Riajul Wahab

Stage dependent expression and tumor suppressive function of FAM134B (JK1) in colon cancer.

The aims of the present study are to investigate sub‐cellular location, differential expression in different cancer stages and functional role of FAM134B in colon cancer development. FAM134B expression was studied and quantified at protein and mRNA levels in cell lines using immunocytochemistry, Western blot and real‐time PCR. In vitro functional assays and an in vivo xenotransplantation mouse models were used to investigate the molecular role of FAM134B in cancer cell biology in response to FAM134B silencing with shRNA lentiviral particles. FAM134B protein was noted in both cytoplasm and nuclei of cancer cells. In cancer cells derived from stage IV colon cancer, FAM134B expression was remarkably reduced when compared to non‐cancer colon cells and cancer cells derived from stage II colon cancer. FAM134B knockdown significantly (P < 0.05) increased the proliferation of colon cancer cells following lentiviral transfection. Furthermore, FAM134B suppression significantly increased (34–52%; P < 0.05) the clonogenic capacity, wound healing potential of and increases the proportion of cells performing DNA synthesis (P < 0.01). Xenotransplantation model showed that larger and higher‐grade tumors were formed in mice receiving FAM134B knockdown cells. To conclude, expression analysis, in vitro and in vivo indicated that FAM134B acts as a cancer suppressor gene in colon cancer.

MicroRNA-186-5p overexpression modulates colon cancer growth by repressing the expression of the FAM134B tumour inhibitor

Objectives: The role and underlying mechanism of miR‐186‐5p in colorectal cancer remain unknown. The present study aims to examine the various cellular effects of miR‐186‐5p in the carcinogenesis of colorectal cancer. Also, the interacting targets and association of clinicopathological factors with miR‐186‐5p expression in patients with colorectal cancer were analysed. Methods: The miR‐186‐5p expression levels in colorectal cancer tissues (n=126) and colon cancer cell lines (n=3) were analysed by real‐time PCR. Matched non‐neoplastic colorectal tissues and a non‐neoplastic colonic epithelial cell line were used as controls. Various in vitro assays such as cell proliferation, wound healing and colony formation assays were performed to examine the miR‐186‐5p specific cellular effects. Western blots and immunohistochemistry analysis were performed to examine the modulation of FAM134B, PARP9 and KLF7 proteins expression. Results: Significant high expression of miR‐186‐5p was noted in cancer tissues (p< 0.001) and cell lines (p<0.05) when compared to control tissues and cells. The majority of the patients with colorectal cancer (88/126) had shown overexpression of miR‐186‐5p. This miR‐186‐5p overexpression was predominantly noted with in cancer with distant metastasis (p=0.001), lymphovascular permeation (p=0.037), microsatellite instability (MSI) stable (p=0.015), in distal colorectum (p=0.043) and with associated adenomas (p=0.047). Overexpression of miR‐186‐5p resulted in increased cell proliferation, colony formation, wound healing capacities and induced alteration of cell cycle kinetics in colon cancer cells. On the other hand, inhibition of endogenous miR‐186‐5p reduced the cancer growth properties. miR‐186‐5p overexpression reduced FAM134B expression significantly in the cancer cells (p<0.01). Also, FAM134B and miR‐186‐5p expressions are inversely correlated in colorectal cancer tissues and cells. Conclusion: The miR‐186‐5p expression promotes colorectal cancer pathogenesis by regulating tumour suppressor FAM134B. Reduced cancer cells growth followed by inhibition of miR‐186‐5p highlights the potential of miR‐186‐5p inhibitor as a novel strategy for targeting colorectal cancer initiation and progression. HighlightsSignificant high expression of miR‐186‐5p was noted in cancer tissues and cells.miR‐186‐5p overexpression was correlated with adverse clinical factors of cancer.Modulation of miR‐186‐5p, regulate cancer cells growth and cell cycle kinetics.miR‐186‐5p overexpression reduced FAM134B expression in colorectal cancer.Anti‐miR‐186‐5p treatment inhibited colon cancer cells growth remarkably.

