Effect of RSK4 on biological characteristics of colorectal cancer

Background

This study aimed to investigate the expression of P90 Ribosomal Protein S6 kinase 4 (RSK4) in colorectal cancer cells and its biological function.

Methods

We selected early SW480 and HCT116 colorectal cancer cell lines, using Lipofectamine? 2000 transfection reagent carrying RSK4 gene transfected into cells to establish the colorectal cancer cell lines with high expression of RSK4. RT-PCR and western blot (WB) analysis confirmed RSK4 expression in SW480 and HCT116 cancer cell lines. We used methylthiazoltetrazolium (MTT) assay and flow cytometry to detect the proliferation of colorectal cancer cells. After transfection of RSK4, the effect of RSK4 on the RNA levels associated with epithelial–mesenchymal transition (EMT) of colorectal cancer cells was analyzed by real-time fluorescence quantitative PCR and the expression of EMT-related protein was detected by WB analysis.

Results

After transfection of RSK4 overexpression, the MTT assay detected that RSK4 could significantly inhibit the growth of colorectal cancer cells in vitro; flow cytometry detected that S-phase cells decreased significantly, and G0/1 cells increased significantly (P?<?0.05). The invasion ability of SW480 and HCT116 cells transfected with RSK4 was markedly lower than that in the control group, and the difference was statistically significant (P?<?0.05). Fluorescent quantitative PCR and WB analysis showed that the expression of EMT-associated molecular E-cadherin was remarkably increased and the expression of Snail was significantly decreased (P?<?0.01).

Conclusion

RSK4 gene in colorectal cancer cell lines with low expression of RSK4 after transfection can inhibit the growth and invasion of tumor cells. RSK4 gene may inhibit EMT and inhibit metastasis of colorectal cancer cells, may be a potential tumor suppressor gene and inhibit tumor distant metastasis, and may provide the biological basis for new therapeutic targets.

     

Lgr5 Methylation in Cancer Stem Cell Differentiation and Prognosis-Prediction in Colorectal Cancer

Objective

Leucine-rich-repeat-containing G-protein-coupled receptor 5 (lgr5) is a candidate marker for colorectal cancer stem cells (CSC). In the current study, we investigated the methylation status within thelgr5 promoter and evaluated its relationship with CSC differentiation, prognosis for colorectal cancer, and its clinicopathological features.

Methods

The methylation status within Lgr5 promoter was detected with a methylation-specific PCR in six colorectal cancer cell lines as well as 169 primary colorectal tumor tissues. Differentiation of CSC was examined with immunofluorescence and immunocytochemistry. Down-regulation of lgr5 was achieved with gene-specific siRNA. The associations between lgr5 methylation and the clinicopathological features as well as survival of patients were analyzed with statistical methods.

Results

The lgr5 promoter was methylated to different degrees for the six colorectal cell lines examined, with complete methylation observed in HCT116 cells in which the lgr5 expression was partially recovered following DAC treatment. The stem-cell sphere formation from HCT116 cells was accompanied by increasing methylation within the lgr5 promoter and decreasing expression of lgr5. Knocking down lgr5 by siRNA also led to stem-cell spheres formation. Among primary colorectal tumors, 40% (67/169) were positive for lgr5 methylation, while none of the normal colon tissues were positive for lgr5 methylation. Furthermore, lgr5 methylation significantly associated with higher tumor grade, and negative distant metastasis (p < 0.05), as well as better prognosis (p = 0.001) in patients with colorectal cancer.

Conclusions

Our data suggests that lgr5 methylation, through the regulation of lgr5 expression and colorectal CSC differentiation, may constitute a novel prognostic marker for colorectal cancer patients.     

Tazarotene-induced gene 1 inhibits prostaglandin E2-stimulated HCT116 colon cancer cell growth

Background  

The tazarotene-induced gene 1 (TIG1) is a putative tumor suppressor gene. We have recently demonstrated both TIG1A and TIG1B isoforms inhibited cell growth and induced the expression of G protein-coupled receptor kinase 5 (GRK5) in colon cancer cells. Because elevated prostaglandin E2 (PGE2) signaling plays a significant role in colorectal carcinogenesis, the objective of this study was to explore the effect of TIG1 on PGE2-induced cellular proliferation and signaling in colon cancer cells.     

