The role of coagulome in the tumor immune microenvironment.

The rising incidence and persistent thrombosis in multiple cancers including those that are immunosuppressive highlight the need for understanding the tumor coagulome system and its role beyond hemostatic complications. Immunotherapy has shown significant benefits in solid organ tumors but has been disappointing in the treatment of hypercoagulable cancers, such as glioblastoma and pancreatic ductal adenocarcinomas. Thus, targeting thrombosis to prevent immunosuppression seems a clinically viable approach in cancer treatment. Hypercoagulable tumors often develop fibrin clots within the tumor microenvironment (TME) that dictates the biophysical characteristics of the tumor tissue. The application of systems biology and single-cell approaches highlight the potential role of coagulome or thrombocytosis in shaping the tumor immune microenvironment (TIME). In-depth knowledge of the tumor coagulome would provide unprecedented opportunities to better predict the hemostatic complications, explore how thrombotic stroma modulates tumor immunity, reexamine the significance of clinical biomarkers, and enable steering the stromal versus systemic immune response for boosting the effectiveness of immune checkpoint inhibitors in cancer treatment. We focus on the role of coagulation factors in priming a suppressive TIME and the huge potential of existing anticoagulant drugs in the clinical settings of cancer immunotherapy.

Cancer-on-a-Chip: Models for Studying Metastasis.

The microfluidic-based cancer-on-a-chip models work as a powerful tool to study the tumor microenvironment and its role in metastasis. The models recapitulate and systematically simplify the in vitro tumor microenvironment. This enables the study of a metastatic process in unprecedented detail. This review examines the development of cancer-on-a-chip microfluidic platforms at the invasion/intravasation, extravasation, and angiogenesis steps over the last three years. The on-chip modeling of mechanical cues involved in the metastasis cascade are also discussed. Finally, the popular design of microfluidic chip models for each step are discussed along with the challenges and perspectives of cancer-on-a-chip models.

The tumour suppressor effects and regulation of cancer stem cells by macrophage migration inhibitory factor targeted miR-451 in colon cancer.

BACKGROUND: This study aimed to investigate the impact of miR-451 on the biological behaviours of colon cancer cells along with its targets interactions. METHOD: The levels of miR-451 were tested in colon cancer cell lines (SW480 and SW48). Multiple functional and immunological assays were performed to analyse miR-451 induced growth changes in-vitro and downstream effects on target proteins. RESULTS: Overexpression of miR-451 in colon cancer cells led to reduced cell proliferation, increased apoptosis and decrease accumulation of the cells at the G0/G1 phase of the cell cycle. In addition, a significant increase in the number of the cells was noted in the G2-M phase of cell cycle. Moreover, miR-451 reduced the expression of Oct-4, Sox-2 and Snail indicating its role in stem cell and epithelial-mesenchymal transition (EMT) regulation. An inverse correlation between miR-451 and macrophage migration inhibitory protein (MIF) protein expression occurred in colon cancer cells. Furthermore, restoration the level of miR-451 in colon cancer cells inhibits tumour spheres formation. CONCLUSION: miR-451 has tumour suppressor effects in vitro, which can inhibit the cancer-related signalling pathways in colon cancer.

Bone Invasive Properties of Oral Squamous Cell Carcinoma and its Interactions with Alveolar Bone Cells: An In Vitro Study.

BACKGROUND: Co-culture of cancer cells with alveolar bone cells could modulate bone invasion and destructions. However, the mechanisms of interaction between oral squamous cell carcinoma (OSCC) and bone cells remain unclear. OBJECTIVE: The aim of this study is to analyse the direct and indirect effects of OSCC cells in the stimulation of osteolytic activity and bone invasion. METHODS: Direct co-culture was achieved by culturing OSCC (TCA8113) with a primary alveolar bone cell line. In the indirect co-culture, the supernatant of TCA8113 cells was collected to culture the alveolar bone cells. To assess the bone invasion properties, in vitro assays were performed. RESULTS: The proliferation of co-cultured cancer cells was significantly (p<0.05) higher in comparison to the monolayer control cells. However, the proliferation rates were not significantly different between direct and indirect co-cultured cells with indirect co-cultured cells proliferated slightly more than the direct co-cultured cells. Invasion and migration capacities of co-cultured OSCC and alveolar bone cells enhanced significantly (p<0.05) when compared to that of control monolayer counterparts. Most importantly, we noted that OSCC cells directly co-cultured with alveolar bone cells stimulated pronounced bone collagen destruction. In addition, stem cells and epithelialmesenchymal transition markers have shown significant changes in their expression in co-cultured cells. CONCLUSION: In conclusion, the findings of this study highlight the importance of the interaction of alveolar bone cells and OSCC cells in co-culture setting in the pathogenesis of bone invasion. This may help in the development of potential future biotherapies for bone invasion in OSCC.

