Role Of TrpC5 In Chemoresistance Of Colorectal Cancer

In recent decades,the incidence and mortality of colorectal cancer(CRC)have been declining due to the decreased smoking rates and the increased use of aspirin and the introduction and dissemination of screening tests.However,CRC is one of the most common forms of malignancy.To date,chemotherapy is the most effective way to treat colorectal cancer.But the long-term survival after treatment is still unsatisfactory.The most important reason is patients resistant to chemotherapy.Although research on resistance has been for several decades,there is no clear mechanism to clarify the resistance to chemotherapy.Thus,exploring molecular mechanism underlying CRC may provide a new direction to CRC treatment.Moreover,it can be used as a target for the development of new types of agents,and design the enteric drug for targeted drug delivery,in order to provide a new drug for better clinical treatment of CRC.In this study,we found a new molecular mechanism and signal pathway that affecting the resistance to CRC.Through a large number of patients with CRC,we found TrpC5,a member of Trp family,was aberrant overexpression in the specimens.Based on these findings,we explored the mechanism of TrpC5 in regulating the development of CRC.The main findings were described as follows:RT-PCR showed that the expression of TrpC5 was significantly higher in cancer samples than in matched adjacent tissue,while TrpC1,TrpC3,TrpC4 and TrpC6 didn't change.Then we used western bolt to further confirm the expression of TrpC5 in carcinoma and matched adjacent tissue.The results showed that the expression of TrpC5 in carcinoma was higher than in adjacent tissue.These results suggested that TrpC5 plays a role in the development of colorectal cancer.To further explore the clinical relevance of TrpC5 in CRC,immunohistochemistry was used to analysis the relationship between TrpC5 expression and the clinical data.The results showed that the expression of TrpC5 was positively correlated with tumor grading,but had no difference with other clinical factors.While in primary cultures of human CRC cells,we found the expression and the function of TrpC5 were significantly elevated in poorly differentiated CRC cells.Then we investigate the correlation between TrpC5 and differentiation in CRC cell lines.RT-PCR and immunostaining results showed the expression of TrpC5 was negatively with the degree of differentiation in 8 CRC cell lines.Inhibition of TrpC5 reduced the Alp1 and Klf4 expression in m RNA level.These results indicated that the expression of TrpC5 is correlated with differentiation in CRC.Combined with cell lines and clinical samples,we investigate the molecular mechanism of TrpC5 regulating differentiation.RT-PCR showed that in all Wnt ligands,only Wnt5 a expression was correlated with TrpC5 expression and the level of cell differentiation.Furthermore,we found TrpC5 knockdown in poorly differentiated cells decreased Wnt 5a expression and the Wnt5 a secretion and the nuclear translocation of ?-catenin,as well as Cyclin D1 and c-myc expression.In contrast,these measures clearly increased when TrpC5 was overexpressed in well differentiated cells.In clinical samples,the lower degree of tumor differentiation,the higher of TrpC5 and Wnt5 a expression in carcinoma,as well as higherconcentrations of Wnt5 a in serum.These results suggested that TrpC5 mediates Wnt5 a expression through Wnt5a/?-catenin signal pathway to regulate differentiation.On the basis of TrpC5 is related to CRC cell differentiation,we further deeply analysis whether TrpC5 will affect cancer cell stemness or drug treatment sensitivity.Immunostaining and FACS showed that inhibited TrpC5 can decrease CD44 and CD133 expression in poorly differentiated cell lines.Moreover,downregulate TrpC5 expression significantly reduced sphere formation and tumor formation in vitro and in vivo.While in terms of resistance,Real-time PCR and western blot showed that inhibited TrpC5 significantly decreased P-gp expression in transcriptional and translational level.Moreover,TrpC5 knockdown in poorly differentiated cells made cells less resistant to 5-Fu.In contrast,overexpression TrpC5 increased P-gp expression and rendered cells less sensitive to 5-Fu.Taken together,these results revealed that TrpC5 regulates differentiation and enhances stemness and drug resistance in CRC cells.To assess the clinical significance of TrpC5 expression in CRC,the survival follow-up information on patients was analyzed.We found patients with tumors that expressed higher levels of TrpC5 showed significantly poorly disease-free and overall survival.More importantly,among the 279 patients who had received 5-Fu-based chemotherapy,patients with tumors that expressed high TrpC5 levels showed less disease-free(DFS)and overall survival(OS)than those with tumors with low TrpC5 expression.In contrast,the remaining67 patients who did not receive 5-Fu-based chemotherapy,showed no difference in DFS and OS regardless of the TrpC5 levels in the tumors.These results provided us initial evidence that TrpC5 is an independent adverse prognostic factor for death in CRC and is a potentially crucial role of TrpC5 in clinical resistance to 5-Fu-based chemotherapy for CRC.