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.
Highlights
Altered 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
SEPT9 is amplified in breast cancer cell lines and human breast adenocarcinomas Using Fluorescence in situ hybridization (FISH) to determine DNA copy number, a locusspecific probe for SEPT9 was cohybridized with a probe for HER2, a well-established oncogenetic biomarker that maps to the same chromosome arm as SEPT9 (Additional file 2, Supplementary Figure S1A)
The nontumorigenic cell line HBL100, carries a higher average number of copies per cell for both genes (8.96 and 8.68), which is consistent with the knowledge that over time, cultured, immortalized cell lines derived from nontumorigenic breast tissue may acquire cytogenetic alterations similar to those of cancer cell lines [40]
Summary
Altered 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. Identification of biomarkers for early detection and new therapeutic targets of breast cancer helps to continuously reduce the morbidity of this frequent pathology in women. Analysis of a limited set of mouse mammary gland (MG) adenocarcinomas proposed SEPT9 as a novel oncogene because of its high level of genomic amplification in the form of double-minute chromosomes and jumping translocations. Such cytogenetic alterations act as a means of amplification for strong oncogenes, resulting in their overexpression and gain of function [4]. SEPT9 downregulation results in the arrest of cell division in human mammary epithelial cells and human cancer cell lines [16,17]
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