AXL is a marker for epithelial‑mesenchymal transition in esophageal squamous cell carcinoma
- Authors:
- Published online on: November 20, 2017 https://doi.org/10.3892/ol.2017.7443
- Pages: 1900-1906
Abstract
Introduction
AXL is a member of the TYRO3-AXL-MER family of receptor tyrosine kinases, which includes two other members: TYRO3 and MER. AXL is activated by its ligand, growth arrest-specific 6, which in turn triggers several downstream signaling pathways depending on the cell type, primarily phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) and mitogen-activated protein kinase/extracellular-signal-regulated kinase, nuclear factor-κB (NF-κB) and signal transducer and activator of transcription 3 (STAT3) signaling pathways. It exerts a wide array of functions including cell survival, proliferation, migration and adhesion (1). AXL has also been implicated in the pathophysiology of a number of types of cancer, including breast, gastric, prostate, ovarian and lung (2–4). AXL expression promotes cancer cell survival, angiogenesis, metastasis and drug resistance (3,5).
AXL is significantly associated with epithelial-mesenchymal transition (EMT) in several tumors. RNA sequencing data of 643 cancer cell lines demonstrated that AXL expression is markedly associated with a mesenchymal phenotype (6). AXL expression is increased in mesenchymal cells compared with epithelial cells in non-small cell lung cancer (NSCLC) cell lines (7). Furthermore, AXL downregulation in A549 and H460 mesenchymal cells leads to enhanced expression of epithelial cadherin (E-cadherin) and decreased expression of vimentin and neural cadherin (N-cadherin), which are features of mesenchymal-epithelial transition (7,8), suggesting that AXL is involved in maintaining EMT. AXL has also been identified to regulate EMT in other types of cancer, including breast (9,10), prostate (11), ovarian (12) and pancreatic (13,14).
China is among the countries at highest risk of esophageal squamous cell carcinoma (ESCC) (15). ESCC is the fourth most common cancer in China, accounting for ~13% of all cancer cases in 2015 (16). ESCC is frequently associated with a high risk of recurrence and high mortality rate and the 5-year survival rate is <20% (17).
The function of AXL in ESCC has been demonstrated only recently (18–20). AXL gene (19) and protein (19,20) expression were upregulated in ESCC cells compared with normal adjacent cells, and were identified to be associated with tumor progression, increased risk of mortality and distant metastasis (20). AXL was also identified to be consistently overexpressed in ESCC cell lines (19). Knockdown of AXL expression inhibited cell proliferation, survival, migration and invasion in vitro and in vivo, and those effects were mediated by the ΑKΤ/NF-κB and ΑKΤ/glycogen synthase kinase-3β signaling pathways (19). Overexpression of AXL also mediated resistance to the PI3Kα inhibitor BYL719 by activating signaling pathways in ESCC cells (18).
However, the function of AXL in EMT in ESCC cells is not well-documented. To the best of our knowledge, the present study provides the first evidence that AXL is a marker for the mesenchymal phenotype in ESCC using online mRNA profile data.
Materials and methods
Gene expression data
GSE47404 (21), GSE23400 (22) and GSE21293 (23) gene expression data were obtained from the Gene Expression Omnibus database (GEO; www.ncbi.nlm.nih.gov/geo). Further information on the sample size and the microarray platforms used for the creation of these datasets is presented in Table I. Processed expression data and information of corresponding platforms were downloaded and used further. Expression values (mRNA log2 intensity) for each gene were examined manually and used for further study.
Gene Set Enrichment Analysis (GSEA)
GSEA is a computational software that determines whether predefined set of genes exhibit statistically significant concordant differences between two biological states (http://software.broadinstitute.org/gsea/index.jsp) (24). AXL expression was determined on the basis of GSE47404 and GSE23400 datasets. If two probe sets correspond to AXL, AXL expression is calculated as the average of the value of the two probe sets. Following determination of the mean expression of AXL, the samples were designated AXL-high ESCC (samples with an above-average AXL value) or AXL-low ESCC (samples with a below-average AXL value). AXL-low samples served as the control samples. This collection of gene sets summarized and represented specific well-defined biological states or processes, and was generated by a computational methodology based on identifying gene set overlaps and retaining genes that display coordinate expression across several datasets (25). All the parameters were set to their default values. A false discovery rate (FDR) value <0.25 was considered to indicate significant enrichment. The procedure followed for the GSEA is presented in Fig. 1.
