Rapid testing of candidate oncogenes and tumour suppressor genes in signal transduction and neoplastic transformation.

The COSMIC database (version 94) lists 576 genes in the Cancer Gene Census which have a defined function as drivers of malignancy (oncogenes) or as tumour suppressors (Tier 1). In addition, there are 147 genes with similar functions, but which are less well characterised (Tier 2). Furthermore, next-generation sequencing projects in the context of precision oncology activities are constantly discovering new ones. Since cancer genes differ from their wild-type precursors in numerous molecular and biochemical properties and exert significant differential effects on downstream processes, simple assays that can uncover oncogenic or anti-oncogenic functionality are desirable and may precede more sophisticated analyses. We describe simple functional assays for PTPN11 (protein-tyrosine phosphatase, non-receptor-type 11)/SHP2 mutants, which are typically found in RASopathies and exhibit potential oncogenic activity. We have also designed a functional test for lysyl oxidase (LOX), a prototypical class II tumour suppressor gene whose loss of function may contribute to neoplastic transformation by RAS oncogenes. Moreover, we applied this test to analyse three co-regulated, RAS-responsive genes for transformation-suppressive activity. The integration of these tests into systems biology studies will contribute to a better understanding of cellular networks in cancer.

Reverse engineering a hierarchical regulatory network downstream of oncogenic KRAS.

RAS mutations are highly relevant for progression and therapy response of human tumours, but the genetic network that ultimately executes the oncogenic effects is poorly understood. Here, we used a reverse-engineering approach in an ovarian cancer model to reconstruct KRAS oncogene-dependent cytoplasmic and transcriptional networks from perturbation experiments based on gene silencing and pathway inhibitor treatments. We measured mRNA and protein levels in manipulated cells by microarray, RT-PCR and western blot analysis, respectively. The reconstructed model revealed complex interactions among the transcriptional and cytoplasmic components, some of which were confirmed by double pertubation experiments. Interestingly, the transcription factors decomposed into two hierarchically arranged groups. To validate the model predictions, we analysed growth parameters and transcriptional deregulation in the KRAS-transformed epithelial cells. As predicted by the model, we found two functional groups among the selected transcription factors. The experiments thus confirmed the predicted hierarchical transcription factor regulation and showed that the hierarchy manifests itself in downstream gene expression patterns and phenotype.

Systematic evaluation of the miRNA-ome and its downstream effects on mRNA expression identifies gastric cancer progression.

We investigated the differential expression of Dicer and Drosha, as well as that of microRNA (miRNA), in adjacent normal and tumour samples of patients with gastric cancer. The expression of Dicer and Drosha was studied by immunohistochemistry in 332 gastric cancers and correlated with clinico-pathological patient characteristics. Differential expression of miRNAs was studied using the Invitrogen NCode(™) Multi-Species miRNA Microarray Probe Set containing 857 mammalian probes in a test set of six primary gastric cancers (three with and three without lymph node metastases). Differential expression was validated by RT-PCR on an independent validation set of 20 patients with gastric cancer. Dicer and Drosha were differentially expressed in non-neoplastic and neoplastic gastric tissue. The expression of Drosha correlated with local tumour growth and was a significant independent prognosticator of patient survival. Twenty miRNAs were up- and two down-regulated in gastric carcinoma compared with non-neoplastic tissue. Six of these miRNAs separated node-positive from node-negative gastric cancers, ie miR-103, miR-21, miR-145, miR-106b, miR-146a, and miR-148a. Five miRNAs expressed differentially in node-positive cancers had conserved binding sites for mRNAs differentially expressed in the same set of tumour samples. Gastric cancer shows a complex derangement of the miRNA-ome, including Dicer and Drosha. These changes correlate independently with patient prognosis and probably influence local tumour growth and nodal spread.

Evaluation of targets for ovarian cancer gene silencing therapy: in vitro and in vivo approaches.

Ovarian cancer is the most lethal neoplasm of the female genital tract. Despite progress with chemotherapy, surgery and supportive care, the death rate remains extremely high. Gene silencing therapy represents a possible opportunity to advance the management of ovarian cancer patients. The concept of gene silencing therapy, which is based on RNA interference (RNAi) phenomenon, requires selection of targeted genes and development of a strategy for genetic drug development. Recently, plenty of research studies in ovarian cancer genetics have been published. Although they can be analyzed regarding candidate gene selection, the therapeutic effect of particular gene silencing can only be evaluated experimentally at this time. Obviously, the correct choice and application of a genetic drug delivery system determines the efficacy of gene silencing. Complexation of therapeutic nucleic acids with cationic polymers, cationic lipids, or their combination, represents a main strategy of non-virus-mediated delivery of genetic drug. Owing to a tendency of ovarian cancer to spread through the abdominal cavity, a delivery system should allow intraperitoneal mode of administration. Therefore, clinical application of RNAi may rely on a combination of biosciences and nanotechnology: in particular, identifying optimal small interfering RNAs (siRNAs) against optimal target genes and developing an efficient system for siRNA delivery into the cancer cells.