Sub-lethal irradiation of human colorectal tumor cells imparts enhanced and sustained susceptibility to multiple death receptor signaling pathways

Background

Death receptors (DR) of the TNF family function as anti-tumor immune effector molecules. Tumor cells, however, often exhibit DR-signaling resistance. Previous studies indicate that radiation can modify gene expression within tumor cells and increase tumor cell sensitivity to immune attack. The aim of this study is to investigate the synergistic effect of sub-lethal doses of ionizing radiation in sensitizing colorectal carcinoma cells to death receptor-mediated apoptosis.

Methodology/Principal Findings

The ability of radiation to modulate the expression of multiple death receptors (Fas/CD95, TRAILR1/DR4, TRAILR2/DR5, TNF-R1 and LTβR) was examined in colorectal tumor cells. The functional significance of sub-lethal doses of radiation in enhancing tumor cell susceptibility to DR-induced apoptosis was determined by in vitro functional sensitivity assays. The longevity of these changes and the underlying molecular mechanism of irradiation in sensitizing diverse colorectal carcinoma cells to death receptor-mediated apoptosis were also examined. We found that radiation increased surface expression of Fas, DR4 and DR5 but not LTβR or TNF-R1 in these cells. Increased expression of DRs was observed 2 days post-irradiation and remained elevated 7-days post irradiation. Sub-lethal tumor cell irradiation alone exhibited minimal cell death, but effectively sensitized three of three colorectal carcinoma cells to both TRAIL and Fas-induced apoptosis, but not LTβR-induced death. Furthermore, radiation-enhanced Fas and TRAIL-induced cell death lasted as long as 5-days post-irradiation. Specific analysis of intracellular sensitizers to apoptosis indicated that while radiation did reduce Bcl-X_L and c-FLIP protein expression, this reduction did not correlate with the radiation-enhanced sensitivity to Fas and/or TRAIL mediated apoptosis among the three cell types.

Conclusions/Significance

Irradiation of tumor cells can overcome Fas and TRAIL resistance that is long lasting. Overall, results of these investigations suggest that non-lethal doses of radiation can be used to make human tumors more amenable to attack by anti-tumor effector molecules and cells.     

The PTEN phosphatase controls intestinal epithelial cell polarity and barrier function: role in colorectal cancer progression

Background

The PTEN phosphatase acts on phosphatidylinositol 3,4,5-triphosphates resulting from phosphatidylinositol 3-kinase (PI3K) activation. PTEN expression has been shown to be decreased in colorectal cancer. Little is known however as to the specific cellular role of PTEN in human intestinal epithelial cells. The aim of this study was to investigate the role of PTEN in human colorectal cancer cells.

Methodology/Principal Findings

Caco-2/15, HCT116 and CT26 cells were infected with recombinant lentiviruses expressing a shRNA specifically designed to knock-down PTEN. The impact of PTEN downregulation was analyzed on cell polarization and differentiation, intercellular junction integrity (expression of cell-cell adhesion proteins, barrier function), migration (wound assay), invasion (matrigel-coated transwells) and on tumor and metastasis formation in mice. Electron microscopy analysis showed that lentiviral infection of PTEN shRNA significantly inhibited Caco-2/15 cell polarization, functional differentiation and brush border development. A strong reduction in claudin 1, 3, 4 and 8 was also observed as well as a decrease in transepithelial resistance. Loss of PTEN expression increased the spreading, migration and invasion capacities of colorectal cancer cells in vitro. PTEN downregulation also increased tumor size following subcutaneous injection of colorectal cancer cells in nude mice. Finally, loss of PTEN expression in HCT116 and CT26, but not in Caco-2/15, led to an increase in their metastatic potential following tail-vein injections in mice.

Conclusions/Significance

Altogether, these results indicate that PTEN controls cellular polarity, establishment of cell-cell junctions, paracellular permeability, migration and tumorigenic/metastatic potential of human colorectal cancer cells.     