Association of AXL expression and cancer cell invasive ability in GSE21293
GSE21293 displayed the mRNA profiles of 34 genetically engineered human esophageal cells with different invasive abilities (23). To determine whether AXL is associated with the invasive ability of ESCC cells, AXL expression in the samples was examined and the mean expression value was calculated, and samples were divided into two groups according to their AXL expression value as aforementioned: AXL-high and AXL-low. AXL-low samples acted as the control samples. The samples were also divided into two groups according to their invasive ability: Invasive and non-invasive. Fisher's exact test was used to examine their association. Furthermore, AXL expression in the invasive and non-invasive groups was also compared using Mann-Whitney U test.
Statistical analysis
Data were analyzed using SPSS (version 13.0; SPSS, Inc., Chicago, IL, USA). Spearman's correlation was used to analyze the association between AXL mRNA and mRNA of mesenchymal markers. Fisher's Exact Test was applied to determine the correlation between AXL expression and invasion ability of genetically engineered esophageal cells. Mann-Whitney U test was used to assess the expression difference of AXL mRNA in invasive and non-invasive esophageal cells. P<0.05 was considered to indicate a statistically significant difference.
Results
AXL mRNA expression is associated with certain mesenchymal markers
To investigate the function of AXL in EMT, the association of AXL with several EMT markers, including E-cadherin, vimentin, fibronectin, snail family transcriptional repressor 1 (SNAI1), snail family transcriptional repressor 2 (SNAI2), twist family basic helix-loop-helix transcription factor 1 (TWIST1), zinc finger E-box-binding homeobox 1 (ZEB1) and N-cadherin, was examined (Fig. 2). It was revealed that AXL mRNA expression in GSE47404 and GSE23400 datasets is associated with several EMT markers, including vimentin (P=1.00×10−6 for GSE47404 and 1.00×10−6 for GSE23400; Fig. 2B and J), fibronectin (P=2.22×10−4 for GSE47404 and 1.00×10−6 for GSE23400; Fig. 2D and L), TWIST1 (P=0.01 for GSE47404 and 1.90×10−4 for GSE23400; Fig. 2G and O) and ZEB1 (P=0.04 for GSE47404 and 1.00×10−6 for GSE23400; Fig. 2H and P). However, no association between AXL expression and E-cadherin (Fig. 2A and I) or SNAI2 (Fig. 2F and N) was identified in either dataset. A significant association was identified between AXL and SNAI1 in GSE47404 (P=2.03×10−3; Fig. 2E), but not in GSE23400 (P=0.67; Fig. 2M). Additionally, the association between AXL and N-cadherin was significant in GSE23400 (P=6.59×10−3; Fig. 2K), but not in GSE47404 (P=0.74; Fig. 2C). These preliminary results suggest that AXL may be involved in EMT in ESCC.
EMT gene set is enriched in the ESCC group with high AXL expression
To further determine the function of AXL in EMT in ESCC, GSEA was performed to examine whether the AXL-high and the AXL-low expression groups display a different EMT state in ESCC. The GSEA of the present study utilized ‘Hallmark gene sets [h.all.v5.2.symbols.gmt (Hallmarks)]’ as the gene sets to be analyzed.
The results revealed that in GSE47404, the ESCC group with high AXL expression levels displayed a more marked mesenchymal state compared with the ESCC group with low AXL expression levels (Fig. 3A, and Table II). Furthermore, transforming growth factor-β signaling, which has a predominant function in EMT in cancer (26,27), is also enriched in the AXL-high expression group (Fig. 3B and Table II). Analyze on GSE23400 also showed similar pattern (Fig. 3C and D and Table II). STAT3 signaling, which is reported to serve a function in EMT in ESCC (28), is also highly enriched in the AXL-high expression group in the two datasets (Table II).
AXL expression is associated with increased invasive ability of ESCC cells
A prominent feature of EMT is to confer enhanced invasive ability on cancer cells. Therefore, the present study assessed the association between AXL expression and the invasive ability of ESCC on the basis of the GSE21293 dataset (23). This dataset includes normal esophageal cells, which are genetically engineered with human telomerase reverse transcriptase, epidermal growth factor receptor overexpression and p53 mutations, in order to immortalize them and display differential invasion. According to their ability to invade, these cells were divided into invasive and non-invasive groups. As presented in Table III, the AXL-high expression group displayed increased ability of invasion (P=2.61×10−4). AXL expression in non-invasive and invasive groups was also compared and it was revealed that the invasive group expressed significantly increased AXL levels (P=1.95×10−4; Fig. 4).
