RNA-binding motif protein 35A is a novel tumor suppressor for colorectal cancer.

The frequent occurrence of inactivating gene mutations in tumors suggests a tumor suppressor function of the mutated gene. The RNA binding motif protein 35A (RBM35A) is mutated in approximately 50% of analyzed primary colon tumors with microsatellite instability. The Tet-off regulated ectopic expression of RBM35A gene in RBM35A-null LS180 colon carcinoma cells inhibited anchorage-independent growth in vitro, suppressed tumorigenic potential in vivo and enhanced adhesiveness of these cancer cells. Using microarray hybridization we found that in response to RBM35A expression a small fraction of genes showed a decrease in polysome-associated mRNA. Experiments using cell-free in vitro translation system demonstrated that RBM35A differentially affects translation of luciferase reporter mediated by various 5'untranslated regions (UTR). We found that Gibbs energy value (DeltaG) of secondary structure formed by 5'UTRs of mRNAs can account for differential effect of RBM35A on reporter translation efficiency. Targeted mutation in the FOS 5'UTR sequence, which increased the DeltaG value of hairpin stem formation, resulted in a stronger inhibitory effect of RBM35A on reporter translation efficiency mediated by this UTR. Immunoblotting revealed that ectopic expression of RBM35A in LS180 cells caused alterations in protein levels for several cancer related genes. Our results demonstrate for the first time that RBM35A functions as a tumor suppressor in colon cancer cells. We propose that RBM35A is involved in posttranscriptional regulation of a number of genes by exerting a differential effect on protein translation via 5'UTRs of mRNAs.

Transformation of MCF-10A cells by random mutagenesis with frameshift mutagen ICR191: a model for identifying candidate breast-tumor suppressors.

BACKGROUND: Widely accepted somatic mutation theory of carcinogenesis states that mutations in oncogenes and tumor suppressor genes in genomes of somatic cells is the cause of neoplastic transformation. Identifying frequent mutations in cancer cells suggests the involvement of mutant genes in carcinogenesis. RESULTS: To develop an in vitro model for the analysis of genetic alterations associated with breast carcinogenesis, we used random mutagenesis and selection of human non-tumorigenic immortalized breast epithelial cells MCF-10A in tissue-culture conditions that mimic tumor environment. Random mutations were generated in MCF-10A cells by cultivating them in a tissue-culture medium containing the frameshift-inducing agent ICR191. The first selective condition we used to transform MCF1-10A cells was cultivation in a medium containing mutagen at a concentration that allowed cell replication despite p53 protein accumulation induced by mutagen treatment. The second step of selection was either cell cultivation in a medium with reduced growth-factor supply or in a medium that mimics a hypoxia condition or growing in soft agar. Using mutagenesis and selection, we have generated several independently derived cultures with various degrees of transformation. Gene Identification by Nonsense-mediated mRNA decay Inhibition (GINI) analysis has identified the ICR191-induced frameshift mutations in the TP53, smoothelin, Ras association (RalGDS/AF-6) domain family 6 (RASSF6) and other genes in the transformed MCF-10A cells. The TP53 gene mutations resulting in the loss of protein expression had been found in all independently transformed MCF-10A cultures, which form large progressively growing tumors with sustained angiogenesis in nude mice. CONCLUSION: Identifying genes containing bi-allelic ICR191-induced frameshift mutations in the transformed MCF-10A cells generated by random mutagenesis and selection indicates putative breast-tumor suppressors. This can provide a model for studying the role of mutant genes in breast carcinogenesis.