Loss Of Rarγ Contributes To Colorectal Tunorigenesis And Metastasis Through Regulating The Hippo-Yap Pathway

Background and ObjectiveWith the development of economy, the improvement of people’s living standard and the change of diet, the colorectal cancer has become one of the most common digestive tumors in the world. Although the development of science and technology in recent years has made great progress in the diagnosis and treatment of cancer, but the incidence and mortality of colorectal cancer in China is still showing a rising trend. At present, metastasis and recurrence of colorectal cancer are the main causes of death. Therefore, the study of the mechanism of the occurrence and metastasis of colorectal cancer is of great significance for early screening, clinical treatment and prognosis of patients with colorectal cancer.Retinoic acid receptor γ(RARγ), a member of the nuclear receptor subfamily, plays a critical role in mediating cell proliferation, differentiation, and apoptosis. Some data of the RARγ from recent studies also show that nongenomic transcription regulation of RARγ plays a key role in carcinogenesis. But the regulating effect of RARγ in colorectal cancer is not described, namely how the RARγ regulate colorectal tumorigenesis and development is still not clear.The Hippo pathway plays a very important role in controlling the size of organ development and maintaining the volume of organ development through regulating cell proliferation and apoptosis. Recently, accumulating evidence has strongly suggested that dysregulation of this pathway contributes to cancer development. Ectopic expression of YAP, a key factor in the Hippo pathway, in cancer cell lines drives tumor growth and metastasis. These findings suggest that the Hippo pathway plays an important role in tumorigenesis.In this study, we sought to investigate the role of nuclear receptor RARγ in CRC development, to illustrate RARγ functions as a novel regulator for the Hippo-Yap pathway to mediate CRC cell growth and metastasis. At the same time, it is proved that the loss of RAR is related to the prognosis of colon cancer patients.Methods1、Expression of RARγ in human colorectal cancer tissues and cells was detected by Western blot;2、The correlation between the expression of RARγ and tumor size, distant metastasis and prognosis was analyzed by immunohistochemistry;3、Using lentivirus vector to construct stable cell lines that knockdown RARγ;4、The changes of cell proliferation were detected by MTT and plate colony formation;5、The ability of cell migration and invasion was detected by Transwell assay;6、The changes of tumor cells were detected by xenograft and lung metastasis model in nude mice;7、Localization of proteins in cells by immunofluorescence and nuclear & cytoplasmic extraction;8、The effect of RARγ in Hipoo-YAP pathway was detected by luciferase reporter system;9、Using RT-PCR to detect the expression changes of downstream gene;10、The interaction between proteins was studied by co-immunoprecipitation.ResultSection 1 To detect the expression of RAR in human colorectal cancer tissuesWe found that the expression of RARγ protein was higher in surrounding tissues compared with their matched carcinoma tissues in 78% primary CRC. Immunohistochemical results also suggest that RARγ express in surrounding tissues higher than carcinoma tissues, and the expression of RARγ was significantly correlated VII with tumor size, distant metastasis, and survival.Section 2 The biological characteristics of colorectal cancer cells in vitro and in vivo were affected by loss of RARγConstructing and screening stable SW480 and HT-29 cell lines of knockdown RARγ. MTT experimental result showed that knocking down of RARγ significantly increased proliferation of SW480 stable cell. Plate colony formation results showed that the ability of SW480 knockdown RARγ cells were raised compared with control cells in vitro. Migration assay showed that knocking RARγ in SW480 increased migratocy cells number. Similarly, knocking RARγ in SW480 increased invasive cells number too. Nude mice xenograft experiment results showed tumor formation of knockdown RARγ cells grew faster than control cells in vivo. Metastasis model in nude mice suggested SW480/RARγ/sh RNA cells formed much metastatic foci in lung than SW480/RARγ/control cells.Section 3 The biological characteristics of colorectal cancer cells in vitro and in vivo were affected by overexpression of RARγConstructing and screening stable DLD-1 and RKO cell lines that overexpression of RARγ. Plate colony formation results showed that the ability of DLD-1 and RKO cells were impaired when RARγ was over expression in vitro. Migration assay showed that overexpression of RARγ in DLD-1 and RKO reduced migratocy cells number. Similarly, overexpression of RARγ in DLD-1 reduced invasive cells number too. Nude mice xenograft experiment results showed tumor formation by DLD-1/RARγ cells grew fast compared to the control cells in vivo. Metastasis model in nude mice suggested DLD-1/RARγ cells formed less metastatic foci in lung than DLD-1/control cells.Section 4 RARγ regulated proliferation and metastasis in colorectal cancer cell through Hippo pathway by changing phosphorylation of YAPWestern blot results showed that the stable knockdown of RARγ in human colorectal cancer cells SW480 and HT-29 decreased phosphorylation of YAP. Conversely, overexpression of RARγ in DLD-1 and RKO increased phosphorylation of YAP. Phosphorylation level of YAP in xenograft that was formed by stable SW480 VIII knockdown of RARγ was lower than control group. ATRA regulated RARγ changing phosphorylation of YAP. Immnofluorescence, immunohistochemical, and cell component separation suggested that YAP located in nucleus of stable knockdown of RARγ cells. Luciferase reporter system results showed that overexpression of RARγ reduced YAP-TEAD transcriptional activity in SW480 and RKO cells. In contrast, knockdown of RARγ in SW480 increased the transcriptional activity of YAP-TEAD. RT-PCR experiments showed that the expressions of AREG and CTGF, as downstream genes in Hippo pathway, were significantly increased in stable knockdown of RARγ SW480 cells.Section 5 RARγ regulated EMT through a YAP-dependent pathwayRT-PCR and Western blot results showed that knockdown of RARγ was downregulated expression of E-cadherin, and upregulated expression of Vimentin. Conversely, overexpression of RARγ was upregulated expression of E-cadherin, and downregulated expression of Vimentin. Western blot results detected YAP interfered by si RNA could aggravate this phenomenon. Migration results showed that while knocking down RARγ and YAP reduced cell migration ability that due to knockdown of RARγ. Invasion results showed that while knocking down RARγ and YAP reduced cell invasion ability that due to knockdown of RARγ.Section 6 RARγ interacted with YAPImmunofluorescence results showed that RARγ, as a nuclear reportor, also existed in the cytoplasm of SW480 and HCT116 cell line that were high expression of RARγ. Co-immunoprecipitation and immunofluorescence experiments indicated that RARγ interacted with Yap either endogenous or exogenous, and this interaction was regulated by ATRA. Through the study of RARγ and Yap deletion mutants showed that the LBD domain of RARγ and the region(102-263aa) of YAP were their interaction area. RARγ promoted the interaction between Lats1 and YAP, and increased the phosphorylation of YAP.Section 7 RARγ expression was positively correlated with E-cadherin and negatively correlated with Vimentin and CTGFBy Spearman’s rank correlation analysis of expression of RARγ, E-cadherin, Vimentin and CTGF in colorectal cancer tissues were found that the expression of RARγ was positively correlated with E-cadherin and negatively correlated with Vimentin and CTGF.