MicroRNA-211 expression promotes colorectal cancer cell growth in vitro and in vivo by targeting tumor suppressor CHD5

Background

Chromodomain-helicase-DNA-binding protein 5 (CHD5) is a newly identified tumor suppressor that is frequently downregulated in a variety of human cancers. Our previous work revealed that the low expression of CHD5 in colorectal cancer is correlated with CHD5 promoter CpG island hypermethylation. In this study, we investigated the effect of microRNA-211 (miR-211)-regulated CHD5 expression on colorectal tumorigenesis.

Methodology/Principal Findings

miR-211 was predicted to target CHD5 by TargetScan software analysis. A stably expressing exogenous miR-211 colorectal cancer cell line (HCT-116^miR-211) was generated using lentiviral transduction and used as a model for in vitro and in vivo studies. The expression level of miR-211 in HCT-116^miR-211 cells was upregulated by 16-fold compared to vector control cells (HCT-116^vector). Exogenous miR-211 directly binds to the 3′-untranslated region (3′-UTR) of CHD5 mRNA, resulting in a 50% decrease in CHD5 protein level in HCT-116^miR-211 cells. The levels of cell proliferation, tumor growth, and cell migration of HCT-116^miR-211 cells were significantly higher than HCT-116^vector cells under both in vitro and in vivo conditions, as determined using the methods of MTT, colony formation, flow cytometry, scratch assay, and tumor xenografts, respectively. In addition, we found that enforced expression of miR-211 in HCT-116 cells was able to alter p53 pathway-associated regulatory proteins, such as MDM2, Bcl-2, Bcl-xL, and Bax.

Conclusion/Significance

Our results demonstrate that CHD5 is a direct target of miR-211 regulation. Enforced expression of miR-211 promotes tumor cell growth at least in part by downregulating the expression level of the CHD5 tumor suppressor. Our results provide a better understanding of the association of between miR-211-regulated CHD5 expression and CHD5 function in colorectal tumorigenesis.     

Anti-tumor activity of a novel monoclonal antibody, NPC-1C, optimized for recognition of tumor antigen MUC5AC variant in preclinical models

Purpose

NPC-1C is a chimeric immunoglobulin IgG1 developed from antigen tested in the Hollinshead tumor vaccine trials that recognizes an immunogenic MUC5AC-related tumor-associated antigen. In this article, we describe the pre-clinical characterization of this antibody that is currently being tested in human clinical trials.

Experimental design

The specificity of NPC-1C for pancreatic and colorectal cancer cell lines was tested by flow cytometry assays and immunohistochemical staining. Antibody-dependent cell cytotoxicity was measured using a tumor cell line lysis assay. Anti-tumor efficacy and biodistribution were assessed in nude mice bearing human pancreatic tumor xenografts.

Results

Human tumor cell binding measured by flow cytometry ranged from 52 to 94 % of cells stained positive with NPC-1C in three colorectal and one pancreatic cell lines, while IHC demonstrated staining of 43 % of colon cancers and 48 % of pancreatic cancer tissues, with little or no cross-reactivity of NPC-1C with normal colon or pancreas tissues. In vitro NPC-1C-mediated tumor cell killing occurred in a median of 44.5 % of four colorectal and three pancreatic tumor cell lines. In vivo anti-tumor efficacy in a human pancreatic CFPAC-1 tumor xenograft model was demonstrated with a twofold to threefold reduction in tumor growth in the NPC-1C-treated mice compared to saline and human IgG controls. Pharmacodynamic studies indicate NPC-1C localizes in antigen-positive tumors and has minimal uptake in normal mouse tissues.

Conclusions

NPC-1C, a chimeric monoclonal antibody that reacts with a MUC5AC-related antigen expressed by pancreatic and colorectal tumor tissues, has promising preclinical activity in pancreatic and colorectal adenocarcinoma.     