Table III.Association between AXL expression and invasive ability of genetically engineered esophageal cells. |
Discussion
EMT is a fundamental process in body development, and in the differentiation of multiple tissues and organs (29). EMT endows cancer cells with invasive properties and with stem-cell like characteristics, prevents drug-induced apoptosis and senescence, and contributes to immunosuppression (29,30). The EMT state defined by the E-cadherin/vimentin ratio has been demonstrated to be associated with a poor 5-year survival rate of patients with ESCC (31).
AXL has been demonstrated to be an important factor in certain tumors including NSCLC, breast cancer, prostate cancer, myeloid leukemia and ovarian cancer (2,3,12,32). AXL enhances tumor proliferation, promotes EMT and induces drug resistance (2,3,32). Clinical trials for melanoma, NSCLC and acute myeloid leukemia are ongoing to determine the safety and efficiency of AXL inhibitors (https://clinicaltrials.gov/ct2/results?cond=&term=BGB324&cntry1=&state1=&Search=Search).
The function of AXL in ESCC has only recently been identified (18–20). However, its function in EMT in ESCC is even less well-elucidated. The present study provides preliminary evidence from three independent mRNA array studies indicating that AXL is a marker for the mesenchymal state of ESCC. According to the results of the present study, it was revealed that AXL expression is associated with certain EMT markers, including vimentin, fibronectin, TWIST1 and ZEB1. However, AXL was not associated with other EMT markers, including E-cadherin. These results are consistent with those in a study by Wilson et al (6) which demonstrated that AXL mRNA is associated with vimentin mRNA expression in a group of 643 human cancer cell lines. In contrast, Sato et al (33) indicated that AXL expression is associated with E-cadherin, but not vimentin, in lung adenocarcinomas. Additionally, the ESCC group with high AXL expression was enriched with the EMT gene set as revealed by GSEA. Another data cohort revealed that AXL is markedly associated with the invasive ability of genetically engineered esophageal cells. Collectively, the results of the present study suggest that AXL is an EMT marker for ESCC. However, further studies, including in vitro and in vivo cancer models, are required to confirm those results. As mRNA and protein expression are not necessarily associated (34), experiments analyzing AXL protein levels in ESCC are required.
Acknowledgements
The present study is supported by a Shandong Province Higher Educational Science and Technology Program (grant no. J13LK14), Natural Science Foundation of Shandong province (grant no. 2013ZRC03007), Jining Scientific and Technological Project (grant nos. 2012jnjc02, 2014jnjc17 and 2015-57-71), Youth Foundation of Jining Medical University (grant nos. JY2013KJ035 and JY2015KJ023), The Research Innovation Program for College Graduates of Shandong Province (grant no. SDYY14014) and Projects of Education Planning Office of Shandong Province (grant no. 2013GG043). The authors thank Dr Liyan Guo (Associate Professor of Epidemiology, Jining Medical University) for assistance with statistical analysis.
References
Hafizi S and Dahlbäck B: Signalling and functional diversity within the Axl subfamily of receptor tyrosine kinases. Cytokine Growth Factor Rev. 17:295–304. 2006. View Article : Google Scholar : PubMed/NCBI | |
Paccez JD, Vogelsang M, Parker MI and Zerbini LF: The receptor tyrosine kinase Axl in cancer: Biological functions and therapeutic implications. Int J Cancer. 134:1024–1033. 2014. View Article : Google Scholar : PubMed/NCBI | |
Graham DK, DeRyckere D, Davies KD and Earp HS: The TAM family: Phosphatidylserine sensing receptor tyrosine kinases gone awry in cancer. Nat Rev Cancer. 14:769–785. 2014. View Article : Google Scholar : PubMed/NCBI | |
