Proteins In Colorectal Cancer Cells Research Articles

Disruption of the c-Myc/miR-200b-3p/PRDX2 regulatory loop enhances tumor metastasis and chemotherapeutic resistance in colorectal cancer

BackgroundMetastasis is a major threat to colorectal cancer (CRC) patients. We have reported that peroxiredoxin-2 (PRDX2) is associated with CRC invasion and metastasis. However, the mechanisms regulating PRDX2 expression remain unclear. We investigate whether microRNAs (miRNAs) regulate PRDX2 expression in CRC progression.MethodsQuantitative real-time polymerase chain reaction (qPCR) was used to measure microRNA-200b-3p (miR-200b-3p) expression. Immunohistochemistry (IHC) was performed to detect c-Myc and PRDX2 protein levels in CRC tissue samples (n = 97). Western blot was used to quantify PRDX2, c-Myc, AKT2/GSK3β pathway-associated proteins and epithelial-mesenchymal transition (EMT)-related proteins in CRC cells. Luciferase reporter assays were used to analyze the interaction between miR-200b-3p and 3′untranslated region (3′UTR) of PRDX2 mRNA and AKT2 mRNA as well as c-Myc and the miR-200b-3p promoter. Chromatin immunoprecipitation (ChIP) assay was used to evaluate binding of c-Myc to the miR-200b-3p promoter. Invasive assay and metastatic model were used to assess invasive and metastatic capacities of CRC cells in vitro and in vivo. Moreover, drug-induced apoptosis was measured by flow cytometry.ResultsWe found that miR-200b-3p was significantly downregulated, whereas c-Myc and PRDX2 were upregulated in metastatic CRC cells and CRC tissues compared to their counterparts. An inverse correlation existed between c-Myc and miR-200b-3p, and between miR-200b-3p and PRDX2. We also found that PRDX2 was a target of miR-200b-3p. Importantly, overexpression of nontargetable PRDX2 eliminated the suppressive effects of miR-200b-3p on proliferation, invasion, EMT, chemotherapeutic resistance and metastasis of CRC cells. Moreover, c-Myc bound to the promoter of miR-200b-3p and repressed its transcription. In turn, miR-200b-3p disrupted the stability of c-Myc protein by inducing c-Myc protein threonine 58 (T58) phosphorylation and serine 62 (S62) dephosphorylation via AKT2/GSK3β pathway.ConclusionsOur findings reveal that the c-Myc/miR-200b/PRDX2 loop regulates CRC progression and its disruption enhances tumor metastasis and chemotherapeutic resistance in CRC.

Polypyrimidine tract-binding protein 1-mediated down-regulation of ATG10 facilitates metastasis of colorectal cancer cells

Autophagy plays complex roles in tumor initiation and development, and the expression of autophagy-related genes (ATGs) is differentially regulated in various cancer cells, depending on their environment. In this study, we analyzed the expressional relationship between polypyrimidine tract-binding protein 1 (PTBP1) and ATG10 in metastatic colorectal cancer. PTBP1 is associated with tumor metastasis in primary colorectal tumors and colorectal cancer liver metastasis (CLM) tissues. In addition, PTPB1 directly interacts with mRNA of ATG10, and regulates ATG10 expression level in colorectal cancer cells. Ectopic expression of PTBP1 decreased ATG10 expression, whereas down-regulation of PTBP1 increased ATG10 level. In contrast to PTBP1, expression of ATG10 was decreased in CLM tissues. Knock down of ATG10 promoted cell migration and invasion of colorectal cancer cells. Moreover, depletion of ATG10 modulated epithelial-mesenchymal transition-associated proteins in colorectal cancer cells: N-cadherin, TCF-8/ZEB1, and CD44 were up-regulated, whereas E-cadherin was down-regulated. Taken together, our findings suggest that expression of ATG10 negatively regulated by PTBP1 is associated with metastasis of colorectal cancer cells.