Target genes suitable for silencing approaches and protein product interference in ovarian epithelial cancer.

Gene expression profiling studies and conventional approaches for the identification of genes involved in the initiation and progression of ovarian cancer have identified a plethora of potential therapeutic targets. This review summarizes the targets that show promise for specific interference in cancer cells, groups them according to their subcellular localization and involved biological processes and discusses their impact on experimental, pre-clinical and clinical therapy.

Prognostic significance of Dicer expression in ovarian cancer-link to global microRNA changes and oestrogen receptor expression.

MicroRNA (miRNA) deregulation is a hallmark of human cancer. However, the mechanisms underlying miRNA alteration and the specific role of proteins involved in miRNA processing remains to be elucidated. Dicer is a key enzyme in the miRNA processing pathway that is essential for the production of mature miRNAs from their precursors. We tested the hypothesis that Dicer has biological and clinical relevance in ovarian cancer, using a range of methods including in vitro manipulation of Dicer expression. We observed down-regulation of Dicer in a subgroup of ovarian carcinomas, and found that decreased Dicer expression correlates significantly with reduced patient survival in serous cancers and advanced disease stages. Moreover, microarray and functional analysis suggest that reduced Dicer expression is connected with a global down-regulation of the microRNAome and with gene expression changes, particularly reduced expression of oestrogen receptor (ER) mRNA and protein in tumour tissue and in cell culture. Our data suggest a common mechanism for miRNAs changes by alterations in the basic machinery controlling miRNA biogenesis, of which Dicer is a central enzyme. These alterations of miRNA processing are of prognostic value and may play a role in the molecular pathogenesis of ovarian carcinoma and, possibly, other tumours. Knowledge of these molecular pathways may help toward new targeted therapeutic approaches for ovarian cancer.

A systems biological approach suggests that transcriptional feedback regulation by dual-specificity phosphatase 6 shapes extracellular signal-related kinase activity in RAS-transformed fibroblasts.

Mitogen-activated protein kinase (MAPK) signaling determines crucial cell fate decisions in most cell types, and mediates cellular transformation in many types of cancer. The activity of MAPK is controlled by reversible phosphorylation, and the quantitative characteristics of MAPK activation determine the cellular response. Many systems biological studies have analyzed the activation kinetics and the dose-response behavior of the MAPK signaling pathway. Here we investigate how the pathway activity is controlled by transcriptional feedback loops. Initially, we predict that MAPK signaling regulates phosphatases, by integrating promoter sequence data and ontology-based classification of gene function. From this, we deduce that MAPK signaling might be controlled by transcriptional negative feedback regulation via dual-specificity phosphatases (DUSPs), and implement a mathematical model to further test this hypothesis. Using time-resolved measurements of pathway activity and gene expression, we employ a model selection approach, and select DUSP6 as a highly likely candidate for shaping the activity of the MAPK pathway during cellular transformation caused by oncogenic RAS. Two predictions from the model were confirmed: first, feedback regulation requires that DUSP6 mRNA and protein are unstable; and second, the activation kinetics of MAPK are ultrasensitive. Taken together, an integrated systems biological approach reveals that transcriptional negative feedback controls the kinetics and the extent of MAPK activation under both physiological and pathological conditions.

HMGA2 gene is a promising target for ovarian cancer silencing therapy.

