Loss Of Smad4in Colorectal Cancer Promotes Tumor Malignancy And Induces Resistance To5-fluorouracil Through Activating Akt Pathway

Objectives:To study the effect and biological mechanism of Smad4on cell proliferation, migration and invasion in colorectal cancer in vitro and in vivo.Methods:We first knocked down Smad4from CT26cells and characterized the tumorigenic properties. Stable knockdown of Smad4in clones was tested by western blotting and downstream reporter luciferase activity. The Smad4stably expressing clones from SW620cells were established previously. Cell proliferation was tested by cell counting, MTT cell proliferation, and Thymidine incorporation assays. We next examined the effects of the loss of Smad4expression on tumorigenicity of these cells using anchorage-independent growth assay. To determine the aggressiveness of the Smad4knockdown cells, we performed wound healing, transwell cell migration and invasion assay. An orthotopic cecal injection model was established to evaluate the effect of Smad4deficiency on CRC tumorigenicity and liver metastasis. In addition, the subcutaneous injection and splenic injection models were performed to evaluate tumorigenicity and liver metastasis, respectively. Immunohistochemistry staining of Ki67was used to distinguish the tumors and evaluate cell proliferation. CT26and SW620cell clones or tumor tissues were examined by western blotting analysis to evaluate the non-Smad pathways, cell cycle relatied proteins, cell proliferation and apoptosis related proteins,. Rsults:Smad4expression is sharply lower in the Smad4knockdown clones compared with CT26parental cells and vector control clones. TGF-β induced p3TP-Lux and (GAGA)9MLP-Luc reporter activities were observed in vector control cells, but not in Smad4knockdown clone. Smad4deficiency promoted cell proliferation, tumorigenicity, migration and invasion in CT26cells. We observed significantly increased primary cecum tumor growth by Smad4deficient clones, in which9out of15mice generated visible liver metastasis and2mice presented splenic metastasis. On the contrary, only one out of10mice injected with control cells presented portal vein metastasis, while no splenic or liver metastasis was observed. We also observed that Smad4knockdown clones showed enhanced tumor growth when compared with the vector control clones in the subcutaneous injection model. Consistent with the data from orthotopic model, we also observed significantly increased liver metastasis in mice injected with Smad4knockdown clones compared to control cells using splenic injection model. The Akt and p38-MAPK phosphorylation was activated when Smad4was deficient in CT26cells, while downregulated in SW620cells when Smad4was overexpressed. In contrast, we did not observe any significant change in the activation of ERK pathway by the presence or absence of Smad4in these two cell lines. In both cell lines, Smad4downregulated c-Myc, Cyclin Dl, while upregulated p21Cipl and p27Kipl. We observed downregulation of CDK2by Smad4in SW620cells, but not in CT26cells. On the other hand, the anti-apoptotic Bcl-2and Bcl-w were down-regulated by Smad4, while the pro-apoptotic Bim and Bad were up-regulated by Smad4in CT26cells. In cecum tumors and in tumor allografts from Smad4deficient clones, Smad4was maintained at lower level, when compared with that in tumors from control cells. p21Cipl and p27Kipl were downregulated, while c-Myc and Cyclin Dl were upregulated in the tumors from Smad4deficient clones.Conclusion:These data suggest that loss of Smad4in CT26cells induces proliferation, migration, invasion and tumorigenicity, and provide a mechanism by which Smad4inhibits cell proliferation through cell cycle arrest and promotes apoptosis by regulating anti-and pro-apoptotic proteins, and activation of Akt may play a role in these processes. Objectives:To study the effect of Smad4on5-FU chemosensitivity and explore the mechamism how Smad4regulate chemosensitivity in colorectal cancer in vitro and in vivo.Methods:We treated Smad4knockdown CT26cells and SW620cells stably expressing Smad4with indicated concentration of5-FU for72h, and MTT assay was performed to evaluate the cell viability. IC50value was calculated and used to evaluate the drug sensitivity. Anchor independent tumorigenicity assay was performed to evaluate sensitivity to5-FU after Smad4knowdown or overexpression in both cells line. We next tested the effect of Smad4on5-FU induced apoptosis in CT26and SW620clones by flow cytometry analysis. A subcutaneous injection model of both cell lines with5-FU treatment was established, and the tumor growth curve and over all survival was analyzed to evaluate the5-FU sensitivity in vivo. We next examine the subcutaneous tumours by immunohistochemistrical staining with Ki67and Cleaved-Caspase3, to test cell proliferation and apoptosis. Akt pathway and its downstream related proteins were examined by western blotting analysis to explore some latent mechanism for Smad4regulating drug sensitivity.Results:CT26parental cells, the vector clone, or SW620clones stably expressing Smad4showed significantly higher sensitivity to5-FU, when compared with that of Smad4deficient CT26clones or SW620control cells. The sensitivity of CT26cells and vector clones to5-FU was more than double compared to Smad4deficient CT26clones based on IC50value. In contrast, SW620clones expressing Smad4were3fold more sensitive to5-FU treatment compared to parental and vector control cells. More evidence of5-FU resistance in Smad4deficient cells was obtained in in vitro tumorigenicity assay in both cells line. Treatment with5-FU decreased the number of colonies by85%in CT26vector control clone, while by only18%in Smad4deficient clone. In contrary,5-FU treatment decreased the number of colonies by46%in SW620vector control clone, while by89%in Smad4stable expressing clone. We next tested the effect of Smad4on5-FU induced apoptosis in CT26and SW620clones. With5-FU treatment, the percentage of apoptotic cells in CT26vector control category