Abstract 1125: FOXM1 and FOXQ1 are novel oncogenes in colorectal cancer and can be therapeutically targeted through miRNA-based therapeutics

Abstract OBJECTIVES: The development of colorectal cancer (CRC) is a highly complex process which needs coordinated regulation in the gene regulatory network. Forkhead box (FOX) proteins constitute a large family of transcriptional regulators. Accumulating evidence indicates that several FOX family members are critical regulators of tumor progression. However, the role of FOX proteins in CRC remains vastly unknown. miRNAs are single stranded small non-coding RNAs which can regulate a variety of biological processes in various human diseases including cancer. AIMS: Herein, we aimed to identify the expression patterns of FOX family members in CRC, followed by extensive characterization for their functional and mechanistic role in this malignancy. In addition, we sought to identify specific miRNAs that are responsible for dysregulated expression of FOX proteins in CRC. METHODS: The web-based database ONCOMINE was used to identify most highly expressed FOX family members in CRC. A cohort of 178 frozen tissues including 134 primary CRC tissues, and 44 matched normal mucosa tissues were collected to analyze the expression levels of FOX genes. For functional analysis, cell proliferation, migration and invasion assays were performed following siRNA knockdown of specific FOX proteins in CRC cells. The potential FOX-targeting miRNAs were initially identified using in-silico approaches, followed by luciferase reporter assays to validate their binding to downstream target genes. RESULTS: We systematically analyzed the expression profiles of FOX family members across 24 studies in cancer and normal tissues, and identified FOXM1, FOXQ1 and FOXA2 as the most highly expressed FOX genes in CRC. The over-expression of FOXM1 and FOXQ1 in CRC was confirmed in a cohort of CRC and matched adjacent normal tissues. Furthermore, the analysis of 134 CRC samples revealed that high FOXM1 and FOXQ1 expression resulted in worse overall survival. We next identified miR-342 as a potential regulator of both FOXM1 and FOXQ1, and subsequently validated its direct binding to the 3’UTR regions of both genes. Furthermore, the expression of miR-342 in CRC tissues inversely correlated with both FOXM1 and FOXQ1 expression. In-vitro functional analysis revealed that suppression of FOXM1 or overexpression of miR-342 inhibited CRC cell proliferation, while FOXM1 and FOXQ1 knockdown or miR-342 overexpression suppressed migration and invasion capacity of CRC cell lines. The stable overexpression of miR-342 reduced FOXM1 and FOXQ1 expression and significantly inhibited tumor growth in CRC xenografts in nude mice. CONCLUSIONS: FOXM1 and FOXQ1 promote cancer progression and act as potential prognostic factors in colon cancer. The downregulation of miR-342 in tumor tissues may contribute to the CRC development through inhibition of FOXM1 and FOXQ1 expression, suggesting that miR-342 could be used as a therapeutic target in colorectal cancer. Citation Format: Wenhao Weng, Yoshinaga Okugawa, Shusuke Toden, Ajay Goel. FOXM1 and FOXQ1 are novel oncogenes in colorectal cancer and can be therapeutically targeted through miRNA-based therapeutics. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1125.

Identification of differential proteins in colorectal cancer cells treated with caffeic acid phenethyl ester.

