The Molecular Mechanism Of FBXO8 In Colorectal Cancer Invasion And Metastasis By Targeting GSTP1 For Ubiquitin Mediated Degradation

Aim and backgroundBesides lung and breast cancer, colorectal cancer (CRC) ranks third among all the malignances worldwide. Most patients have developed metastasis when definitely diagnosed, thus miss the proper time for surgical resection. Patients with metastasis always have bad prognosis and low survival rate.Colorectal cancer burdens a lot to patients’ families and even the whole society, so it is important to research pathogenesis of colorectal cancer and its mechanism related to metastasis, which may make significance in early diagnosis and treatment of CRC and therefore reduce patients’pain and improve survival rate.Ubiquitin protease system consists of E1 (ubiquitin kinase), E2 (ubiquitin desmoenzyme), E3 (ubiquitin ligase), which are engaged in protein ubiquitination and induce protein inactivation or degradation together. F-box family, which belonging to ubquitin protease system E3 (ubiquitin ligase), mainly contributes to protein ubiquitination and thus regulates progression, apoptosis, metabolism, motion, damage and repair, and almost all the body vital movement. F-box family contains three kinds of F-box proteins:Fbls, Fbws, Fbxs. Fbls, containing 12 protein members, is the biggest component of F-box family and enrolled two new members of FBL3 and FBL7. FBX08, F-box protein, also named FBX8, is a new member of Fbxs family and only reported in several articles. C-myc promote cell invasion by inhibiting its new binding protein FBXO8. Abnormal expression of FBXO8 promotes breast cancer cell invasion by regulating ubiquitination of Arf6 abnormally.Previous work of our research group indentified that FBXO8 is low-expressed in hepatocyte carcinoma and inhibits progression and invasion of hepatocyte cancer cells, which may provided genetic target for clinical treatment of hepatocyte carcinoma[6]. Based on these researches, we conclude that FBXO8 has close relationship with tumor progression, invasion and metastasis.In this project, FBXO8, a ubiquitination protein, is choosed as entry point to explore metastasis related mechanism in CRC by screening out its downstream ubiquitination target protein.1, Screening out downstream ubiquitination target protein of FBXO8;2, Exploration of relationship between FBXO8 and GSTP1 and demonstrating ubiquitination function of FBXO8 on GSTP1;3, Rescue role detection of GSTP1 in alteration of colorectal invasion induced by FBX08;4, Detection of GSTP1 expression in CRC tissues and its biological function in CRC cell lines.Methods:1, Screening out downstream ubiqitination target protein of FBXO8(1) FBXO8 over-expression plasmid was constructed and transfected into 293 T cell. qRT-PCR and Western-Blot were performed to detect FBXO8 expression.(2)FBXO8 binding proteins were separated by IP and then digested peptide segments were analyzed by mass spectrographic. The final analysed protein might be downstream ubiqitination target protein of FBX08.2, Exploration of relationship between FBXO8 and GSTP1 and demonstrating ubiquitination function of FBXO8 on GSTF1(1)Co-IP and con-focal were performed to verify the combination of FBXO8 and GSTP1.(2)CRC cell lines were treated with MG132, the inhibitor of proteasome, and Western-Blot was performed to detect protein level of GSTP1.(3)Levels of GSTP1 were detected after over-expression or knock-down of FBXO8.(4) Influence of FBXO8 expression on GSTP1 ubiquitination was analyzed by transfecting over-expression ubquitin vector into CRC cell lines.(5) GSTP1 truncate vectors were constructed based on secondary structure presented in Uniprot.3, Exploration of GSTP1 function in alteration of CRC cell invasion induced by FBXO8(l)Co-overexpression and co-depletion of FBX08 and GSTP1 were executed in CRC cell lines. Western-Blot and qRT-PCR were performed to ananlyse FBXO8and GSTP1 expression.