Septin 9 isoform expression, localization and epigenetic changes during human and mouse breast cancer progression

Abstract

IntroductionAltered expression of Septin 9 (SEPT9), a septin coding for multiple isoform variants, has been observed in several carcinomas, including colorectal, head and neck, ovarian and breast, compared to normal tissues. The mechanisms regulating its expression during tumor initiation and progression in vivo and the oncogenic function of its different isoforms remain elusive.MethodsUsing an integrative approach, we investigated SEPT9 at the genetic, epigenetic, mRNA and protein levels in breast cancer. We analyzed a panel of breast cancer cell lines, human primary tumors and corresponding tumor-free areas, normal breast tissues from reduction mammoplasty patients, as well as primary mammary gland adenocarcinomas derived from the polyoma virus middle T antigen, or PyMT, mouse model. MCF7 clones expressing individual GFP-tagged SEPT9 isoforms were used to determine their respective intracellular distributions and effects on cell migration.ResultsAn overall increase in gene amplification and altered expression of SEPT9 were observed during breast tumorigenesis. We identified an intragenic alternative promoter at which methylation regulates SEPT9_v3 expression. Transfection of specific GFP-SEPT9 isoforms in MCF7 cells indicates that these isoforms exhibit differential localization and affect migration rates. Additionally, the loss of an uncharacterized SEPT9 nucleolar localization is observed during tumorigenesis.ConclusionsIn this study, we found conserved in vivo changes of SEPT9 gene amplification and overexpression during human and mouse breast tumorigenesis. We show that DNA methylation is a prominent mechanism responsible for regulating differential SEPT9 isoform expression and that breast tumor samples exhibit distinctive SEPT9 intracellular localization. Together, these findings support the significance of SEPT9 as a promising tool in breast cancer detection and further emphasize the importance of analyzing and targeting SEPT9 isoform-specific expression and function.

Analysis of K-Ras Interactions by Biotin Ligase Tagging

“…K-Ras itself was readily biotinylated as a domain of the chimeric BirA*Ras proteins ( Figure 1) and served as a cell sample biotinylation control. Another GTPase identified at roughly 10% the levels of K-Ras, was Septin-9, a breast cancer marker involved in cytokinesis (74). We also detected a number of GTPase-activating proteins, including the Ras GAPs, GAPD1 and nGAP (RASAL2), and GAPs for Rho, Rac, and Ral proteins (DOCK7, RHG21, SLIT-ROBO, RGPA1, RHG01).…”

Septin 9 negatively regulates ubiquitin-dependent downregulation of epidermal growth factor receptor

“…1G). Of note, long SEPT9 isoforms have been shown to be upregulated in various tumors and cancer cell lines (Amir et al, 2010;Connolly et al, 2014;Connolly et al, 2011b;Stanbery et al, 2010). It remains to be determined whether and to what extent their interaction with CIN85 might contribute to cancer progression.…”

Septin roles in tumorigenesis

“…The role of septins in migration in relation to tumor metastasis and invasion, as illustrated by preliminary data on SEPT9 (Chacko et al, 2005;Gonzalez et al, 2007;Connolly et al, 2011), will require detailed functional analysis to establish the molecular mechanisms involved in this process including further investigation of the septin interactome in normal and cancer cells. Although septin localization along actin microfilaments and microtubules has been observed by several laboratories (Surka et al, 2002;Nagata et al, 2003;Spiliotis et al, 2005) little is known about its link to promigratory functions.…”