Overexpression of the Interferon regulatory factor 4-binding protein in human colorectal cancer and its clinical significance

Background: IFN regulatory factor 4-binding protein (IBP) is a novel type of activator of Rho GTPases. It has been linked with differentiation and apoptosis of lymphocytes, but its function in oncogenesis remains unclear. Here we studied the expression of endogenous IBP in four human colorectal cancer cell lines, normal, adenoma and tumor colorectal tissues. Methods: Molecular (Western blot and RT-PCR), and confocal analyses were used to investigate IBP expression in human colorectal cancer cell lines. Matched normal and tumor tissue sections of 63 patients and 15 adenoma tissue sections were analyzed for IBP expression by immunohistochemistry (IHC). Results: IBP was ubiquitely expressed in human colorectal cancer cell lines. The expression of IBP can be detected at both the mRNA and protein level in SW480, SW620 and HT29 cells. Clinically, IBP were elevated in human colorectal cancer specimens in comparison to normal colorectal tissues. Substantial high expression of IBP was observed in colorectal cancer tissues (67%), whereas corresponding normal tissues and 15 adenoma tissues showed consistently absent immunoreactivity of IBP. Moreover, IBP expression is correlated with the differentiation level of colorectal cancer cells (p < 0.05) and clinical stage of patients (p < 0.01). Conclusions: Our data show, for the first time, a dysregulated expression of IBP in human colorectal cancer, offering new perspectives for its role in cancer development and progression. IBP may be a novel tumor marker and a therapeutic target for colorectal cancer.     

Persistent left superior vena cava: Review of the literature, clinical implications, and relevance of alterations in thoracic central venous anatomy as pertaining to the general principles of central venous access device placement and venography in cancer patients

Introduction

Bcl-xL, an important member of anti-apoptotic Bcl-2 family, plays critical roles in tumor progression and development. Previously, we have reported that overexpression of Bcl-xL was correlated with prognosis of colorectal cancer (CRC) patients. The aim of this study was to investigate the association of Bcl-xL expression with invasion and radiosensitivity of human CRC cells.

Methods

RT-PCR and Western blot assays were performed to determine the expression of Bcl-xL mRNA and protein in CRC cells and normal human intestinal epithelial cell line. Then, adenovirus-mediated RNA interference technique was employed to inhibit the expression of Bcl-xL gene in CRC cells. The proliferation of CRC cells was analyzed by MTT and colony formation assay. The migration and invasion of CRC cells was determined by wound-healing and tranwell invasion assays. Additionally, the in vitro and in vivo radiosensitivity of CRC cells was determined by clonogenic cell survival assay and murine xnograft model, respectively.

Results

The levels of Bcl-xL mRNA and protein expression were significantly higher in human CRC cells than in normal human intestinal epithelial cell line. Ad/shBcl-xL could significantly reduce the expression of Bcl-xL protein in CRC cells. Also, we showed that adenovirus-mediated siRNA targeting Bcl-xL could significantly inhibit proliferation and colony formation of CRC cells. Ad/shBcl-xL could significantly suppress migration and invasion of CRC cells. Moreover, Ad/shBcl-xL could enhance in vitro and in vivo radiosensitivity of CRC cells by increasing caspase-dependent apoptosis.

Conclusions

Targeting Bcl-xL will be a promising strategy to inhibit the metastatic potential and reverse the radioresistance of human CRC.     

Identification of the sAPRIL Binding Peptide and Its Growth Inhibition Effects in the Colorectal Cancer Cells

Background

A proliferation-inducing ligand (APRIL) is a member of the tumor necrosis factor (TNF) super family. It binds to its specific receptors and is involved in multiple processes during tumorigenesis and tumor cells proliferation. High levels of APRIL expression are closely correlated to the growth, metastasis, and 5-FU drug resistance of colorectal cancer. The aim of this study was to identify a specific APRIL binding peptide (BP) able to block APRIL activity that could be used as a potential treatment for colorectal cancer.

Methods

A phage display library was used to identify peptides that bound selectively to soluble recombinant human APRIL (sAPRIL). The peptides with the highest binding affinity for sAPRIL were identified using ELISA. The effects of sAPRIL-BP on cell proliferation and cell cycle/apoptosis in vitro were evaluated using the CCK-8 assay and flow cytometry, respectively. An in vivo mouse model of colorectal cancer was used to determine the anti-tumor efficacy of the sAPRIL-BP.

