Abstract 2347: RNAi-mediated diminution of myoferlin reduces invasiveness of breast cancer cells

Abstract

Abstract Introduction: Ferlins are an evolutionarily conserved family of proteins that play a variety of roles associated with plasma membrane dynamics in eukaryotes, including membrane repair and endocytosis. Myoferlin (MYOF) is a mammalian ferlin protein with homology to ancestral Fer-1, a C. elegans protein that regulates Ca2+-dependent spermatic membrane fusion, a key mechanism underlying the amoeboid-like movements of C. elegans sperm. Consequently, mutations in fer-1 lead to immotile C. elegans sperm and infertility. This immotility phenotype in C. elegans spermatids is of interest since metastatic cancer cells are often associated with an amoeboid appearance as they invade surrounding tissues and blood and lymphatic vessels. Given this, we hypothesized that MYOF plays a role in cancer cell biology by contributing to the stability of the plasma membrane during proliferation, migration and/or invasion. Methods and Results: We first demonstrated the expression of MYOF protein and mRNA in the malignant, invasive breast cell line MDA-MB-231 using immunoblotting and real time polymerase chain reaction (RT-PCR) methods, respectively. Using lentiviral-mediated RNA interference (RNAi) gene silencing technology, we then generated stable MYOF-deficient MDA-MB-231 (231) cells and confirmed MYOF reduction with immunoblotting. RNAi control cells were generated in tandem with a non-human gene targeting construct. Interestingly, when MYOF-deficient 231 cells were grown in culture, a reversion from a mesenchymal to a more epithelial morphology was observed, and sustained through subsequent passages. This suggests that MYOF may be critical for one or more events during epithelial-mesenchymal transition (EMT). Using a commercial colorimetric assay for the quantification of viable cells, we did not detect any significant effect of MYOF deficiency on the proliferation of the 231 cells. Using the Boyden Chamber assay, MYOF-deficient 231 cells show a reduced invasive capacity through Matrigel compared to RNAi control cells, while no difference was observed for the migration capability of MYOF-deficient 231 cells. The invasive potential of 231 cells was further evaluated with a real-time invasion assay (xCELLigence, Roche Applied Science) that also showed a reduction in the invasive potential of the MYOF-deficient cells. These results suggest that MYOF may be important for cancer cell invasion. PCR array screening of extracellular matrix related genes (SABiosciences) in MYOF-deficient and RNAi control 231 cells indicates that matrix metalloproteinase levels may be affected by MYOF-diminution, and thereby contribute to the mechanism behind the altered invasion of MYOF-deficient cells. Conclusion: Myoferlin may play a role in cancer progression by promoting cellular invasion, and therefore be a potentially important prospective target for cancer therapy and a marker for prognosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2347. doi:10.1158/1538-7445.AM2011-2347