was increased by15.3±3.8%, while in Smad4deficient clone by6.4±1.6%. This effect is more obvious in SW620cells, in which5-FU increased apoptosis by26.3±3.4%in vector control clone, while by47.3±6.2%in Smad4stable expressing clones. Smad4expressing CT26tumors and SW620tumors stably expressing Smad4were sensitive to5-FU treatment in the model model. Allografts from Smad4knockdown CT26clone or xenografts from SW620vector clone not expressing Smad4showed resistance to5-FU treatment. We observed similar effects of5-FU on tumor from other clones. Mice bearing tumors from CT26or SW620cell clones with less or no expression of Smad4showed shorter survival, when compared with those from Smad4expressing or overexpressing clones. In addition,5-FU significantly improved overall survival of mice bearing CT26vector clone or SW620clone transfected with Smad4, while it had almost no effect on those mice bearing Smad4deficient tumors. Immunostaining showed Smad4suppressed Ki67but increased cleaved-Caspase3expression in CT26vector control tumors, and these differences were amplified by5-FU treatment.5-FU treatment did not have much effect on tumors from Smad4knockdown clone. The cleaved-PARP and cleaved-Caspase3were sharply increased in the CT26vector control tumors after5-FU treatment, but there were low or no levels of these in Smad4deficient tumors even after5-FU treatment. The activation of Akt was marginally reduced in tumors from Smad4knockdown clone by5-FU treatment and upregulation of Bcl-w in CT26deficient clone was maintained in vivo, which is consistent with the in vitro study. Interestingly, while the expression of Survivin in control tumors was decreased by5-FU, its expression was increased in knockdown tumors by5-FU. Conclusion:These results suggest that loss of Smad4desensitizes CRC cells to5-FU through activation of Akt pathway and regulation of downstream apoptotic proteins. These observations suggest a mechanism by which loss of Smad4results in drug resistance of CRC cells, and poor survival of mice bearing CRC tumors. Objectives:To study the exact molecular mechanism of Smad4deficiency induced5-FU chemoresistance in colorectal cancer.Methods:Cell viability assay was performed after treating CT26and SW620cell clones with5-FU and/or the PI3K inhibitor, LY294002, to evaluate the effect of Akt pathway in5-FU chemoresistance induced by Smad4deficiency. We next used flowcytometry analysis of cell apoptosis to examine the effect of Akt activation induced by Smad4deficiency in5-FU induced apoptosis. Western blotting analysis was applied to examine the Akt/Bcl-2and Akt/Survivin pathway, and Cleavage of Caspase3and PARP. We examined the tumor samples after5-FU treatment macroscopically and microscopically, and CD31immunostaining was performed to analysis the vessel density and numbers in the tumor. VEFG expression in the cells or tumours from animal models with or without5-FU treatment was examined by Western blotting analysis. We finally tested the Smad4and Akt activation in8paired normal and colorectal caner specimens from the patients. Tissue microarray was performed to examine the correlation between Smad4and Bcl-2/Bcl-w, and to analyze survival. Results:The Smad4inactivated clones, which were less sensitive to5-FU treatment alone, showed significantly higher sensitivity to5-FU in presence of LY294002. On the contrary, the sensitivity to5-FU was marginally changed after LY294002treatment in CT26vector control and SW620Smad4expressing cells. LY294002alone had marginal effect on cell apoptosis in both CT26and SW620cells. However, it significantly amplified the5-FU-induced apoptosis in Smad4deficient cells compared to that in Smad4expressing cells. At protein level, LY294002inhibited Akt activation in Smad4deficient cells to the similar level as that in Smad4expressing cells. The expression of Bcl-w in both cell lines and expression of Bcl-2in SW620cells positively correlated with the activation of Akt depending on the Smad4status. The expression of Bim was mostly restored in Smad4knockdown cells by LY294002. In addition, Survivin expression was downregulated by LY294002in Smad4deficient cells, though its expression was increased by5-FU alone. There was not much change in the cleavage of Caspase-3or PARP in Smad4deficient cells by5-FU. However, treatment of these cells by LY294002and5-FU synergistically induced cleaved-Caspase3and cleaved-PARP. We observed that VEGF expression in CT26cells and tumors was upregulated when Smad4was knocked down compared to vector control clone. On the other hand, Smad4downregulated VEGF expression in SW620cells when Smad4was overexpressed. Tumor masses from Smad4deficient clones presented significantly more visible vessels, compared with tumors from Smad4expressing cells. CD31immunostaining showed significantly higher in number and density of vessels in the tumors generated by CT26Smad4deficient clones when compared with those from vector control clone, either in orthotopic or heterotopic inoculation model. In human colorectal tumors, Smad4expression was significantly decreased in5out of8patients and was slightly downregulated in two other patients compared to corresponding normal colon tissue. Phosphorylation of Akt was upregulated in those5tumor specimens with significantly lower Smad4expression, whereas Survivin expression was increased in all tumor specimens. Bcl-w expression was inversely correlated with Smad4nuclear staining significantly. Bcl-2expression was more than2times higher in Smad4negative CRC specimen compared to Smad4positive group. Patients with positive Smad4nuclear staining showed significantly longer overall survival, when compared with Smad4negative group.Conclusions:These results suggest that LY294002with5-FU can sensitize the Smad4deficient cells up to the same extent as5-FU alone does to the Smad4expressing cells, through the inhibition of PI3K/Akt pathway. These findings suggest that loss of Smad4in CRC cells induces angiogenesis through upregulation of VEGF and may promote5-FU resistance.