Study Of The Role Of CaMKⅡ In TGF-β1 Induced Fibroblasts Activation And Its Crosstalk With Colon Cancer Cells

Background:Tumor microenvironment(TME)which constitutes of different kinds of cells involving fibroblasts,endothelial cells,immune cells,and complex ex-tracellular matrix(ECM)plays a pivotal role in the cancer sustained growth,metas-tasis and invasion.TME can directly influence cancer cell proliferation,migration and differentiation.Cancer associated fibroblasts(CAFs),as the most important functional cells in TME,can directly or indirectly regulate cancer cell proliferation,migration and inva-sion by secreting a mass of growth factors and ECM through cell-cell contaction.The present studies suggest that CAFs derived from bone marrow mesenchyme stem cells,outer membrane cells(including outer membrane of cells and vascular smooth muscle cells),endothelial cells and resident fibroblasts,but the main source of CAFs are resi-dent fibroblasts.Research shows that transforming growth factor-β1(TGF-β1)is the most important modulator in fibroblasts activation and transformation to CAFs.Resi-dent fibroblasts can be activated to CAFs by the TGF-β1 stimulation,and then promote cancer growth and progression.Preliminary study shows that Ca2+ which is the downstream target of TGF-β1 take part in this process.As the most vital modulator protein of Ca2+ signal,Ca2+/Calmodulin dependent protein kinase(CaMKⅡ)plays a role in a variety of physiological processes such as cycle regulation,apoptosis,gene expression,and cell secretion.Although studies have confirmed that CaMK Ⅱ plays an important role in tumor development,migration and invasion,but the effect and mechanism of CaMK Ⅱ in TGFβ1-induced fibroblasts activation and its crosstalk with colon cancer cells are not clear.So we plan to investigate the role of CaMK Ⅱ in above process and search for new targets and strategies for colon cancer diagnosis and treatment.Part 1:Research on the expression of CaMKⅡ in normal human colon mucosa,paracancerous tissue and colon cancer samplesObjective:Our research aim to characterize and investigate the expression of CaMKⅡ in normal human colon mucosa,paracancerous tissue and colon cancer samples.Methods:48 formalin-fixed,paraffin-embedded samples including normal human colon mucosa,paracancerous tissue and colon cancer were obtained.Each group contains16 cases.Immunohistochemistry staining and double immunofluorescence staining were used to detect the expression of CaMK Ⅱ.Results:CaaMK Ⅱ significantly overexpressed in the stroma of human colon cancer compared to paracancerous tissues and normal colon mucosa(P<0.05).a-SMA magnificently overexpressed in colon cancer stroma while less expression and very weak signal were observed in colon paracancerous samples and normal colon mucosa.Conclusion:The expression of CaMK Ⅱ in different colon tissue is various,and overexpressed CaMK Ⅱ mainly derives from CAFs.CaMK Ⅱ may be a potential modulator in colon fibroblasts activation and CAFs-like differentiation.Part 2:Research on the effect and mechanism of CaMKⅡ in TGFβ1-induced fibroblasts activationObjective:To study the effect and mechanism of CaMK Ⅱ in TGFβ1-induced fibroblasts activation.Methods:Specific a-CaMK Ⅱ shRNAs(CaMK Ⅱ shRNA)and one nonspecific scrambled control shRNAs(control shRNA)lentiviral vectors were constructed and used to infect human colon cell line CCD-18Co.CCD-18Co cells were treated with TGF-β1(5ng/mL)and relevant signal pathway inhibiters(SIS3、PD98059、SP600125、SB203580.LY294002),then the protein expression and mRNA levels of CaMKⅡα,a-SMA,fibroblast activation protein(FAP),fibronectin(FN),Smad3,p-AKT/AKT,P-ERK1/2/ERK1/2,p-JNK/JNK,p-p38/p38 were detected by western blot and Real-time quantitative PCR(qRT-PCR).Cell proliferation was measured by a CCK-8(Cell Counting Kit 8)assay.Transwell assay and Wound-healing assay were used to examine migratory and invasive ability in vitro.The concentration of platelet-derived growth factor(PDGF),epidermal growth factor(EGF)and fibroblast growth factors(FGF)in supernatants were measured by enzyme-linked immunosorbent assay(ELISA).Results:The CaMKⅡα expression of CCD-18Co in CaMK II a-shRNA group was significantly knockdown compare with control group(P<0.05).We found in human colon fibroblasts CCD-18Co,TGFβ1 dramatically induced CaMK II,a-SMA,FAP,FN expression and PDGF,EGF,FGF secretion and cell proliferation,migration(P<0.05).CaMKII knockdown significantly attenuated TGFβ1-induced alteration in CCD-18Co cells(P<0.05).The expression of Smad3,p-AKT/AKT,p-ERK1/2/ERK1/2,p-p38/p38 in CCD-18Co cells were significantly decreased after TGFp1 treatment(P<0.05),but p-JNK/JNK expression were not altered(P>0.05).CaMK Ⅱ knockdown also significantly attenuated these alteration.The inhibiters(except SP600125)SIS3,PD98059,SB203580,LY294002 weakened TGβ1-induced cell proliferation anda-SMA,FAP,FN,PDGF,EGF,FGF expression(P<0.05).Conclusion:CaMK Ⅱ is a pivotal regulator for TGFβ1-induced fibroblasts activation and CAFs associated transformation.Smad3,AKT and MAPK pathways may were involved in TGFβ1-CaMK Ⅱ-mediated fibroblasts activation and CAFs-like differentiation.Part 3:Research on the effect and mechanism of CaMKⅡ in cross talk between fibroblasts and colon cancer cellsObjective:To investigate the role and mechanism of CaMKⅡ in crosstalk between fibroblasts and colon cancer cells.Methods:We construct a coculture system between human colon fibroblasts CCD-18Co and human colon cancer cells HCT116 by transwell in vitro.After these two kinds of cells were co-cultured 72 hours,the protein expression and mRNA levels of CaMK Ⅱα,a-SMA,FAP,FN in CCD-18Co cells and matrix metalloproteinases(MMP2,MMP9),tissue inhibitor matrix metalloproteinase-1(TIMP-1)in HCT116 cells were measured by western blot and qRT-PCR.MTT assay was applied to detect the proliferation of CCD-18Co cells and HCT116 cells in coculture system.Transwell was used to measure migratory and invasive ability of CCD-18Co cells and HCT116 cells in vitro after co-cultured 24 hours.Results:The protein expression of CaMKⅡα,a-SMA,FAP,FN in CCD-18Co cells were significantly increased when co-cultured with HCT116 cells.The migratory and proliferation ability of CCD-18Co cells were also enhanced.In comparison with control fibroblasts(P<0.05),little alteration was observed in CaMKⅡ knockdown fibroblasts in coculture system.Proliferation,migration and invasion of HCT 116 cells was significantly increased(P<0.05)when co-cultured with CCD-18Co cells whereas little alteration was detected in those co-cultured with CaMK Ⅱ knockdown fibroblasts.Compared with fibroblasts,CaMKⅡ knockdown fibroblasts dramatically decreased MMP2 and MMP9 expression and increased TIMP-1 levels in HCT 116 cancer cells.Conclusion:Colon cancer cells can induce colon fibroblasts activation and CAFs-like transformation,in the other hand colon fibroblasts can promote the proliferation,migration and invasion of colon cancer cells.This process rely on the modulation of CaMKⅡ.