Par3 Promotes Tumorigenesis And Metastasis In Colorectal Cancer/AhR Activation Prevents Intestinal Barrier Dysfunction Through Regulation Of Claudin-2 Expression

Par3 promotes tumorigenesis and metastasis in colorectal cancerBackgroundColorectal cancer(CRC)is the second most common type of diagnosed cancer,with approximately 1.36 million new cases and close to 694 000 deaths per year.Almost 40% of the patients who present with colorectal cancer will develop metastasis during the course of the disease.Although CRC is highly treatable if diagnosed and surgically removed at an early stage,5-year survival is <10% in patients with unresectable metastatic disease.The liver,peritoneum,and lungs are the most frequent sites of metastases in CRC patients.Despite advances in early diagnosis and multidisciplinary therapeutic approaches,including surgery and chemotherapy,the clinical outcome and prognosis of CRC patients remains poor.Thus,it is necessary to clarify the underlying mechanisms of colorectal cancer metastasis in order to develop new,effective treatments.Cell polarity,which is essential for epithelial cells to exert their physiological functions and for the development of various organisms,is involved in the processes of morphogenesis,proliferation and differentiation.In mammals,dysfunctional epithelial cell polarity leads to carcinoma invasion and metastasis.The partitioning defective(Par)complex,which is localized at apical regions to specify the apical membrane,is necessary for the establishment and maintenance of epithelial cell polarization.The Par complex is composed of three proteins: Par3,Par6,and atypical protein kinase C(aPKC),which plays important roles in the regulation of many cell processes,such as apical-basal polarity,proliferation,and migration.We have focused on Par3,a multi-domain scaffolding protein that contains three PSD-95/Discslarge/ZO-1(PDZ)domains,an N-terminal dimerization domain and a C-terminal interaction domain.The PDZ domains bind with cell surface proteins such as Par6,junctional adhesion molecules and the phosphatase and tensin homologue(PTEN).The N-terminal domain of Par3 is very important for the regulation of cellular polarity.The C-terminal domain binds with aPKC to suppress its kinase activity and with Tiam1(T lymphoma invasion and metastasis)to restrain its exchange activity.Many reports have shown that overexpression or depletion of PAR3 in epithelial cells leads to mislocalization of PAR6,aPKC and tight junction markers.Recent evidence demonstrated an important role of Par3 in tumor progression and metastasis.In breast cancer,loss of Par3 facilitated cancer cell invasion and tumor metastasis by inhibiting E-cadherin junction stability and compromising cell-cell cohesion.Additionally,downregulation of Par3 could significantly promote lung adenocarcinoma cell proliferation,tumor formation,and metastasis through 14-3-3ζ protein.By contrast,elevated expression of Par3 promoted prostate cancer metastasis and invasion via inactivation of the hippo pathway.In human hepatocellular carcinoma,increased Par3 expression was significantly associated with cancer metastasis and poor survival rates.Thus,Par3 may function as a tumor suppressor or protumorigenic protein in different types of cancer.However,the function of Par3 in colorectal cancer metastasis has not been investigated.In this study,we aimed to determine the possible roles of Par3 in colorectal cancer metastasis and to investigate the Par3-related pathways that might be relevant to the clinical outcome.MethodsFirstly,Colorectal tumor and normal tissue samples from 51 patients were acquired from the operating room following the routine pathological examine.The expressions of Par3 were detected by Western-blot,RT-qPCR and immunohistochemistry method.Secondly,Knockdown of Par3 by shRNA in HCT116 Cells,Western-blot and RT-qPCR analysis were used to detect the expression of Par3,E-cadherin.Colony formation assay was performed to explore the effect of Par3 on thegrowth of CRC cells.Cell migration and invasion assays were performed to examine the roles of Par3 in the metastasis in CRC cellsThirdly,to identify the mechanism by which Par3 controls CRC cell growth and metastasis,we examined potential downstream signaling pathways.Western blot was performed to determine the effect of Par3 knockdown on STAT3,ERK1/2 and JNK.Results1.The expression of Par3 was increased in CRC tumors compared with their corresponding adjacent normal tissues.Elevated expression of Par3 was significantly associated with an older age(≥50 years)and poor differentiation.2.The specific shRNA targeting Par3 mRNA is able to effectively knockdown Par3 expression at both mRNA and protein levels in CRC cells.Par3 knockdown facilitated the expression of E-cadherin in CRC cells3.Par3 contributes to the tumorigenic potential and promotes cell proliferation,invasion and migration in HCT116 cells.4.STAT3 signaling pathway may involved in the mechanism of Par3 regulating CRC cell