To investigate the molecular mechanisms of the anti-cancer activity of caffeic acid phenethyl ester (CAPE). Protein profiles of human colorectal cancer SW480 cells treated with or without CAPE were analysed using a two-dimensional (2D) electrophoresis gel-based proteomics approach. After electrophoresis, the gels were stained with Coomassie brilliant blue R-250. Digital images were taken with a GS-800 Calibrated Densitometer, and image analysis was performed using PDQuest 2-D Analysis software. The altered proteins following CAPE treatment were further identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry following a database search. The identified proteins were validated by Western blot and immunofluorescence assay. CAPE induced human colorectal cancer cell apoptosis. Four up-regulated proteins and seven down-regulated proteins in colorectal cancer cells treated with CAPE were found. The identified down-regulated proteins in CAPE-treated colorectal cancer cells were Triosephosphate Isomerase (Tim), Proteasome subunit alpha 4 (PSMA4) protein, Guanine nucleotide binding protein beta, Phosphoserine aminotransferase 1 (PSAT1), PSMA1, Myosin XVIIIB and Tryptophanyl-tRNA synthetase. Notably, CAPE treatment led to the down-regulation of PSAT1 and PSMA1, two proteins that have been implicated in tumorigenesis. The identified up-regulated proteins were Annexin A4, glyceraldehyde-3-phosphate dehydrogenase, Glucosamine-6-phosphate deaminase 1 (GNPDA1), and Glutathione peroxidase (GPX-1). Based on high match scores and potential role in cell growth control, PSMA1, PSAT1, GNPDA1 and GPX-1 were further validated by Western blotting and immunofluorescence assay. PSMA1 and PSAT1 were down-regulated, while GNPDA1 and GPX-1 were up-regulated in CAPE-treated colorectal cancer cells. These differentiated proteins in colorectal cancer cells following CAPE treatment, may be potential molecular targets of CAPE and involved in the anti-cancer effect of CAPE.

Ouabain-Induced Alterations of the Apical Junctional Complex Involve α1 and β1 Na,K-ATPase Downregulation and ERK1/2 Activation Independent of Caveolae in Colorectal Cancer Cells

Studies have reported that Na,K-ATPase interacts with E-cadherin to stabilize (AJs) and regulate the expression of claudins, the main proteins present in the tight junction (TJ) in epithelial cells containing caveolae. However, the role of this ATPase in the regulation of the AJ and TJ proteins in colorectal cancer cells as well as the molecular events underlying this event in a caveolae-independent system remain undefined. In the present study, we used ouabain, a classic drug known to inhibit Na,K-ATPase, and Caco-2 cells, which are a well-established human colorectal cancer model that does not exhibit caveolae. We demonstrated that ouabain treatment resulted in a reduction of the β1 Na,K-ATPase protein and cell redistribution of the AJ proteins E-cadherin and β-catenin, as well as the α1 Na,K-ATPase subunit. Furthermore, ouabain increased claudin-3 protein levels, impaired the TJ barrier function and increased cell viability and proliferation during the early stages of treatment. Additionally, the observed ouabain-induced events were dependent on the activation of ERK1/2 signaling; but in contrast to previous studies, this signaling cascade was caveolae-independent. In conclusion, our findings strongly suggest that α1 and β1 Na,K-ATPase downregulation and ERK1/2 activation induced by ouabain are interlinked events that play an important role during cell-cell adhesion loss, which is an important step during the tumor progression of colorectal carcinomas.

Nimbolide, a Limonoid Triterpene, Inhibits Growth of Human Colorectal Cancer Xenografts by Suppressing the Proinflammatory Microenvironment

Extensive research over the past decade has revealed that the proinflammatory microenvironment plays a critical role in the development of colorectal cancer. Whether nimbolide, a limonoid triterpene, can inhibit the growth of colorectal cancer was investigated in the present study. The effect of nimbolide on proliferation of colorectal cancer cell lines was examined by MTT assay, apoptosis by caspase activation and poly-ADP ribose polymerase cleavage, NF-κB activation by DNA-binding assay, and protein expression by Western blotting. The effect of nimbolide on the tumor growth in vivo was examined in colorectal cancer xenografts in a nude mouse model. Nimbolide inhibited proliferation, induced apoptosis, and suppressed NF-κB activation and NF-κB-regulated tumorigenic proteins in colorectal cancer cells. The suppression of NF-κB activation by nimbolide was caused by sequential inhibition of IκB kinase (IKK) activation, IκBα phosphorylation, and p65 nuclear translocation. Furthermore, the effect of nimbolide on IKK activity was found to be direct. In vivo, nimbolide (at 5 and 20 mg/kg body weight), injected intraperitoneally after tumor inoculation, significantly decreased the volume of colorectal cancer xenografts. The limonoid-treated xenografts exhibited significant downregulation in the expression of proteins involved in tumor cell survival (Bcl-2, Bcl-xL, c-IAP-1, survivin, and Mcl-1), proliferation (c-Myc and cyclin D1), invasion (MMP-9, ICAM-1), metastasis (CXCR4), and angiogenesis (VEGF). The limonoid was found to be bioavailable in the blood plasma and tumor tissues of treated mice. Our studies provide evidence that nimbolide can suppress the growth of human colorectal cancer through modulation of the proinflammatory microenvironment.