(2) MTT assay, transwell invasion assay and nude mice tail vein metastasis assay were performed to analyse GSTP1 function in alteration of CRC cell invasion induced by FBXO8.4, Detection of GSTP1 expression in CRC tissues and its function in biological behavior of CRC cell lines.(1) mRNA expressions of GSTP1 in 20 pairs matched fresh CRC tissues and their corresponding normal tissues were detected by qRT-PCR.(2)Immunohistochemistry (IHC) was performed in 136 patients to detect expression of GSTP1 in paraffin imbedded CRC tissues. SPSS 13.0 was applied in survival analysis and GSTP1 expression between tumor and normal areas.(3)Endogenous expressions of GSTP1 in six CRC cell lines were detected by qRT-PCR and Western-Blot. High expression cell lines were selected for knock-down and low expression cell lines were selected for over-expression of GSTP1. Western-Blot and qRT-PCR were performed to ananalyse GSTP1 expression.(4) MTT assay, Borden transwell assay and nude mice tail vein assay were performed to analyze influence of GSTP1 on proliferation and invasion ability of CRC cell lines in vitro. Flow cytometry was performed to analyze influence of GSTP1 on CRC apopsis.Results:1, Screening out downstream ubiqitination target protein of FBXO8FBXO8 vector was inserted into pEGFP-C1 empty plasmid and the length of vector was 960bp. Sequecing result and agarose gel electrophoresis showed FBXO8 over-expression plasmid was construted successfully. pEGFP-C1/FBXO8 and its corresponding empty plasmid were separately transfected into 293T cell, independent samples T test showed that FBXO8 was high expressed 2736 times in 293T/FBXO8 group than in mock group (t=665.8, P<0.001). IP assay and mass spectrographic analysis inicially demonstated three protein molecules:NFX1, GGT7 and GSTP1. We choosed GSTP1 as proper ubiquitination target protein for GSTP1 had the highest score in peptide fragment blast and good repeatability.2, GSTP1 was a ubiquitination target protein of FBXO82.1 Co-localization of FBXO8 and GSTP1(1) Con-focal immunofluorescence was performed to identify cellular co-localization of FBXO8 and GSTP1 by staining FBXO8 as green and GSTP1 as red. Con-focal result showed that FBXO8 and GSTP1 had co-localization in cytoplasm.(2) Co-IP was performed in 293T cell. The results showed that GSTP1 could be pulled down by FBXO8 and FBXO8 also could be pulled down by GSTP1. These results identified the interaction between the two proteins.2.2. Influence of FBXO8 expression on GSTP1(1)CRC cell lines SW480 was selected for transfecting FBXO8 over-expression plasmid. Western-Blot result showed that GSTP1 decreased when FBXO8 was over-expressed.(2)CRC cell lines SW620 was selected for transfecting siRNA targeted FBXO8. Western-Blot result showed that GSTP1 content increased when FBXO8 was knocked-down.2.3 Influence of proteasome inhibitor MG132 on GSTP1 protein.Western-Blot was performed to detect GSTP1 protein level of SW620 and SW620/FBXO8 after being treated with MG132. Results showed that GSTP1 was increased both in SW620 and SW620/FBXO8 groups after treatment of MG132.2.4 Construction of GSTP1 truncatesSequencing results showed that we successfully constructed three truncate vectors GSTP1-C, GSTP1-N and GSTP1-PTZ. (Co-IP of truncate vector is on progress.)3, Rescue role of GSTP1 in alteration of CRC cell invasion induced by FBXO8(1) SW480/FBXO8/GSTP1 double over-expression cell line was established. Functional results analyzed by variance of factorial design showed that over-expression of GSTP1 could rescue proliferation inhibition induced by FBXO8 over-expression (FTime=453.149, PTimer<0.001;FCells=197.89,PCells<0.001;FTime×Cells= 26.039, PTime×Cells<0.001). Over-expression of GSTP1 could also rescue invasion inhibition induced by FBXO8 over-expression (F=68.235, P< 0.001). Results showed that over-expression of GSTP1 could rescue metastasis inhibition induced by FBXO8 over-expression (F=5.477,P=0.016).