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Molecular and Functional Characterization of JK1 (FAM134B) Gene in Human Colorectal Cancer

By Md. Farhadul Islam

Abstract

Family with sequence similarity 134, member B (FAM134B) (also called JK1/RETREG1) was first identified in 2001 using comparative genomic hybridization analysis in cancer. Subsequent studies characterized this gene in biological processes and human diseases. FAM134B regulates endoplasmic reticulum-turnover by selective autophagy and loss or dysregulation of FAM134B impairs endoplasmic reticulum-turnover. In addition, aberrant FAM134B expression encompasses in the pathogenesis of a number of human diseases, including, neuronal disorder, vascular disease, allergic rhinitis, viral infections and cancer. In cancer, FAM134B acts as a tumour suppressor or promoter in different types of cancer. For example, in oesophageal squamous cell carcinoma, FAM134B acts as an oncogene and stimulates cancer cell growth and proliferation, whereas in breast and colorectal cancer (CRC) it acts as a tumour inhibitor. In CRC, FAM134B copy number deletion, loss or reduced mRNA and protein expressions associated with the biological aggressiveness of cancer. However, indepth functional insights, genetic and epigenetic regulation, interacting signalling pathways and expression pattern of FAM134B in different stages of CRC have never been studied. Thus, the present study aims to explore in vitro and in vivo functional role, and molecular characterization of FAM134B through mutational, micro-RNAs, promoter methylation screening and identifying partners to which FAM134B interacts in CRC. The clinical significance and validation of FAM134B in a large cohort of patients with CRC also investigated. Detection of FAM134B expression and quantified at the protein and mRNA levels in cell lines derived from different stages of colon cancer and non-neoplastic colon epithelial cells was performed using immunocytochemistry, immunofluorescence, western blot and real-time PCR. Functional assays and mouse xenotransplantation model were used to investigate the role of FAM134B in cancer cell biology followed by FAM134B silencing with shRNA lentiviral particles. In cells derived from stage IV colon cancer, FAM134B expression was remarkably reduced when compared to non-neoplastic colon cells and cells derived from stage II colon cancer. Suppression of FAM134B increased the proliferation of colon cancer cells following lentiviral transfection (p < 0.05). Additionally, FAM134B silencing induced increased clonogenic capacity (34-52 %; p < 0.05), wound healing potential and enhanced the proportion of cells in S-phase of the cell cycle (p < 0.01). In vivo xenotransplantation mouse model showed that larger and higher-grade tumours were formed in mice receiving FAM134B knockdown cells. Following in vitro, in vivo functional studies, we have screened FAM134B mutations in CRC tissue samples (n = 88) and cell lines (n = 4) using high-resolution melt-curve (HRM) analysis and subsequently confirmed by Sanger sequencing. The FAM134B mutation was noted in 46.5 % (41/88) of patients with CRC. Thirty-one novel pathogenic mutations were identified in coding and intronic sites of FAM134B in CRC. Of the 31 mutations, eight novel frameshift mutations showed the potential to cause nonsense-mediated mRNA decay (NMD) in computational analysis. The identified mutations were associated with different clinicopathological factors, including, sex of the patients, the presence of metachronous cancer, the larger size of a tumour, advanced T stages, the presence of distant metastases and positivity of microsatellite instability (MSI) in cancer (p < 0.05). Also, the expressions of FAM134B mRNA and protein were downregulated in FAM134B mutated cancers. Afterwards, this study investigated the epigenetic (methylation and micro-RNA mediated) regulation of FAM134B expression in CRC. Methylation-specific high-resolution melt-curve (MS-HRM) analysis followed by sequencing was used to identify FAM134B promoter methylation in colorectal adenomas (n = 32), CRC (n = 164), adjacent non-neoplastic (n = 83) tissue samples and cell lines (n = 4). FAM134B promoter methylation was more frequent in CRC (52 %; 85/164) compared to that of adenomas (28 %; 9/32) and non-neoplastic (35 %; 29/83) tissue samples. FAM134B promoter methylation was inversely correlated with low FAM134B copy numbers and mRNA/protein