growth and metastasis.ConclusionsIn this study we found that Par3 expression was increased in CRC tumors,and this elevated expression of Par3 was associated with an older age(≥50 years)and poor differentiation.Loss of Par3 in CRC cells restrained colony formation and decreased the metastatic and invasive ability of colorectal cancer cells in vitro.Par3 regulating CRC cell growth and metastasis though STAT3 signaling pathway.Aryl hydrocarbon receptor activation prevents intestinal barrier dysfunction through regulation of claudin-2 expressionThe intestinal epithelial barrier(IEB)plays a key role in resisting toxins and pathogenic organisms.Tight junctions(TJs)are the most apical organelle of the junctional complexes and work as the major determinants of intestinal barrier function.The damage of IEB function caused by various factors is accompanied by the abnormal expression and integrity of TJ protein.Inflammatory bowel disease(IBD)is a chronic non-specific inflammatory condition of the gastrointestinal tract,including Crohn’s disease and ulcerative colitis.A large number of studies have shown that TJ dysfunction plays an indispensable role in the pathological process of IBD.The pore-forming protein claudin-2 is a member of the TJ claudins family,and its increased expression significantly impairs the intestinal TJ barrier function.The expression of claudin-2 is Upregulated in the colons of IBD patients.Interleukin-6(IL-6)is a pleiotropic cytokine with a variety of pro-inflammatory effects.IL-6 plays an important role in the pathogenesis of IBD.Recent evidence suggests that IL-6 regulates intestinal epithelial barrier function by increasing the expression of claudin-2.The aryl hydrocarbon receptor(Ah R)is a ligand-dependent transcription factor.Ah R enters the nucleus of the nucleus in a ligand-dependent manner and then forms a heterodimer with the Ah R nuclear translocator.6-Formylindolo(3,2-b)carbazole(FICZ),a tryptophan photoproduct,is one of the most potent natural activators of Ah R.Studies have shown that FICZ-induced aryl hydrocarbon receptor activation can inhibit gastrointestinal inflammation.Therefore,this study will investigate the role and regulatory mechanism of FICZ induced Ah R activation on IL-6 induced intestinal barrier dysfunction,utilizing RNA interference,western blot,immunohistochemistry,laser confocal microscopy technique and so on.Methods1.A DSS-induced mouse colitis model was established and FICZ(1 μg/mouse)was used by intraperitoneal injection.The expression of IL-6 was detected by RT-q PCR,and detect the expression of Claudin-2 by immunohistochemistry.The Ussing Chamber was used to detect colonic transepithelial electrical resistance.2.Two well-established in vitro intestinal epithelial model systems were established.After Ah R si RNA or FICZ treatment,RT-q PCR,Western blot,and immunofluorescence techniques were used to detect the expression of Claudin-2 and CYP1A1.3.The intestinal epithelial cell line was treated with HNF-1α si RNA or CDX-2 si RNA,with or without FICZ,the expression of HNF-1α,CDX-2 and claudin-2 was detected by Western blotting.Results1.FICZ improved colonic inflammation in a mouse model of DSS-induced colitis.Histological examination showed that mice treated with FICZ developed less severe colitis.And lower levels of IL-6 was detected in mice treated with FICZ.2.FICZ down-regulated Claudin-2 expression and maintains intestinal barrier function in a DSS-induced colitis mouse model.FICZ prevented DSS-induced TER decrease,and FICZ prevented DSS-induced increase in Claudin-2 m RNA and protein expression.Immunohistochemical(IHC)result showed that Claudin-2 was significantly down-regulated compared to the DSS-treated group after administration of FICZ.3.FICZ improved IL-6-induced intestinal barrier dysfunction and reduced the expression of Claudin-2 in vitro.FICZ prevented the decrease of TER induced by IL-6 in Caco-2 monolayer.Immunofluorescence results showed that the intensity of Claudin-2 in the junction region of the Caco-2 cell monolayer was significantly reduced after FICZ treatment.4.Claudin-2 expression was increased following IL-6 treatment,and FICZ-induced Ah R activation inhibited IL-6-induced increase in Claudin-2.However,Ah R si RNA treatment significantly blocked the effects of FICZ.5.HNF-1α and CDX-2 are involved in the regulation of Claudin-2.Silencing of HNF-1α or CDX-2 inhibits IL-6-induced upregulation of Claudin-2.Expression of HNF-1α and CDX-2 was also increased in DSS-treated mice,and administration of FICZ inhibited upregulation of HNF-1α and CDX-2.ConclusionsFICZ-induced Ah R activation maintains intestinal barrier function and down-regulated Claudin-2 expression in DSS-induced colitis.FICZ improved IL-6-induced intestinal barrier dysfunction and reduced the expression of Claudin-2 in vitro.Silencing of Ah R prevented FICZ-induced down-regulation of Claudin-2.HNF-1α and CDX-2 were involved in the regulation of claudin-2 expression after FICZ and IL-6 treatment.