An integrative network biology analysis identifies miR-508-3p as the determinant and a prognosis biomarker of the mesenchymal subtype ovarian cancer
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ABSTRACT: Recently, several research groups have identified the mesenchymal subtype of serous OvCa on the basis of transcriptome data and its potential correlation with poor prognosis. We set out to define the regulatory mechanisms underlying the distinct gene expression profiles of serous OvCa using a network-based approach involving multiple molecular modalities such as gene expression and microRNA (miR) expression. Our study demonstrated that the mir-508-3p presented as the most powerful determinant of mesenchymal subtype-specific gene expression, tuning the majority of genes differentially expressed in the poor prognosis subtype, including genes associated with the epithelial–mesenchymal transition (EMT) program. Consequent functional experiments illustrate that miR-508-3p inhibition promoted EMT process, in vitro cell migration and invasion, and in vivo cancer metastasis.
Project description:Unsupervised classification of gene expression profiles has resulted in the identification of biologically and clinically distinct colon cancer subtypes (CCSs). The subtype that associates with poor clinical outcome displays a mesenchymal gene expression profile. No driver mutation has been identified for this category and patients are heterogeneous with regard to commonly used clinical markers. Here we report a regulatory network consisting of the miR-200 family members that tunes the majority of genes differentially expressed in the poor prognosis CCS, including genes involved in the epithelial-mesenchymal transition (EMT) process. Our data indicate that the epigenetic silencing of the miR-200 family by promoter methylation is identifying the mesenchymal CCS and is predictive of disease-free survival in this malignancy. We demonstrate that the molecular features of poor prognosis colon cancer - expression of EMT-associated genes and miR-200 promoter methylation - can already be installed at the premalignant stage, suggesting a highly malignant potential of specific colon cancer precursor lesions. Four colorectal cancer cell lines that display methylated miR-200 loci have been used to overexpress miR-200 family members from both loci separatedly or simultaneously.
Project description:Unsupervised classification of gene expression profiles has resulted in the identification of biologically and clinically distinct colon cancer subtypes (CCSs). The subtype that associates with poor clinical outcome displays a mesenchymal gene expression profile. No driver mutation has been identified for this category and patients are heterogeneous with regard to commonly used clinical markers. Here we report a regulatory network consisting of the miR-200 family members that tunes the majority of genes differentially expressed in the poor prognosis CCS, including genes involved in the epithelial-mesenchymal transition (EMT) process. Our data indicate that the epigenetic silencing of the miR-200 family by promoter methylation is identifying the mesenchymal CCS and is predictive of disease-free survival in this malignancy. We demonstrate that the molecular features of poor prognosis colon cancer - expression of EMT-associated genes and miR-200 promoter methylation - can already be installed at the premalignant stage, suggesting a highly malignant potential of specific colon cancer precursor lesions.
Project description:Increasing evidence suggests that global downregulation of miRNA expression is a hallmark of human cancer, potentially due to defects in the miRNA processing machinery. In this study, we found that the protein expression of Argonaute 2 (AGO2), a key regulator of miRNA processing, was downregulated in colorectal cancer (CRC) tissues, which was also consistent with the findings of the Clinical Proteomic Tumor Analysis Consortium (CPTAC). Furthermore, the correlation between the levels of AGO2 and epithelial-mesenchymal transition (EMT) markers (E-cadherin and vimentin) indicated that reduced levels of AGO2 promoted EMT in CRC. Low expression of AGO2 was an indicator of a poor prognosis among CRC patients. Knockdown of AGO2 in CRC cells promoted migration, invasion and metastasis formation in vitro and in vivo but had no influence on proliferation. To provide detailed insight into the regulatory roles of AGO2, we performed integrated transcriptomic, quantitative proteomic and microRNA sequencing (miRNA-seq) analyses of AGO2 knockdown cells and the corresponding wild-type cells and identified neuropilin 1 (NRP1) as a new substrate of AGO2 via miR-185-3p. Our data provided evidence that knockdown of AGO2 resulted in a reduction of miR-185-3p expression, leading to the upregulation of the expression of NRP1, which is a direct target of miR-185-3p, and elevated CRC cell metastatic capacity. Inhibition of NRP1 or treatment with a miR-185-3p mimic successfully rescued the phenotypes of impaired AGO2, which suggested that therapeutically targeting the AGO2/miR-185-3p/NRP1 axis may be a potential treatment approach for CRC.
Project description:An integrative network biology analysis identifies miR-508-3p as the determinant and a prognosis biomarker of the mesenchymal subtype ovarian cancer
Project description:This experiment captures expression over 60,000 well-annotated RefSeq human transcripts over RNA samples from SH-SY5Y neuroblastoma cells transfected with human and non-human primate microRNA mimic variants of miR-299-3p, miR-503-3p, miR-508-3p and miR-541-3p, as well as a RNA duplex negative control (C2 mimic, Dharmacon).