Protein interactions of FAM134B with EB1 and APC/beta-catenin in vitro in colon carcinoma

FAM134B is an autophagy regulator of endoplasmic reticulum and acts as a cancer suppressor in colon cancer. However, the molecular signaling pathways by which FAM134B interacts within colon carcinogenesis is still unknown. Herein, this study aims to determine the interacting partners of FAM134B for the first time in colon cancer and to explore the precise location of FAM134B in cancer signalling pathways. Liquid chromatography coupled with tandem mass spectrometry (LC‐MS/MS) followed by anti‐FAM134B co‐immune precipitation of FAM134B interacting complex was used to identify the potential interactors of FAM134B in colon cancer cells. Western blot and confocal microscopic analysis were used to validate the physical interactions of FAM134B with the interactors. Lentiviral shRNA mediated silencing of FAM134B was used to examine the modulation of FAM134B interactors in cells. We have identified 29 novel binding partners, including CAP1, RPS28, FTH1, KDELR2, MAP4, EB1, PSMD6, PPIB/CYPB etc. Subsequent immunoassays confirmed the direct physical interactions of FAM134B with CAP1, EB1, CYPB, and KDELR2 in colon cancer cells. Exogenous suppression of FAM134B has led to significant upregulation of EB1 as well as reduction of KDELR2 expression. It was noted that overexpression of EB1 promotes WNT/β‐catenin signaling pathways via inactivating tumor suppressor APC followed by activating β‐catenin in colorectal carcinogenesis. This study has first time reported the gene signaling networks with which FAM134B interacts and noted that FAM134B is involved in the regulation of WNT/β‐catenin pathway by EB1‐mediated modulating of APC in colon cancer cells.

Expression of GAEC1 mRNA and protein and its association with clinical and pathological parameters of patients with colorectal adenocarcinoma

AIM GAEC1 (Gene amplified in esophageal cancer 1) is an oncogene with key regulatory roles in the pathogenesis of oesophageal and colorectal carcinomas. The aim of this study was to investigate expression profiles and clinicopathological significance of GAEC1 mRNA and protein in patients with colorectal carcinomas. METHOD Matched cancer and non-cancer fresh frozen tissues were prospectively collected from 80 patients diagnosed with colorectal adenocarcinoma (39 men and 41 women). The tissues were sectioned for RNA extraction and cDNA conversion and quantified by a real-time polymerase chain reaction. GAEC1 protein expression was analysed by immunohistochemistry using a custom made GAEC1 antibody. RESULT GAEC1 mRNA was upregulated in majority (52%, n=42/80) of the colorectal carcinomas when compared to the matched non-neoplastic tissues. High expression of GAEC1 mRNA as correlated with patients of younger age (p=0.008), with lower grade carcinoma (p=0.028), presence of synchronous adenocarcinomas (p=0.034) and without any associated adenomas (p=0.047). In addition, patients with high GAEC1 mRNA overexpression had a shorter survival time. Furthermore, high GAEC1 protein expression was noted among patients having perforated colorectal carcinoma (p=0.04). CONCLUSION The high expression of GAEC1 mRNA/protein as well as its correlation with multiple clinicopathological characteristics in patients with colorectal carcinoma strongly suggests that GAEC1 is a key regulator in the initiation of colorectal carcinogenesis.

GAEC1 mutations and copy number aberration is associated with biological aggressiveness of colorectal cancer

GAEC1 (gene amplified in oesophageal cancer 1) is a transforming oncogene with tumorigenic potential observed in both oesophageal squamous cell carcinoma and colorectal cancer. Nonetheless, there has been a lack of study done on this gene to understand how this gene exert its oncogenic properties in cancer. This study aims to identify novel mutation sites in GAEC1. To do so, seventy-nine matched colorectal cancers were tested for GAEC1 mutation via Sanger sequencing. The mutations noted were investigated for the correlations with the clinicopathological parameters of the patients with the cancer. Additionally, GAEC1 copy number aberration (CNA), mRNA and protein expression were determined with the use of droplet digital (dd) polymerase chain reaction (PCR), real-time PCR and Western blot (confirmed with immunofluorescence analysis). GAEC1 mutation was noted in 8.8% (n = 7/79) of the cancer tissues including one missense mutation, four loss of heterozygosity (LOH) and two substitutions. These mutations were significantly associated with cancer perforation (p = 0.021). GAEC1 mutation is frequently associated with increased GAEC1 protein expression. Nevertheless, GAEC1 mRNA and protein are only weakly associated. Taken together, GAEC1 mutation affects GAEC1 expression and is associated with poorer clinical outcomes. This further strengthens the role of GAEC1 as an oncogene.