Altered Expression Of ETV1and Its Contribution To Tumorigenic Phenotypes In Gastrointestinal Stromal Tumor

Background:Gastrointestinal stromal tumor (GIST) is the most common mesenchymal neoplasm of the gastrointestinal tract which accounts for1-4%of all GI tumors. The incidence of GIST is estimated to be approximately14.5cases per million and now gradually increases year by year. It appears to arise from interstitial cells of Cajal (ICC) or their mesenchymal stem cell precursors. It is reported that ICCs are the neural pacemaker cells responsible for gut peristalsis and are important for the autonomous movement of the GI tract. The majority of GISTs are primarily defined by activating mutations in the KIT (85%) or PDGFRA (10%) receptor tyrosine kinases. Surgical resection is the preferred management of GISTs. However,55-90%of the patients tend to recur after the surgery. What’s more, the median survival time of the patients with unresectable tumors is less than12months, and traditional chemotherapy and radiation therapy provide little help. Imatinib, a small molecule tyrosine kinase inhibitor, has proven useful in the treatment of recurrent and metastatic gastrointestinal stromal tumor by targeting the activating mutations of KIT and PDGFRA. While complete remission could be achieved by less than3%of patients, approximately50%of the patients treated by imatinib acquired secondary resistance within2years and the median survival time is only15months. Furthermore, GISTs with widetype or PDGFRA D842V mutation are insensitive to imatinib. Therefore, resistance to tyrosine kinase inhibitors is also problematic. KIT inhibiton alone is unlikely to cure GISTs. It is extremely urgent to explore the resistant mechanism and look for novel target therapy.ETS transcription factors constitute the largest family that is involved in signaling-dependent transcriptional regulation. Overexpression of ETS proteins modulates pathways relevant to tumor progression and has become the concern of international scholars in recent years. Researchers have found more than30ETS family members, all of them contain a specific and conserved DNA-binding domain (DBD), the ETS domain. Via the DBD domain consisted of85amino acids, ETS factors interact with the GGAA/T sequence and regulate the genetic transcription. ETS members participate in the diverse process including cell proliferation, differentiation, apoptosis and cell-cell/cell-matrix interactions.ETS variant1(ETV1, ER81) is one of the members of ETS family which locates in the chromosome of7p21.3. It has been defined as an oncoprotein overexpressed in breast cancer, prostatic carcinoma, melanoma, Ewing sarcoma as well as gastrointestinal stromal tumor in previous studies, playing a key role in the neoplastic formation, development and progression. Chi P highlighted the observation that ETV1was a lineage-specific survival factor which cooperated with KIT in GIST. ETV1expressed at a higher level compared with other kinds of tumors such as gastric cancer, liver carcinoma, colorectal cancer, prostate cancer, melanoma and ovarian cancer. Furthermore, results demonstrated that strong ETV1expression and constitutively activated KIT were implicated in hyperplasia ICC set and collaborated to promote tumorigenesis. If we can confirm ETV1regulate target molecule or pathways in the tumorigenesis of GIST, we could find out a novel targeted therapy to solve the imatinib-resisitant problem. Objective:To investigate the expression level of ETV1in gastrointestinal stromal tumor and its cell line (GIST882), analysis the expression difference between the tumor tissue and the corresponding normal tissue, explore the potential relationship between expression, clinicopathologic parameters as well as risk stratification in GIST patients. Down-regulation of ETV1is to find the change of GIST882cell proliferation, cell cycle and apoptosis. Explore the potential mechanism of ETV1in the regulation of tumorigenesis of GIST, providing the basis for the novel targeted therapy.Methods:The expression of ETV1expression in72cases of GIST, its adjacent normal tissues and GIST882cell line was quantified by using qRT-PCR and Western Blot. Immunohistochemical staining in GIST tissue microarray which contains156cases was used to detect the expression distribution and intensity of ETV1. The correlations of ETV1expression with clinicopathological characteristics and KIT expression were analyzed.Three chemical synthesis siRNAs were transiently transfected into GIST882cell line to down-regulate ETV1expression. Using qRT-PCR and Western blot to detect ETV1expression, screen and determine which siRNA to be used as follow-up studies on the basis of gene silencing efficiency. The effect of down-regulation of ETV1on GIST882cell proliferation, apoptosis and cell cycle were detected by using CCK-8kit and flow cytometer, respectively.Western Blot was carried out to detect relevant expression and changes of Ras/Raf/MEK/ERK signaling pathway in the transfected GIST882cell.Results:Altered ETV1expression was evaluated in the72GISTs by using qRT-PCR and Western Blot. The results indicated that ETV1was highly expressed in GIST both in the transcription and protein level. Little or no expression of ETV1was detected in other sarcomas and their corresponding tumor-adjacent normal specimens (P<0.01). Further analysis in GIST882cell line revealed strong positive KIT and ETV1expression. In addition, immunohistochemical staining in GIST TMA sections which contained156cases also confirmed previous results, ETV1was positive diffusely in the nuclei of the tumor cells and was prone to localize in stomach (P<0.05). The data showed that ETV1up-regulation has nothing to do with age or gender (P>0.05). Moreover, the frequencies of ETV1and KIT were mainly found in GIST and they cooperated with each other in the tumorigenesis. With respect to risk stratification, the frequency of ETV1positivity was lower compared to KIT expression in GIST with low-risk and intermediate-risk but higher in the high-risk group.In vitro experiment, we transfected3siRNAs into GIST882cell transiently and qRT-PCR was used to detect the level of ETV1mRNA in each group after transfected24h,48h,72h. The inhibition ratios of3siRNAs in different periods were:siRNA-A57.4%,63.0%,78.4%; siRNA-B37.5%,52.7%,65.5%; siRNA-C29.6%,46.1%,55.7%. ETV1mRNA expression was significantly lowered compared with negative control group and siRNA-A transfected72h was better in comparison with another two sequences. Western Blot further confirmed the above result, thus, siRNA-A was chosen for subsequent studies. CCK-8detected that cell proliferation was significantly inhibited after siRNA intervened. In addition, GIST882cell was arrested at G0/G1phase, the proportion was (84.60±1.22)%, and the difference was statistically significant compared with the Control group as well as Negative group. As ETV1siRNA was transfected into GIST882, the number of apoptotic cells significantly increased. However, cell apoptosis was not obvious compared with neither the negative group nor the control group.We also examined the effects of ETV1down-regulation on proliferation-associated signaling pathway by using Western Blot. It is impressive that once ETV1was inhibited after siRNA transfection, the protein level of Ras, p-c-Raf, p-MEK, p-ERK in the siRNA group was down-regulated compared with the negative control group while the total protein isoforms of Raf, MEK and ERK in each group were similar.Conclusions:ETV1is normally up-regulated in GIST tumor tissues as well as GIST882cell line. Experiments in vitro proved that down-regulation of ETV1could inhibit GIST882cell growth, block the cell cycle process and pormote cell apoptosis. As ETV1gene was silenced, the expression of Ras、p-c-Raf、p-MEK、p-ERK was significantly inhibited, suggesting that ETV1participated in the Ras/Raf/MEK/ERK pathway and integrated into cellular signaling network, then play important role in the development of tumorigenesis in gastrointestinal stromal tumor.