Results

Three candidate peptides were characterized from eight phage clones with high binding affinity for sAPRIL. The peptide with the highest affinity was selected for further characterization. The identified sAPRIL-BP suppressed tumor cell proliferation and cell cycle progression in LOVO cells in a dose-dependent manner. In vivo in a mouse colorectal challenge model, the sAPRIL-BP reduced the growth of tumor xenografts in nude mice by inhibiting proliferation and inducing apoptosis intratumorally. Moreover, in an in vivo metastasis model, sAPRIL-BP reduced liver metastasis of colorectal cancer cells.

Conclusions

sAPRIL-BP significantly suppressed tumor growth in vitro and in vivo and might be a candidate for treating colorectal cancers that express high levels of APRIL.     

Different cDNA microarray patterns of gene expression reflecting changes during metastatic progression in adenoid cystic carcinoma

Background  

The metastatic ability of tumor cells is determined by level of expression of specific genes that may be identified with the aid of cDNA microarray containing thousands of genes and can be used to establish the expression profile of disease related genes in complex biological system.     

Dissection of a metastatic gene expression signature into distinct components

Background  

Metastasis, the process whereby cancer cells spread, is in part caused by an incompletely understood interplay between cancer cells and the surrounding stroma. Gene expression studies typically analyze samples containing tumor cells and stroma. Samples with less than 50% tumor cells are generally excluded, thereby reducing the number of patients that can benefit from clinically relevant signatures.     

Up-Regulation of 91H Promotes Tumor Metastasis and Predicts Poor Prognosis for Patients with Colorectal Cancer

Background

Long noncoding RNAs (lncRNAs) play widespread roles in gene regulation and cellular processes. However, the functional roles of lncRNAs in colorectal cancer (CRC) are not yet well elucidated. The aim of the present study was to measure the levels of lncRNA 91H expression in CRC and evaluate its clinical significance and biological roles in the development and progression of CRC.

Methods

91H expression and copy number variation (CNV) were measured in 72 CRC tumor tissues and adjacent normal tissues by real-time PCR. The biological roles of 91H were evaluated by MTT, scratch wound assay, migration and invasion assays, and flow cytometry.

Results

91H was significantly overexpressed in cancerous tissue and CRC cell lines compared with adjacent normal tissue and a normal human intestinal epithelial cell line. Moreover, 91H overexpression was closely associated with distant metastasis and poor prognosis in patients with CRC, except for CNV of 91H. Multivariate analysis indicated that 91H expression was an independent prognostic indicator, as well as distant metastasis. Our in vitro data indicated that knockdown of 91H inhibited the proliferation, migration, and invasiveness of CRC cells.

Conclusions

91H played an important role in the molecular etiology of CRC and might be regarded as a novel prognosis indicator in patients with CRC.     

Normal Fibroblasts Induce E-Cadherin Loss and Increase Lymph Node Metastasis in Gastric Cancer

Background

A tumor is considered a heterogeneous complex in a three-dimensional environment that is flush with pathophysiological and biomechanical signals. Cell-stroma interactions guide the development and generation of tumors. Here, we evaluate the contributions of normal fibroblasts to gastric cancer.

Methodology/Principal Findings

By coculturing normal fibroblasts in monolayers of BGC-823 gastric cancer cells, tumor cells sporadically developed short, spindle-like morphological characteristics and demonstrated enhanced proliferation and invasive potential. Furthermore, the transformed tumor cells demonstrated decreased tumor formation and increased lymphomatic and intestinal metastatic potential. Non-transformed BGC-823 cells, in contrast, demonstrated primary tumor formation and delayed intestinal and lymph node invasion. We also observed E-cadherin loss and the upregulation of vimentin expression in the transformed tumor cells, which suggested that the increase in metastasis was induced by epithelial-to-mesenchymal transition.

Conclusion

Collectively, our data indicated that normal fibroblasts sufficiently induce epithelial-to-mesenchymal transition in cancer cells, thereby leading to metastasis.