Project description:In many solid tumors, tissue of mesenchymal subtype is frequently associated with epithelial-mesenchymal transition (EMT) state, strong stromal infiltration, and poor prognosis. Emerging evidence from tumor ecosystem studies indicate that the stromal components, including cancer associated fibroblast (CAFs), actively participate in cancer growth and progression. Transcriptomic analysis of xenograft tissues provides a unique advantage in dissecting hallmark genes of tumor (human) or stromal (murine) origins.
Project description:A variety of human cancers demonstrate alterations in microRNA expression. We hypothesized that regulatory defects in microRNAs play a central early role in organizing the molecular changes involved in ovarian cancer (OvCa). Using both gene arrays and deep sequencing, we comprehensively profiled mRNA and microRNA expression, respectively, in human serous epithelial OvCa cell lines, serous tumors, and short-term primary cultures of normal ovarian surface epithelium (NOSE). We expected that over-expression of a specific microRNA would lead to lower expression of its mRNA targets, and under-expression of a specific microRNA would lead to higher expression of its target genes. Using our expression data in conjunction with established in silico algorithms, we found putative microRNA:mRNA functional pairs. Furthermore, gene expression profiles were taken of serous cultures having functional knockdown or over-expression of specific microRNAs of interest. Over-expression of mir-31 (found under-expressed in serous OvCa) resulted in down-regulation in vitro of a significant number of the in silico predicted mir-31 target genes. Keywords: two group comparison
Project description:Oxaliplatin (oxPt) resistance in colorectal cancers (CRC) is a major unsolved problem. Consequently, predictive markers and a better understanding of resistance mechanisms are urgently needed. To investigate if the recently identified predictive miR-625-3p is functionally involved in oxPt resistance, stable and inducible models of miR-625-3p dysregulation were analyzed. Ectopic expression of miR-625-3p in CRC cells led to increased resistance towards oxPt. The mitogen-activated protein kinase (MAPK) kinase 6 (MAP2K6/MKK6) – an activator of p38 MAPK - was identified as a functional target of miR-625-3p, and, in agreement, was down-regulated in patients not responding to oxPt therapy. The miR-625-3p resistance phenotype could be reversed by anti-miR-625-3p treatment and by ectopic expression of a miR-625-3p insensitive MAP2K6 variant. Transcriptome, proteome and phosphoproteome profiles revealed inactivation of MAP2K6-p38 signaling as a possible driving force behind oxPt resistance. We conclude that miR-625-3p induces oxPt resistance by abrogating MAP2K6-p38 regulated apoptosis and cell cycle control networks.
Project description:Using RNA-seq, we recently developed the 52-gene-based Oxford Classifier of Carcinoma of the Ovary (Oxford Classic, OxC) for molecular stratification of serous ovarian cancers (SOCs) based on the molecular profiles of their cell-of-origin in the fallopian tube epithelium. Here, we developed a 52-gene NanoString panel for the OxC to test the robustness of the classifier. We measured the expression of the 52 genes in an independent cohort of prospectively collected SOC samples (n = 150) from a homogenous cohort who were treated maximal debulking surgery and chemotherapy. We performed data mining of published expression profiles of SOCs and validated the classifier results on tissue arrays comprising 137 SOCs. We found evidence of profound non-genetic heterogeneity in SOC. ~20% of SOCs were classified as epithelial-mesenchymal-transition-high (EMT-high) tumors, that were associated with poor survival. This was independent of established prognostic factors such as tumor stage, tumor grade and residual disease after surgery (HR = 3.3, p = 0.02). Mining expression data of 593 patients revealed a significant association between the EMT scores of tumors and the estimated fraction of alternatively activated macrophages (M2) (p < 0.0001) suggesting a mechanistic link between immunosuppression and poor prognosis in EMT-high tumors. The OxC-defined EMT-high serous ovarian cancers carry particularly poor prognosis independent of established clinical parameters. These tumors are associated with high frequency of immunosuppressive macrophages suggesting a potential therapeutic target to improve clinical outcome.
Project description:Introduction: Ampullary cancer is a relatively rare entity and usually treated by pancreatoduodenectomy followed by adjuvant therapy. The intestinal subtype is associated with markedly improved prognosis after resection. Only few cell lines are available for in vitro studies of ampullary cancer and they have not been collectively characterized. Methods: We characterized available ampullary cancer cell lines by subtype maker expression, epithelial-mesenchymal transition (EMT) features, growth and invasion, drug sensitivity and response to cancer-associated fibroblast conditioned medium (CAF-CM). Results: On the basis of EMT features, subtype marker expression, growth, invasion and drug sensitivity three types of cell lines could be distinguished: mesenchymal-like, pancreatobiliary-like and intestinal-like. In response to CAF-CM, enhanced growth, EMT induction as well as suppression of intestinal differentiation markers were observed, but in a heterogenous pattern. Also proteomic analysis of the CAF response distinguished intestinal-like from other cell lines. Discussion: Most of the available AMPAC cell lines seem to reflect a poorly differentiated pancreatobiliary or mesenchymal-like phenotype, consistent with their origin. We suggest that the best cell line model for intestinal-like AMPAC is the SNU869 cell line, while others seem to reflect aggressive AMPAC subtypes.