Ovarian cancer is one of the most lethal gynecological malignancies and the small success rate of routine therapeutic methods justifies efforts to develop new approaches. Evaluation of targets for effective inhibition of ovarian cancer cell growth should precipitate clinical application of gene silencing therapy. In our previous work, we showed upregulation of HMGA2 gene expression as a result of Ras-induced rat ovarian surface epithelial cell transformation. This gene codes the HMGA2 protein, a member of the high-mobility group AT-hook (HMGA) family of nonhistone chromatin proteins. Genome-wide studies revealed upregulation of the HMGA2 gene in human ovarian carcinomas. Herein we have evaluated over-expression of the HMGA2 gene, relevant to ovarian cancer, in subsets of human specimens and cell lines by in situ RNA hybridization and RT-PCR. Transient silencing of HMGA2 gene by means of siRNA inhibited proliferation of those ovarian cancer cells, which over-express this gene initially. Growth suppression was mediated by cell-cycle arrest. Stable silencing of highly expressed HMGA2 gene by shRNAi in A27/80, Ovcar-3 and OAW-42 ovarian cancer cell lines resulted in growth inhibition because of G1 arrest and increase of apoptosis as well. The tumor growth inhibition effect of HMGA2 silencing for Ovcar-3 cells was validated in vivo. Our findings revealed that the HMGA2 gene represents a promising target for gene silencing therapy in ovarian cancer.

Oncogenic signaling pathways and deregulated target genes.

A limited number of somatic mutations are known to trigger malignancy via chronic activation of cellular signaling pathways. High-throughput analysis of gene expression in cancer cells has revealed a plethora of deregulated genes by far exceeding the number of known genetic alterations. Targeted tumor therapy takes advantage of deregulated signaling in cancer. However, cancer cells may evade successful therapy, e.g., targeting oncogenic kinases, due to mutation of the target protein or to resistance mechanisms acting downstream of or parallel to the therapeutic block. To improve therapy and molecular diagnostics, we need detailed information on the wiring of pathway components and targets that ultimately execute the malignant properties of advanced tumors. Here we review work on Ras-mediated signal transduction and Ras pathway-responsive targets. We introduce the concept of signal-regulated transcriptional modules comprising groups of target genes responding to individual branches of the pathway network. Furthermore, we discuss functional approaches based on RNA interference for elucidating critical nodes in oncogenic signaling and the targets essential for malignancy.

Effects of Ras signaling on gene expression analyzed by customized microarrays.

Many signal transduction processes converge on Ras proteins that serve as molecular switches to couple external stimuli with cytoplasmic and nuclear targets. Oncogenic mutations lock Ras proteins in their activated state. Cellular responses to permanent Ras activation such as the induction of neoplastic phenotypes are mediated by distinct transcriptional alterations. A number of studies have reported alterations of the genetic program because of short-term or long-term activation of Ras signaling pathways. However, a consistent pattern of Ras-related transcriptional alterations has not yet emerged, because currently available investigations were based on different methods for assessing mRNA expression profiles, on different types of cells, and on heterogeneous experimental conditions. Here we describe the "Ras signaling target array" (RASTA) representing approximately 300 Ras-responsive target genes. This customized oligonucleotide array is a universal tool for assessing transcriptional patterns of cells or tissues expressing oncogenic Ras genes, as well as upstream and downstream effectors. To validate the results obtained by array-based expression profiling, we have compared the data with those obtained by suppression subtractive hybridization and conventional expression analysis by Northern blotting. Target RNAs were prepared from preneoplastic rat ovarian surface epithelial cells (ROSE) and the KRAS-transformed derivative A2/5. By interrogating Ras signaling target arrays with mRNAs prepared from the same types of cells as hybridization target, we correctly recognized 85% of genes differentially expressed on conversion of normal ovarian epithelial cells to the Ras-transformed state.

Transcriptional basis of KRAS oncogene-mediated cellular transformation in ovarian epithelial cells.

To understand the relationship between oncogenic signaling and the reprogramming of gene expression, we performed transcriptional profiling in rat ovarian surface epithelial cells (ROSE), in which neoplastic transformation is driven by a mutated KRAS oncogene. We identified >200 genes whose expression was elevated or reduced following permanent KRAS expression. Deregulated KRAS-responsive genes encode transcriptional regulators, signaling effectors, proteases, extracellular matrix and adhesion proteins, transformation-suppressing proteins and negative growth regulators. Many of them have not been previously identified in cells expressing oncogenic RAS genes or in other well-studied models of oncogenic signaling. The number of critical genes related to the execution of anchorage-independent proliferation and epithelial-mesenchymal transition was narrowed down to 79 by selectively inhibiting the mitogen-activated protein kinase (MAPK/ERK) and phosphatidylinositol 3-kinase (PI3K) pathways. Blocking MAPK/ERK-signaling caused reversion to the normal epithelial phenotype in conjunction with the reversal of deregulated target transcription to pretransformation levels. In addition, silencing of the overexpressed transcriptional regulator Fra-1 by RNA interference resulted in growth reduction, suggesting that this factor partially contributes to, but is not sufficient for the proliferative capacity of KRAS-transformed epithelial cells.