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/ß-catenin signaling pathways via inactivating tumor suppressor APC followed by activating ß-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/ß-catenin pathway by EB1-mediated modulating of APC in colon cancer cells.

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.

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.

Clinical and biological significance of miR-193a-3p targeted KRAS in colorectal cancer pathogenesis.

This study was to investigate the expression pattern, mechanisms and clinicopathological implications of miR-193a-3p in colorectal cancer. Fresh-frozen tissues from 70 matched colorectal adenocarciomas and the adjacent non-neoplastic mucosae were prospectively collected. Two colorectal cancer cell lines (SW480 and SW48) and a non-neoplastic colon cell line (FHC) were also used. The expression levels of miR193a-3p in the cells and tissues were measured by quantitative real-time polymerase chain reaction. The expression of KRAS protein as a predicted downstream target for miR-193a was studied by immunohistochemistry. Restoration of the miR-193a level in the cell lines by permanent transfection was achieved and multiple functional and immunological assays were performed to analyze the functions of miR-193a in vitro. Down-regulation of miR-193a-3p was noted in 70% of the colorectal cancer tissues when compared to non-neoplastic colorectal tissues. In addition, down-regulation of miR-193a was significantly correlated with carcinoma of early stages (P<.05). Significant inverse correlation between miR-193a-3p and its target KRAS protein was determined (P<.05). Overexpression of miR-193a in colon cancer cells resulted in reduced cell proliferation, increased apoptosis, induced significant changes in cell cycle events and decreased the expression of epithelial-mesenchymal transition marker TWIST. This study confirms the tumor suppressor roles of miR-193a-3p, its downstream target affinity to KRAS and clinical significance in patients with colorectal adenocarcinoma.

Cellular expression, in-vitro and in-vivo confirmation of GAEC1 oncogenic properties in colon cancer.

GAEC1 (Gene amplified in esophageal cancer 1) alterations have oncogenic properties in oesophageal squamous cell carcinomas and frequent amplifications of the gene were noted in colorectal adenocarcinomas. However, the subcellular localization and expression of GAEC1 at the protein level have never been reported in human cancer cells. The present study aimed to investigate whether GAEC1 is differentially expressed in different stages of colon cancer and to elucidate its underlying cellular and molecular mechanism in colon cancer progression. We found differential expression of GAEC1 protein and mRNA in different pathological stages of colon cancer cells (SW480-Stage II, SW48-Stage III and HCT116-Stage IV) when compared to non-neoplastic colon cells (FHC cells) by immunocytochemistry, immunofluorescence, western blot analysis and real-time polymerase chain reaction. GAEC1 protein was predominantly expressed in the cytoplasm of colon cancer cells (SW480, SW48, and HCT116) and in the nucleus of non-neoplastic colon epithelial cells (FHC cells). The transient knockdown of GAEC1 using siRNA induced apoptosis in SW480 and SW48 cells, which was associated with G2/M phase arrest and decreased expression of bcl-2 and K-ras proteins and increased expression of p53. In addition, down-regulation of GAEC1 significantly inhibited (p<0.05) cell proliferation, reduced migration capacity and decreased clonogenic potentiality of colon cancer cells (SW480 and SW48 cells). Furthermore, a xenotransplantation model showed that stable knockdown of GAEC1 using shRNA constructs in colon cancer cells fully suppressed xenograft tumour growth in mice. Collectively, the expression analysis, in vitro and in vivo data indicated that GAEC1 is differentially expressed in cancer cells and act as an oncogene in colon cancer progression.