expressions, whereas in vitro demethylation has restored FAM134B expression in colon cancer cells. FAM134B promoter methylation was associated with adverse clinicopathological factors, including, high histological grade (p = 0.025), presence of peri-neural infiltration (p = 0.012), lymphovascular invasion (p = 0.021), lymph node metastasis (p = 0.0001), distant metastasis (p = 0.0001) and advanced pathological stages (p = 0.0001). Also, FAM134B promoter methylation correlated with cancer recurrence (p = 0.001) and poor survival (p = 0.009) of patients with CRC. Furthermore, the present study investigated micro-RNA mediated repression of FAM134B in CRC. It was noted that microRNA-186-5p (miR-186-5p) represses the endogenous FAM134B expression in colon cancer cells and tissues, which in turn, modulated various cellular events such as cell proliferation, colony formation, cell migration and altered cell cycle kinetics. Therefore, this project explored the epigenetic silencing of FAM134B in CRC, which might lead the initiation and progression of CRC. Simultaneously, the current study identified potential binding proteins partner of FAM134B in colon cancer cells using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and western blot analysis followed by co-immunoprecipitation (co IP). Interacting partners, including, CAP1, RPS28, FTH1, KDELR2, PSMD6, MAP4, MAPRE1/EB1, PPIB/CYPB of FAM134B were detected by LC-MS/MS analysis. Subsequent western blot analysis followed by co-IP confirmed the interactions of FAM134B with CAP1, EB1, CYPB (onco-proteins), and KDELR2 (ER-resided protein). In addition, immunofluorescence microscopic analysis exhibited co-localization of FAM134B with CAP1, KDELR2, EB1, CYPB proteins in colon cancer cells. Furthermore, upregulation of EB1 as well as downregulation of KDELR2 proteins followed by knockdown of FAM134B in colon cancer cells further indicated the potential interactions between themselves. Activation of EB1 via FAM134B downregulation could inactivate tumour inhibitor APC or activate aurora B kinase, thereby could modulate the WNT/β-catenin pathway in adenoma-carcinoma sequence of colon cancer pathogenesis. Finally, this study developed an electrochemical approach for rapid, sensitive, and specific detection of FAM134B protein in biological and clinical samples. The method usages a differential pulse voltammetry (DPV) in the presence of the [Fe(CN)6]3-/4- redox system to quantify the FAM134B protein in a two-step approach that involves (i) initial attachment of anti-FAM134B antibody on the surface of extravidin-modified screen-printed carbon electrode, and (ii) subsequent detection of FAM134B protein present in the samples. This method has detected FAM134B protein at a concentration down to 10 pg μl-1 in phosphate buffered saline (pH 7.4) with a good inter-assay reproducibility (% RSD = <8.64, n = 3). Also, this method showed excellent sensitivity and specificity for the detection of FAM134B protein in a panel of colon cancer cell lines and serum samples of patients with CRC. The assay performance was validated with ELISA and it is believed that this method could potentially lead a low-cost alternative to conventional immuno-based assays for target antigens analysis in point-of-care applications. Altogether, from genetic to epigenetic, functional to molecular studies herein, has provided critical insights of tumour suppressive properties of FAM134B in CRC pathogenesis. FAM134B executes cancer inhibitory function by interacting with oncoproteins and proteins involved in vesicle-mediated transport in cells. Genetic and epigenetic alterations of FAM134B regulates its expression in colon cancer cells and clinical samples of patients with CRC, which in turn contributes to the biological aggressiveness of carcinomas. The association of the FAM134B promoter methylation and mutations, as obvious loss or reduced FAM134B expression, with adverse clinicopathological factors are in concur with the tumour suppressive property of the gene, which in turn, might act as a potential marker in predicting clinical prognosis in colorectal carcinomas.Thesis (PhD Doctorate)Doctor of Philosophy (PhD)School of MedicineGriffith HealthFull Tex

Topics: Molecular characterization, Colorectal cancer, Family sequence similarity
Publisher: Griffith University
Year: 2017
OAI identifier: oai:research-repository.griffith.edu.au:10072/371217
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