Qu X, Liu J, Zhong X, Li X and Zhang Q: Role of AXL expression in non-small cell lung cancer. Oncol Lett. 12:5085–5091. 2016.PubMed/NCBI | |
Zhang Z, Lee JC, Lin L, Olivas V, Au V, LaFramboise T, Abdel-Rahman M, Wang X, Levine AD, Rho JK, et al: Activation of the AXL kinase causes resistance to EGFR-targeted therapy in lung cancer. Nat Genet. 44:852–860. 2012. View Article : Google Scholar : PubMed/NCBI | |
Wilson C, Ye X, Pham T, Lin E, Chan S, McNamara E, Neve RM, Belmont L, Koeppen H, Yauch RL, et al: AXL inhibition sensitizes mesenchymal cancer cells to antimitotic drugs. Cancer Res. 74:5878–5890. 2014. View Article : Google Scholar : PubMed/NCBI | |
Byers LA, Diao L, Wang J, Saintigny P, Girard L, Peyton M, Shen L, Fan Y, Giri U, Tumula PK, et al: An epithelial-mesenchymal transition gene signature predicts resistance to EGFR and PI3K inhibitors and identifies Axl as a therapeutic target for overcoming EGFR inhibitor resistance. Clin Cancer Res. 19:279–290. 2013. View Article : Google Scholar : PubMed/NCBI | |
Wu F, Li J, Jang C, Wang J and Xiong J: The role of Axl in drug resistance and epithelial-to-mesenchymal transition of non-small cell lung carcinoma. Int J Clin Exp Pathol. 7:6653–6661. 2014.PubMed/NCBI | |
Asiedu MK, Beauchamp-Perez FD, Ingle JN, Behrens MD, Radisky DC and Knutson KL: AXL induces epithelial-to-mesenchymal transition and regulates the function of breast cancer stem cells. Oncogene. 33:1316–1324. 2014. View Article : Google Scholar : PubMed/NCBI | |
Gjerdrum C, Tiron C, Høiby T, Stefansson I, Haugen H, Sandal T, Collett K, Li S, McCormack E, Gjertsen BT, et al: Axl is an essential epithelial-to-mesenchymal transition-induced regulator of breast cancer metastasis and patient survival. Proc Natl Acad Sci USA. 107:pp. 1124–1129. 2010; View Article : Google Scholar : PubMed/NCBI | |
Mishra A, Wang J, Shiozawa Y, McGee S, Kim J, Jung Y, Joseph J, Berry JE, Havens A, Pienta KJ and Taichman RS: Hypoxia stabilizes GAS6/Axl signaling in metastatic prostate cancer. Mol Cancer Res. 10:703–712. 2012. View Article : Google Scholar : PubMed/NCBI | |
Antony J, Tan TZ, Kelly Z, Low J, Choolani M, Recchi C, Gabra H, Thiery JP and Huang RY: The GAS6-AXL signaling network is a mesenchymal (Mes) molecular subtype-specific therapeutic target for ovarian cancer. Sci Signal. 9:ra972016. View Article : Google Scholar : PubMed/NCBI | |
Koorstra JB, Karikari CA, Feldmann G, Bisht S, Rojas PL, Offerhaus GJ, Alvarez H and Maitra A: The Axl receptor tyrosine kinase confers an adverse prognostic influence in pancreatic cancer and represents a new therapeutic target. Cancer Biol Ther. 8:618–626. 2009. View Article : Google Scholar : PubMed/NCBI | |
Kirane A, Ludwig KF, Sorrelle N, Haaland G, Sandal T, Ranaweera R, Toombs JE, Wang M, Dineen SP, Micklem D, et al: Warfarin blocks gas6-Mediated Axl activation required for pancreatic cancer epithelial plasticity and metastasis. Cancer Res. 75:3699–3705. 2015. View Article : Google Scholar : PubMed/NCBI | |
Jemal A, Bray F, Center MM, Ferlay J, Ward E and Forman D: Global cancer statistics. CA Cancer J Clin. 61:69–90. 2011. View Article : Google Scholar : PubMed/NCBI | |
Chen W, Zheng R, Baade PD, Zhang S, Zeng H, Bray F, Jemal A, Yu XQ and He J: Cancer statistics in China, 2015. CA Cancer J Clin. 66:115–132. 2016. View Article : Google Scholar : PubMed/NCBI | |
Allum WH, Stenning SP, Bancewicz J, Clark PI and Langley RE: Long-term results of a randomized trial of surgery with or without preoperative chemotherapy in esophageal cancer. J Clin Oncol. 27:5062–5067. 2009. View Article : Google Scholar : PubMed/NCBI | |
Elkabets M, Pazarentzos E, Juric D, Sheng Q, Pelossof RA, Brook S, Benzaken AO, Rodon J, Morse N, Yan JJ, et al: AXL mediates resistance to PI3Kα inhibition by activating the EGFR/PKC/mTOR axis in head and neck and esophageal squamous cell carcinomas. Cancer Cell. 27:533–546. 2015. View Article : Google Scholar : PubMed/NCBI | |