(2) SW620/siFBXO8/siGSTPl double depletion cell line was established. Results analyzed by variance of factorial design showed that GSTP1 depletion could rescue proliferation promotion induced by FBXO8 depletion (FTime=819.974,PTimer< 0.001;FCells=81.03,PCells<0.001;FTime×Cells= 10.296.PTime×Cells<0.001). GSTP1 depletion could also rescue invasion promotion induced by FBXO8 deletion (F=27.447, P<0.001). 4, Detection of GSTP1 expression in CRC tissues and its function in biological behavior of CRC cell lines. 4.1 Expression of GSTPl in CRC tissues(1) GSTP1 expression in 20 pairs matched fresh CRC tissues and their adjacent normal tissues were detected by qRT-PCR. Value of 2-ΔΔCt was analyzed by paired sample t test. The result showed that mRNA expression of GSTP1 was significantly higher in CRC tissues than in adjacent normal tissues (t=2.477, P<0.05).(2) IHC results showed that:GSTP1 located in nucleus of normal tissues while both nucleus and cytoplasm were colored in CRC tissues, and its expression in CRC tissues was obviously higher than in adjacent normal tissues (P<0.01). Survival analysis indicated that patients with high expression of GSTP1 had low survival rate while patients with low expression of GSTP1 had high survival rate (P=0.007).4.2 Effects of GSTP1 on biological behavior of CRC cell in vitro(1)Endogenous expression of GSTP1 showed that GSTP1 was high-expressed in HCT116 and SW620 and low-expressed in SW480, RKO and Lovo.(2)Endogenous low-expression cell lines SW480 and RKO were selected for over-expression of GSTP1.Results analyzed by independent sample t test showed that GSTP1 in SW480/GSTP1 was high-expressed for 401.9 times compared to SW480/mock (t=19.43, P<0.001), GSTP1 in RKO/GSTP was high-expressed for 53.32 times (t=13.43, P=0.0002). Endogenous high-expression cell lines SW620 and HCT116 were selected for depletion of GSTP1. Results analyzed by one-way ANOVA showed that the relative expression of siRNA-1/-2/-3 in SW620 was 0.465, 0.3603,0.769 (F=121.056, P<0.001), the relative expression of siRNA-1/-2/-3 in HCT116 was 0.2042,0.1749,0.9681 (F=300.961,P<0.001).(3) In vitro functional assay results analyzed by variance of factorial design showed that over-expression of GSTP1 significantly increased proliferation of SW480 and RKO in vitro (SW480 FTime=741.627, PTime< 0.001;FCells=186.139, P cells<0.001;FTime×Cells= 47.391,PTime×Cells<0.001; RKO FTime=283.834, PTime< 0.001;FCells=117.041, PCells<0.001;FTime×Cells= 13.922,PTime×Cells<0.001). Results analyzed by independent sample t test showed that over-expression of GSTP1 significantly increased invasion of SW480 and RKO in vitro (t=10.17, P<0.0001; t=11.14, P<0.0001).(4) In vitro functional assay results analyzed by variance of factorial design showed that depletion of GSTP1 significantly decreased proliferation of SW620 and HCT116 in vitro (FTime=806.115,PTime<0.001;FCells=292.199,PCells<0.001;FTime×Cells= 44.787, PTime×Cells<0.001;FTime=156.783,PTime<0.001;FCells=152.425,PCells<0.001;FTime×Cells=10.635, PTimexCells<0.001).Results analyzed by one-way ANOVA showed that depletion of GSTP1 significantly decreased invasion of SW620 and HCT116 in vitro (F=72.831,P<0.001; F=608.011, P<0.001). Flow cytometry results analysed by indepent t test showed that GSTP1 inhibited CRC apopsis (t=4.456, P=0.0112).(5) In vivo functional assay results analyzed by independent t test showed that GSTPl was high-expressed over 55.44 times in lovo/GSTP1 than in mock group. Results of tail vein injection assay analyzed by independent t test showed that GSTP1 increased metastasis of lovo in vivo (t=3.522,P=0.0244).Conclusion:1,GSTPl is a new target protein of FBXO8, FBXO8 regulates proliferation and metastasis of CRC by targeting GSTP1 for ubiquitination;2, GSTP1 is high-expressed in CRC tissues and promotes proliferation and metastasis of CRC cell lines in vitro and in vivo.