961 Toll-Like Receptor 4 Activation May Promote Development of Esophageal Adenocarcinoma in Barrett's Esophagus Through Induction of COX2

Background: Limited studies indicate that VEGF expression is upregulated in colorectal cancer (CRC). Since normal colonic epithelial cells have minimal or no expression of VEGF, increased VEGF in CRC represents its aberrant expression. The mechanisms of this phenomenon are not explained. Importins mediate in some cells (e.g., HeLa) the nuclear transport of transcription factors and signaling proteins that potentially can activate the VEGF gene. We hypothesized that upregulation of importins α and/or β is the underlying mechanism for the aberrant VEGF expression in CRC and cell proliferation by facilitating the nuclear transport of transcription factors (P-STAT3 and P-CREB) and P-MAPK/Erk1/2, respectively. Methods: We used: (1) human colon tissue arrays (CRC and normal specimens, n=60), and (2) human CRC cell lines HCT116 & HT29 and normal colonic epithelial cells NCM356 & NCM460 cultured in nutrient media. Studies: 1) Expressions of VEGF, its receptors (VEGFR1 & VEGFR2), importins, P-CREB, P-STAT3 and P-MAPK/Erk1/2 proteins in CRC cells in tissue array, and in cultured CRC and normal epithelial cells by immunostaining and/or western blotting; 2) In cultured CRC and normal epithelial cells: a) downregulation of importins with specific siRNAs, b) expression of VEGF mRNA by real time RTPCR, c) cell proliferation by BrdU assay, d) apoptosis by TUNEL staining.Results: Expression of VEGF and its receptor was aberrant and was significantly increased by 2-4 fold (P 80% (p<0.001). Conclusions: 1) Expression of VEGF and its receptors is upregulated in CRC cells, 2) Aberrant VEGF expression in CRC cells is dependent on upregulation of importin and is mediated by increased nuclear transport of P-CREB and P-STAT3, which bind to and activate VEGF gene promoter, 3) Increased expression of VEGF and its receptors in CRC cells suggests a direct regulation of their growth by VEGF through an autocrine mechanism.

960 Familial Clustering of Barrett's Esophagus and Esophageal Adenocarcinoma in the Netherlands

Background: Limited studies indicate that VEGF expression is upregulated in colorectal cancer (CRC). Since normal colonic epithelial cells have minimal or no expression of VEGF, increased VEGF in CRC represents its aberrant expression. The mechanisms of this phenomenon are not explained. Importins mediate in some cells (e.g., HeLa) the nuclear transport of transcription factors and signaling proteins that potentially can activate the VEGF gene. We hypothesized that upregulation of importins α and/or β is the underlying mechanism for the aberrant VEGF expression in CRC and cell proliferation by facilitating the nuclear transport of transcription factors (P-STAT3 and P-CREB) and P-MAPK/Erk1/2, respectively. Methods: We used: (1) human colon tissue arrays (CRC and normal specimens, n=60), and (2) human CRC cell lines HCT116 & HT29 and normal colonic epithelial cells NCM356 & NCM460 cultured in nutrient media. Studies: 1) Expressions of VEGF, its receptors (VEGFR1 & VEGFR2), importins, P-CREB, P-STAT3 and P-MAPK/Erk1/2 proteins in CRC cells in tissue array, and in cultured CRC and normal epithelial cells by immunostaining and/or western blotting; 2) In cultured CRC and normal epithelial cells: a) downregulation of importins with specific siRNAs, b) expression of VEGF mRNA by real time RTPCR, c) cell proliferation by BrdU assay, d) apoptosis by TUNEL staining.Results: Expression of VEGF and its receptor was aberrant and was significantly increased by 2-4 fold (P 80% (p<0.001). Conclusions: 1) Expression of VEGF and its receptors is upregulated in CRC cells, 2) Aberrant VEGF expression in CRC cells is dependent on upregulation of importin and is mediated by increased nuclear transport of P-CREB and P-STAT3, which bind to and activate VEGF gene promoter, 3) Increased expression of VEGF and its receptors in CRC cells suggests a direct regulation of their growth by VEGF through an autocrine mechanism.