Paccez JD, Duncan K, Vava A, Correa RG, Libermann TA, Parker MI and Zerbini LF: Inactivation of GSK3β and activation of NF-kB pathway via Axl represents an important mediator of tumorigenesis in esophageal squamous cell carcinoma. Mol Biol Cell. 26:821–831. 2015. View Article : Google Scholar : PubMed/NCBI | |
Hsieh MS, Yang PW, Wong LF and Lee JM: The AXL receptor tyrosine kinase is associated with adverse prognosis and distant metastasis in esophageal squamous cell carcinoma. Oncotarget. 7:36956–36970. 2016. View Article : Google Scholar : PubMed/NCBI | |
Sawada G, Niida A, Uchi R, Hirata H, Shimamura T, Suzuki Y, Shiraishi Y, Chiba K, Imoto S, Takahashi Y, et al: Genomic landscape of esophageal squamous cell carcinoma in a japanese population. Gastroenterology. 150:1171–1182. 2016. View Article : Google Scholar : PubMed/NCBI | |
Su H, Hu N, Yang HH, Wang C, Takikita M, Wang QH, Giffen C, Clifford R, Hewitt SM, Shou JZ, et al: Global gene expression profiling and validation in esophageal squamous cell carcinoma and its association with clinical phenotypes. Clin Cancer Res. 17:2955–2966. 2011. View Article : Google Scholar : PubMed/NCBI | |
Michaylira CZ, Wong GS, Miller CG, Gutierrez CM, Nakagawa H, Hammond R, Klein-Szanto AJ, Lee JS, Kim SB, Herlyn M, et al: Periostin, a cell adhesion molecule, facilitates invasion in the tumor microenvironment and annotates a novel tumor-invasive signature in esophageal cancer. Cancer Res. 70:5281–5292. 2010. View Article : Google Scholar : PubMed/NCBI | |
Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, Paulovich A, Pomeroy SL, Golub TR, Lander ES and Mesirov JP: Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA. 102:pp. 15545–15550. 2005; View Article : Google Scholar : PubMed/NCBI | |
Liberzon A, Birger C, Thorvaldsdóttir H, Ghandi M, Mesirov JP and Tamayo P: The molecular signatures database (MSigDB) hallmark gene set collection. Cell Syst. 1:417–425. 2015. View Article : Google Scholar : PubMed/NCBI | |
Xu J, Lamouille S and Derynck R: TGF-beta-induced epithelial to mesenchymal transition. Cell Res. 19:156–172. 2009. View Article : Google Scholar : PubMed/NCBI | |
Lamouille S, Xu J and Derynck R: Molecular mechanisms of epithelial-mesenchymal transition. Nat Rev Mol Cell Biol. 15:178–196. 2014. View Article : Google Scholar : PubMed/NCBI | |
Cui Y, Li YY, Li J, Zhang HY, Wang F, Bai X and Li SS: STAT3 regulates hypoxia-induced epithelial mesenchymal transition in oesophageal squamous cell cancer. Oncol Rep. 36:108–116. 2016. View Article : Google Scholar : PubMed/NCBI | |
Thiery JP, Acloque H, Huang RY and Nieto MA: Epithelial-mesenchymal transitions in development and disease. Cell. 139:871–890. 2009. View Article : Google Scholar : PubMed/NCBI | |
Singh A and Settleman J: EMT, cancer stem cells and drug resistance: An emerging axis of evil in the war on cancer. Oncogene. 29:4741–4751. 2010. View Article : Google Scholar : PubMed/NCBI | |
Niwa Y, Yamada S, Koike M, Kanda M, Fujii T, Nakayama G, Sugimoto H, Nomoto S, Fujiwara M and Kodera Y: Epithelial to mesenchymal transition correlates with tumor budding and predicts prognosis in esophageal squamous cell carcinoma. J Surg Oncol. 110:764–769. 2014. View Article : Google Scholar : PubMed/NCBI | |
Bivona TG and Okimoto RA: AXL receptor tyrosine kinase as a therapeutic target in NSCLC. Lung Cancer (Auckl). 6:27–34. 2015.PubMed/NCBI | |
Sato K, Suda K, Shimizu S, Sakai K, Mizuuchi H, Tomizawa K, Takemoto T, Nishio K and Mitsudomi T: Clinical, pathological and molecular features of lung adenocarcinomas with AXL expression. PLoS One. 11:e01541862016. View Article : Google Scholar : PubMed/NCBI | |
Vogel C and Marcotte EM: Insights into the regulation of protein abundance from proteomic and transcriptomic analyses. Nat Rev Genet. 13:227–232. 2012.PubMed/NCBI |