Role And Regulation Of Hedgehog Signaling On Chemo-resistance In Pancreatic Cancer

Background：Gemcitabine chemotherapy is still the mostly used method in comprehensivetreatment on pancreatic cancer. However, gemcitabine doesn’t improve the treatmentof patients significantly while brings chemotherapy side effects to patients withpancreatic cancer. The main reason of that is the natural and (or) acquiredgemcitabine tolerance in pancreatic cancer. Hedgehog (HH) pathway is considered tobe the "core" signaling in pancreatic cancer. The inhibitor of HH signaling canincrease the gemcitabine sensitivity in pancreatic cancer and suppress the naturalchemo-resistance of pancreatic cancer. But there is no report on the role of HH signalin acquired drug resistance of pancreatic cancer. SMO is a signal transductioncomponent in HH pathway. Results of a Phase I trial showed that SMO inhibitor hadgood effects in58%patients with advanced basal cell carcinoma, but had nosignificant effect in9patients with pancreatic cancer included in the study, suggestingthat the mechanism of HH signaling in pancreatic cancer is more complex. It is veryimportant to explore new regulation mechanism and new targets.Objective:1、To study the expression of HH signaling components in human pancreaticgemcitabine-resistant cell line, and to explore whether the acquiredgemcitabine-resistance in pancreatic cancer cells could be reversed by inhibition ofHH signaling. 2、To study MAP3K10expression status in pancreatic cancer tissues, and to explorethe effect of MAP3K10on gemcitabine sensitivity in pancreatic cancer cell and itsregulation on HH signaling.3、To study DYRK2expression status in pancreatic cancer tissues, and to explore theeffect of MAP3K10on gemcitabine sensitivity in pancreatic cancer cell and itsregulation on HH pathway.Methods:1、To establish a human pancreatic cancer gemcitabine-resistant cell line SW1990/GR,quantitative PCR and Western blot were used to test the expression of HH pathwaycomponents SMO and Gli-1in SW1990/GR cells; To treat SW1990/GR cells withcyclopamine (inhibitor of HH pathway), quantitative PCR and Western blot wereused to analyze the effects of cyclopamine on the expression of SMO and Gli-1;Combined treatment of cyclopamine and gemcitabine on SW1990/GR, Annexin Vand PI double staining assay was performed to detect cell apoptosis.2、Quantitative PCR、Western-blot and immunohistochemistry were performed todetect MAP3K10expression status in26pairs of pancreatic cancer tissues、adjacenttissues and normal pancreatic tissues. MAP3K10over-expression/RNA interferenceplasmids vectors were constructed and transduced into pancreatic cancer cells. MTTand EDU assay were performed to detect cell proliferation before and aftertransfection; Flow cytometry was used to test cell cycle before and after transfection;Transwell assay was performed to measurement cell invasion ability before and aftertransfection; Flow cytometry apoptosis assay was used to analyze gemcitabinesensitivity in pancreatic cancer cells before and after transfected cells; QuantitativePCR and Western-blot were performed to detect the expression of HH pathwaycomponents (Gli-1/2)、DYRK2and GSK3β in pancreatic cancer cells before and aftertransfection. 3、Quantitative PCR、Western-blot and immunohistochemistry were performed todetect DYRK2expression status in26pairs of pancreatic cancer tissues、adjacenttissues and normal pancreatic tissues. DYRK2over-expression/RNA interferenceplasmids vectors were constructed and transduced into pancreatic cancer cells. MTTand colony formation assay were performed to detect cell proliferation before andafter transfection; Flow cytometry was used to test cell cycle before and aftertransfection; Flow cytometry apoptosis assay was used to analyze gemcitabinesensitivity in pancreatic cancer cells before and after transfected cells; QuantitativePCR and Western-blot were performed to detect the expression of Gli-2, c-Jun, c-Mycand Cyclin-E in pancreatic cancer cells before and after transfection.Results:1、Compared with human pancreatic cancer cell line SW1990, gemcitabine resistantcell line SW1990/GR showed a high expression of SMO and Gli-1; Cyclopaminereduced SMO and Gli-1expression levels in SW1990/GR, increased cell apoptosisinduced by gemcitabine.2、Of the26paired samples,21showed significantly higher levels of MAP3K10inthe cancer tissues than in the adjacent tissues. Compared with the control group,MAP3K10over-expression improved cell proliferation、elevated the percentage ofcells in the S+G2phase and decreased gemcitabine sensitivity, while showed nosignificant affects in cell invasion. Results of quantitative PCR and Western-blotshowed a reduction of mRNA and protein levels of DYRK2and GSK3β byMAP3K10overexpression, and an elevation of mRNA and protein levels of Gli-1andGli-2. MAP3K10knock-down upregulated the mRNA and protein levels of DYRK2and GSK3β, and downregulated mRNA and protein levels of Gli-1and Gli-2.3、Of the26paired samples,22showed significantly higher levels of DYRK2in thecancer tissues than in the adjacent tissues. Compared with the control group, DYRK2 over-expression suppressed cell proliferation、decreased the percentage of cells in theS+G2phase and elevated gemcitabine sensitivity. Results of quantitative PCR andWestern-blot showed a reduction of mRNA and protein levels of Gli-2by DYRK2overexpression, and an elevation of mRNA and protein levels of Gli-2by DYRK2knock-down. DYRK2overexpression had no effect on the mRNA level of c-Jun,c-Myc and Cyclin-E. DYRK2knock-down had no effect on the mRNA level of c-Junand c-Myc, but elevated the mRNA level of the cyclin-E; DYRK2overexpression hadno significant effect on the protein level of c-Jun, c-Myc and Cyclin-E, while DYRK2knock-down elevated protein levels of c-Jun, c-Myc and Cyclin-E.Conclusion:1、Inhibition of HH pathway can reverse the acquired gemcitabine resistance ofpancreatic cancer cells.2、MAP3K10promoted the proliferation, as well as decreased the gemcitabinesensitivity, of pancreatic cancer cells. A possible mechanism of these effects may bethrough the up-regulation of HH signaling.3、DYRK2inhibited the proliferation as well as improved the gemcitabine sensitivityof pancreatic cancer cells. A possible mechanism of these effects may be through theregulation of Gli-2, c-Jun, c-Myc and Cyclin-E.