Effect of (−)‐epigallocatechin gallate on cyclin D1 down‐regulation at the post‐translational level

The potential anti‐carcinogenic effect of green tea catechins has been well documented, especially for one of the major components, (−)‐epigallocatechin gallate (EGCG). Cyclin D1, an oncogenic protein, has been shown to be up‐regulated in various cancer cells, including colorectal cancer. It has been reported that EGCG inhibited cyclin D1 at the transcriptional and translational levels; however, the detailed mechanisms by which EGCG suppresses cyclin D1 expression have not been elucidated. Our objective was to elucidate the molecular mechanism(s) and biological consequence of EGCG‐induced cyclin D1 down‐regulation in colorectal cancer cells. We examined the effect of EGCG on the expression of cell growth related proteins in colorectal cancer cells. As a result, we found that EGCG inhibited cell growth and induced cell cycle arrest and apoptosis. The expression of cylin D1 and D3, but not cyclin E was down‐regulated, while p21 was up‐regulated in the presence of EGCG. Moreover, the cyclin D1 down‐regulation by EGCG was mediated by the 26S proteasome pathway dependent of glycogen synthase kinase‐3 beta (GSK‐3β). Collectively, EGCG facilitates the proteasomal degradation of cyclin D1, which could, at least in part, be responsible for the anti‐cancer effect of EGCG in colorectal cancer.Supported by: NIH R01CA108975 and The University of Tennessee Center of Excellence in Livestock Diseases and Human Health grantGrant Funding Source: NIH R01CA108975

Expressions of two adenomatous polyposis coli and E-cadherin proteins on human colorectal cancers.

Mutations in the adenomatous polyposis coli (APC) gene contribute to the progression of colorectal tumorigenesis. Despite the importance, few studies regarding the localization of this protein on surgically resected human colorectal cancer specimens using immunohistochemistry have been reported so far because of the unavailability of the antibodies for this use. The goal of this study has been to provide the APC protein expression and to validate the APC molecular studies. We took advantage of an immunohistochemistry procedure of applying the unique detergent-mediated antigen retrieval technique to frozen sections and examined the expressions of one amino (N)-terminal (AC4) and one carboxy (C)-terminal APC antibody (HG2). Further, we compared the stainings of APC antibodies with those of the E-cadherin antibody using a quantitative image analysis. E-cadherin is a critical morphogenetic regulator during embryogenesis and recent evidence strongly suggests that downregulation of E-cadherin expression in cancers is associated with a high rate of invasion and metastasis. The analysis indicated statistically that normal epithelia showed stronger staining than cancer cells ( P<0.05). Further, in normal epithelia, the amino (N)-terminal APC antibody (AC4) showed a positive correlation with another carboxy (C)-terminal APC antibody (HG2). E-cadherin showed no positive correlation with other APCs in either the normal epithelia or cancer cells. This study verified reduced expressions of APCs and E-cadherin proteins in colorectal cancer cells. This suggests that the normal APC and E-cadherin protein expressions in benign epithelium are progressively and independently lost in the sporadic colorectal cancers.