The Role Of Small Molecule Inhibitors Of JAK2Kinase In Gastric Cancer And Its Reiated Molecular Mechanism

Gastric cancer (GC) is one of the most common malignant tumor with high mortality worldwide. Along with the advance of medical science, tremendous development has been made in the diagnosis and treatment of GC. However, early detection rate of GC is still very low (about10%), and therapeutic strategy of advanced GC mainly relies on comprehensive treatment in China. Currently, the prognosis of advanced GC is very poor with a low5-year survival rate at about20%-30%. With the development of our understanding on gastric cancer related cell biology and molecular biology, molecular targeted therapy for GC which targeted tumor cell growth, apoptosis and cell cycle et al. has already become the research hotspot and it is urgent for us to do more work on it.Janus kinase2(JAK2) is one member of the JAK family. It is reported that aberrant JAK2signaling has been linked to a wide variety of tumors, such as colorectal cancer, head and neck cancer, lung cancer, breast cancer, leukemia, lymphoma and multiple myeloma. So far, little has been reported on JAK2and GC. Recent results from our laboratory proposed that JAK2may act as an oncogene in GC and small molecule inhibitors of JAK2kinase may be useful in GC treatment. Therefore, the aim of this study was to clarify the effects involving the JAK2signaling on the presence of AG490or JAK2shRNA on GC cell lines in vitro and in vivo; To explore and reveal the potential therapeutic value of AG490on GC and the possible mechanisms of nuclear JAK2signaling, and further clarify the role of JAK2in the development and process of GC, and the possible molecular mechanisms underlying it.Materials and Methods1. The expression of JAK2in GC cell lines and tissues, and its correlation with pathological characteristics of GC patients①The expression of JAK2protein in75GC samples and paired tumor-adjacent normal stomach tissues were determined by immunohistochemical methods;②The expression of JAK2mRNA, JAK2and pJAK2protein in four different differentiated gastric cancer cell lines MKN28, SGC7901, AGS, MKN45and human gastric epithelial cell line GES-1were examined by realtime quantitative RT-PCR and Western blot analysis;③The correlation between JAK2expression and pathological characteristics of GC patients was analyzed by Spearman’s rho test.2. The functional role of JAK2inhibitor AG490in GC①The GC cells were treated with different concentrations of AG490at different time, and the effects of AG490on cell proliferation were measured by MTS assay; Cell apoptosis were detected by Annexin V-FITC/PI Assays; The expressions of apoptosis and cell cycle-related protein were measured by Western blotting;②Xenografted gastric tumor model was used to validate the tumor suppressing effect by AG490in vivo, compared to control.3. Involvement of nuclear JAK2signaling in AG490-induced apoptosis of GC cells③Nuclear protein expressions of JAK2in four different differentiated gastric cancer cell lines and human gastric epithelial cell line GES-1were detected by Western blotting;②The effects of AG490on total cellular, cytoplasmic, and nuclear protein expressions of JAK2and pJAK2were measured by Western blotting. The effects of AG490on expression and distribution of JAK2were also detected by immunofluorescence;③JAK2-STAT3-Hesl complex formation in nucleus of SGC7901cells was verified by Co-Immunoprecipitation (Co-IP);④The effects of AG490combination with Hesl siRNA on SGC7901cells were detected by morphological observation and flow cytometry.4. The functional role of silence of JAK2by shRNA in SGC7901cells①SGC7901cells were transfected with JAK2-specific shRNA vectors (JAK2shRNA), and the expression of JAK2in stably transfected cells was assessed by quantitative RT-PCR and Western blot;②The effects of JAK2silencing on cell proliferation were observed by cell colony formation assay and MTS assay;③The effects of JAK2silencing on cell apoptosis and cell cycle were detected by flow cytometry;④Xenografted gastric tumor model was used to validate the tumor suppressing effect by JAK2silencing in vivo.5. The effects of combination of JAK2shRNA and ERK inhibitor on SGC7901cells in vitro and in vivo①The effects of JAK2silencing on expression and activation of ERK1/2were measured by Western blotting;②The effects of combination of JAK2shRNA and ERK inhibitor on cell proliferation, apoptosis and cell cycle distribution in SGC7901cells were observed;③Xenografted gastric tumor model was used to validate the tumor suppressing effect by combination of JAK2shRNA and ERK inhibitor in vivo. Results and conclusions1. The activation of JAK2may manifest as tumor promotion in GC①The expression of JAK2was significantly upregulated in66.67%(50/75) GC tissues. Moreover, JAK2was mainly expressed in the cell cytoplasm of75specimens, with a positive expression of22specimens in the nucleus;②The expression of JAK2was not corelated with pathological characteristics of GC patients as gender, age, diameter of tumor, depth of tumor invasion, lymph node metastasis and UICC staging (p>0.05);③JAK2and pJAK2was predominantly expressed in whole-cell protein extracts of four GC cell lines MKN28, SGC7901, AGS and MKN45, among which SGC7901expressed highest level of them. JAK2phosphorylation didn’t exist in whole-cell protein extracts of GES-1.2. The antitumor effects of AG490were cell type-dependent①AG490caused a significant reduction in cell viability dose-and time-dependently in AGS cells (p<0.05), but it only caused a significant reduction in cell viability dose-dependently in SGC7901cells (p<0.05);②AG490caused significant cell apoptosis in AGS cells (p<0.05), but not in SGC7901cells (p>0.05);③AG490didn’t suppress the xenografted SGC7901tumor growth in vivo (p>0.05).3. The nuclear translocation of JAK2may contribute to AG490-induced non-apoptotic response in SGC7901cells①JAK2and pJAK2was predominantly expressed at different levels in nuclear protein extracts of four GC cell lines MKN28, SGC7901, AGS and MKN45, while JAK2phosphorylation didn’t exist in nuclear protein extracts of GES-1;②Nuclear translocation of JAK2in AGS cells was decreased by AG490, but nuclear localization of JAK2in SGC7901cells was significantly enhanced by AG490;③AG490enhanced JAK2-STAT3-Hesl complex formation in nucleus of SGC7901 cells, which was inhibited by Hesl siRNA transfection;④Hesl siRNA enhanced apoptosis induced by AG490in SGC7901cells (p<0.05).4. Silence of JAK2by shRNA in SGC7901cells inhibited cell proliferation, but induced no cell apoptosis①SGC7901cells were stably transfected with JAK2-specific shRNA vectors, and the expression of JAK2was significantly downregulated (p<0.05);②Inhibition of cell proliferation in JAK2shRNA-transfected SGC7901cells might not be attributed to induction of apoptosis, but to G2/M cell cycle arrest;③JAK2silencing didn’t suppress the xenografted SGC7901tumor growth in vivo (p>0.05).5. ERK1/2inhibition enhances antitumor effects induced by JAK2silencing in human GC cells①JAK2silencing induced activation of ERK1/2;②Combination of JAK2shRNA and ERK1/2inhibitor PD98059resulted in suppression of tumor growth in vitro and in vivo by enhancing apoptosis and cell cycle arrest (p<0.05).