P. (2005). Expression of the carcinoembryonic antigen gene is inhibited by SOX9 in human colon carcinoma cells. Cancer Research, 65(6), 2193–2198. 7. Thomsen, M. K., et al. (2010). SOX9 elevation in the prostate promotes proliferation and cooperates with PTEN loss to drive tumor formation. Cancer Research, 70(3), 979–987. 8. Kimura, M. S., Mutoh, H., & Sugano, K. (2011). SOX9 is expressed in normal stomach, intestinal metaplasia, and gastric carcinoma in humans. Journal of Gastroenterology, 46(11), 1292–1299. 9. Chakravarty, G., et al. (2011). Prognostic significance of cytoplasmic SOX9 in invasive ductal carcinoma and metastatic breast cancer. Experimental Biology and Medicine (Maywood), 236(2), 145–155. 10. Sutter, R., et al. (2010). Cerebellar stem cells act as medulloblastoma-initiating cells in a mouse model and a neural stem cell signature characterizes a subset of human medulloblastomas. Oncogene, 29(12), 1845–1856. 11. Schlierf, B., et al. (2007). Expression of SoxE and SoxD genes in human gliomas. Neuropathology and Applied Neurobiology, 33(6), 621–630. 12. Wang, L., et al. (2012). Oncogenic role of SOX9 expression in human malignant glioma. Medical Oncology, 29(5), 3484– 3490. 13. Shi, G., et al. (2013). Expression and functional role of Sox9 in human epidermal keratinocytes. PLoS One, 8(1), e54355. 14. Jung, M., et al. (2007). In search of suitable reference genes for gene expression studies of human renal cell carcinoma by realtime PCR. BMC Molecular Biology, 8, 47. 15. Zhao, J. H., et al. (2011). Knockdown of beta-catenin through shRNA cause a reversal of EMT and metastatic phenotypes induced by HIF-1 alpha. Cancer Investigation, 29(6), 377–382. 16. Wang, H., et al. (2013). SOX9 regulates low density lipoprotein receptor-related protein 6 (LRP6) and T-cell factor 4 (TCF4) expression and Wnt/beta-catenin activation in breast cancer. Journal of Biological Chemistry, 288(9), 6478–6487. 17. Galera-Ruiz, H., et al. (2012). WNT pathway in laryngeal squamous cell carcinoma and nasopharyngeal carcinoma. Acta Otorhinolaryngologica Italica, 32(2), 122–123. 18. Fu, Y., et al. (2011). Beta-catenin as a potential key target for tumor suppression. International Journal of Cancer, 129(7), 1541–1551.
123
Cell Biochem Biophys 19. Valenta, T., Hausmann, G., & Basler, K. (2012). The many faces and functions of beta-catenin. EMBO Journal, 31(12), 2714–2736. 20. Capaccione, K. M., et al. (2014). Sox9 mediates Notch1-induced mesenchymal features in lung adenocarcinoma. Oncotarget, 5(11), 3636–3650. 21. Yang, J., & Weinberg, R. A. (2008). Epithelial–mesenchymal transition: At the crossroads of development and tumor metastasis. Developmental Cell, 14(6), 818–829.
123
22. Polette, M., et al. (2007). Beta-catenin and ZO-1: Shuttle molecules involved in tumor invasion-associated epithelial–mesenchymal transition processes. Cells Tissues Organs, 185(1–3), 61–65. 23. Brabletz, T., et al. (2001). Variable beta-catenin expression in colorectal cancers indicates tumor progression driven by the tumor environment. Proceedings of the National Academy Sciences USA, 98(18), 10356–10361.
