Abstract 72: Amino terminus of SEPT9_i1 impacts cellular localization: Implications for mammary tumorigenesis

Abstract

Abstract Septins were first discovered in Saccharomyces cerevisiae as proteins essential for faithful cell division. They are highly conserved and found in all eukaryotes with the exception of plants. One septin, SEPT9, was cloned from a region of allelic imbalance in breast and ovarian cancers on chromosome 17q25. Further analysis showed that SEPT9 encoded multiple protein isoforms. Retroviral expression of one isoform, SEPT9_i1, promoted oncogenic phenotypes including, increased cell proliferation, invasion, motility, and genomic instability in mammary epithelial cells. In addition, abnormal nuclear localization of SEPT9_i1 was observed. SEPT9_i1 is highly expressed in many cancers including, breast, ovarian, prostate, and head and neck. SEPT9_i1 differs from other SEPT9 isoforms by 25 unique amino acids at the N terminus. SEPT9_i3, which lacks these 25 amino acids, does not exhibit the same pro-oncogenic phenotypes associated with SEPT9_i1. The goal of this study is to determine the functional relevance of these amino acids with respect to cellular localization as well as phenotypes related to mammary tumorigenesis and metastasis. First, bioinformatic analysis revealed a bipartite nuclear localization signal within the 25 unique amino acids but no other recognized functional domains. To determine the effects of these 25 amino acids on nuclear localization, FLAG-tagged deletion (ΔN)-SEPT9_i1, full-length (FL) -SEPT9_i1, and empty vector control constructs were transiently transfected into two immortalized human mammary epithelial cell lines, HME and MCF-10a. Immunofluorescence analyses of FL-SEPT9_i1 and ΔN-SEPT9_i1 localization revealed a dramatic decrease in the amount of SEPT9_i1 localized to the nucleus in the ΔN-SEPT9_i1 versus FL- SEPT9_i1 lines. Stably expressing FL-SEPT9_i1 and ΔN-SEPT9_i1 HS578t cell lines were also generated using nucleofection for examination of the effects of ΔN-SEPT9_i1 on cellular phenotypes including cell proliferation, cell motility and invasion. We present data indicating that the deletion of the N terminal 25 amino acids decreases cell motility and invasion as compared to SEPT9_i1 in HS578t cells. In addition, we show increased cell proliferation in cells expressing ΔN-SEPT9_i1 as compared to both EV and SEPT9_i1. To date our results implicate an oncogenic role for high SEPT9_i1 in mammary tumorigenesis where the unique 25 amino acids of SEPT9_i1 may impact tumorigenesis. Further investigation could provide valuable insight into developing targeted chemotherapeutic agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 72. doi:1538-7445.AM2012-72