Abstract
BackgroundThe Ras association domain family 1 (RASSF1) gene is a Ras effector encoding two major mRNA forms, RASSF1A and RASSF1C, derived by alternative promoter selection and alternative mRNA splicing. RASSF1A is a tumor suppressor gene. However, very little is known about the function of RASSF1C both in normal and transformed cells.MethodsGene silencing and over-expression techniques were used to modulate RASSF1C expression in human breast cancer cells. Affymetrix-microarray analysis was performed using T47D cells over-expressing RASSF1C to identify RASSF1C target genes. RT-PCR and western blot techniques were used to validate target gene expression. Cell invasion and apoptosis assays were also performed.ResultsIn this article, we report the effects of altering RASSF1C expression in human breast cancer cells. We found that silencing RASSF1C mRNA in breast cancer cell lines (MDA-MB231 and T47D) caused a small but significant decrease in cell proliferation. Conversely, inducible over-expression of RASSF1C in breast cancer cells (MDA-MB231 and T47D) resulted in a small increase in cell proliferation. We also report on the identification of novel RASSF1C target genes. RASSF1C down-regulates several pro-apoptotic and tumor suppressor genes and up-regulates several growth promoting genes in breast cancer cells. We further show that down-regulation of caspase 3 via overexpression of RASSF1C reduces breast cancer cells' sensitivity to the apoptosis inducing agent, etoposide. Furthermore, we found that RASSF1C over-expression enhances T47D cell invasion/migration in vitro.ConclusionTogether, our findings suggest that RASSF1C, unlike RASSF1A, is not a tumor suppressor, but instead may play a role in stimulating metastasis and survival in breast cancer cells.
Highlights
The Ras association domain family 1 (RASSF1) gene is a Ras effector encoding two major mRNA forms, RASSF1A and RASSF1C, derived by alternative promoter selection and alternative mRNA splicing
Prior to silencing RASSF1C mRNA, the endogenous RASSF1A and RASSF1C mRNA levels were measured in MDA-MB231 and T47D breast cancer cells
Cells transfected with small interfering RNA (siRNA)-RASSF1C plasmid showed a significant decrease (p < 0.05) in cell proliferation compared to cells transfected with control plasmid as judged by the alamar blue and the 3H-thymidine incorporation assays (Figure 1B and 1C)
Summary
The Ras association domain family 1 (RASSF1) gene is a Ras effector encoding two major mRNA forms, RASSF1A and RASSF1C, derived by alternative promoter selection and alternative mRNA splicing. The Ras association domain family 1 (RASSF1) proteins are postulated to function as Ras effectors and to affect cell growth. The RASSF1 gene encodes multiple isoforms derived by alternative promoter selection and alternative mRNA splicing [1,2], with two major isoforms called RASSF1A and RASSF1C. The RASSFIA protein (340 amino acids) contains an amino-terminal diacyl glycerol binding domain (C1 domain), an ataxia telangiectasia mutated (ATM) phosphorylation site, and a carboxy-terminal putative Ras association (RA) domain. RASSF1A is a tumor suppressor gene which is epigenetically inactivated by cytidine methylation in many human solid tumors. Studies of RASSF1A knockout mice showed that RASSF1A -/- and RASSF1A+/- mice exhibit enhanced tumor multiplicity and tumor size compared to wild type animals upon exposure to the chemical carcinogens benzo(a) pyrene and urethane [13]
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