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.

Novel FAM134B mutations and their clinicopathological significance in colorectal cancer.

FAM134B is a putative tumour suppressor gene and no mutations in FAM134B have been reported in colorectal cancer (CRC) to date. This study aims to identify FAM134B mutation sites and the clinicopathological significance of the gene in patients with CRC. Eighty-eight colorectal cancers were studied for FAM134B mutations by Sanger sequencing. The mutations in these cancers were then tested for correlations with the clinical and pathological parameters of the studied cancers. In addition, mRNA and protein expression of FAM134B in colorectal cancers was examined by polymerase chain reaction, Western blots, and immunofluorescence analysis. FAM134B mutation was noted in 46.5% (41/88) of patients with CRC. Thirty-one novel potentially pathogenic mutations were noted in coding and intronic regions of FAM134B in CRC, the majority of which were single-nucleotide substitutions. Of the 31 mutations, eight novel frameshift mutations showed potential to cause non-sense-mediated mRNA decay (NMD) in computational analysis. In addition, FAM134B mutations were associated with various clinical and pathological variables, including sex of the patients, presence of metachronous cancer, size, T staging, presence of distant metastases, and positivity of microsatellite instability (MSI) in the cancer (p < 0.05). FAM134B mRNA and protein expression was decreased in FAM134B mutated cancers. To conclude, FAM134B mutation is common in colorectal cancer. The association of the mutation of this gene with adverse clinical and pathological parameters is congruent with the tumour suppressive properties of the gene.

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. © 2016 Wiley Periodicals, Inc.

Deregulation of miR-126 expression in colorectal cancer pathogenesis and its clinical significance.

In this study, we investigated the expression profiles and clinicopathological significance of miR-126 in large cohort of patients with colorectal cancers as well the cellular repercussions of miR-126 in colon cancer cells along with its targets in-vitro. Down regulation of miR-126 expression was associated with histological subtypes, peri-neural tumour infiltration, microsatellite instability and pathological staging of colorectal cancers (p<0.05). Low miR-126 expression was also associated with poorer survival in patients with colorectal cancer. Analysis of matched tissues from the same patient revealed that approximately 70% of the tested patients had similar levels of expression of miR-126 in primary cancer and cancer metastases in both lymph node and distant metastases. In addition, induced overexpression of miR-126 showed reduced cell proliferation, increased apoptosis and decreased accumulation of cells in the G0-G1 phase of the colon cancer cells. Furthermore, SW480(+miR-126) cells showed reduced BCL-2 and increased P53 protein expression. To conclude, deregulation of miR-126 in colorectal cancer at the tissue and cellular levels as well as its correlation with various clinicopathological parameters confirm the cancer suppressive role of miR-126 in colorectal cancer.

Cancer stem cells in oesophageal squamous cell carcinoma: Identification, prognostic and treatment perspectives.

Cancer stem cells (CSCs) are a vital subpopulation of cells to target for the treatment of cancers. In oesophageal squamous cell carcinoma (ESCC), there are several markers such as CD44, ALDH, Pygo2, MAML1, Twist1, Musashi1, Side population (SP), CD271 and CD90 that have been proposed to identify the cancer stem cells in individual cancer masses. It has also been demonstrated that stem cell markers like ALDH1, HIWI, Oct3/4, ABCG2, SOX2, SALL4, BMI-1, NANOG, CD133 and podoplanin are associated with patient's prognosis, pathological stages, cancer recurrence and therapy resistance. Finding new cancer stem cell targets or designing drugs to manipulate the known molecular targets in CSCs could be useful for improvements in clinical outcomes of the disease. To conclude, data suggest that CSCs in oesophageal squamous cell carcinoma are related to resistance to therapy and poor prognosis of patients with ESCC. Therefore, innovative insights into CSC biology and CSC-targeted therapies will help to achieve more effective management of patients with oesophageal squamous cell carcinoma.