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01.08.2011 | Research article | Ausgabe 4/2011 Open Access

Breast Cancer Research 4/2011

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

Zeitschrift:
Breast Cancer Research > Ausgabe 4/2011
Autoren:
Diana Connolly, Zhixia Yang, Maria Castaldi, Nichelle Simmons, Maja H Oktay, Salvatore Coniglio, Melissa J Fazzari, Pascal Verdier-Pinard, Cristina Montagna
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​bcr2924) contains supplementary material, which is available to authorized users.
Diana Connolly, Zhixia Yang contributed equally to this work.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

DC carried out the methylation, real-time qRT-PCR and migration studies, as well as the immunofluorescence analysis, cell line and tissue sample collection and preparation, and edited and revised the manuscript. ZY performed the FISH and luciferase experiments and generated the GFP fusion clones. MC is the surgeon and NS and MHO are the pathologists who provided samples for analysis. SC supervised the migration assays. PVP performed the Western blot analysis and gave advice on manuscript preparation and revision. CM supervised the study and is responsible for the writing of the manuscript. All authors read and approved the final version of the manuscript.

Abstract

Introduction

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. The mechanisms regulating its expression during tumor initiation and progression in vivo and the oncogenic function of its different isoforms remain elusive.

Methods

Using 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.

Results

An 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.

Conclusions

In 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.
Zusatzmaterial
Additional file 1: Supplementary Table 1. List of breast tissue samples. Supplementary Table 2. List of cell lines used in the study. Supplementary Table 3. List of primer sequences. Supplementary Table 4. Western blot analysis protocol. Supplementary Table 5. Molecular weight of SEPT9 isoforms in human and mouse. Supplementary Table 6. Nuclear versus cytoplasmic localization. (DOC 131 KB)
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Additional file 2: Supplementary Figure S1. SEPT9 is amplified in human breast cancer cell lines. (A) Mapping of BAC clones selected for FISH analysis for SEPT9, HER2 and the subcentromeric region of chromosome 17. (B) Raw counts of the number of SEPT9 (green) and HER2 (red) signals detected by FISH in each of the 25 cells analyzed in our cell line panel. (C) The number of analyzed cells shown to exhibit increased SEPT9 copies (green), HER2 copies (red) or a balanced number of SEPT9 and HER2 copies (black) in human cell lines. (TIFF 485 KB)
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Additional file 3: Supplementary Figure S2. Sequence alignment of mouse and human SEPT9_v1 and mapping of antigens used to generate the antibody. (TIFF 553 KB)
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Additional file 4: Supplementary Figure S3. GFP- SEPT9 isoform construct and expression. (A) Vector map and ideogram depicting the cloning strategy used to generate GFP-SEPT9 fused isoforms. (B) Western blots of untransfected MCF7 and GFP_v1 through GFP_v5 fused clones. The membrane was probed with an anti-GFP antibody (top panel), with the anti-SEPT9 antibody provided by Dr Nagata (middle panel) and with α-tubulin (bottom panel). (C) Isoform-specific primers suitable for cloned cDNA were designed to uniquely amplify the _v1 through _v5 isoforms. These primers were used to confirm the specific overexpression of each isoform. (D) Real-Time qRT-PCR was performed to determine the level of SEPT9 overexpression of the clones (gray bars) compared to the parental MCF7 (white bar). (TIFF 541 KB)
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Additional file 5: Supplementary Figure S4. SEPT9 expression in human cell lines and breast tissues. (A) SEPT9 expression in human breast cancer cell lines detected by Western blot analysis using Dr Nagata's antibody. (B) Western blot of matching human primary breast tissues and adjacent tumor-free area showing the expression of SEPT9 isoforms detected with Dr Cossart's antibody (top panel: Ponceau red, middle panel: SEPT9, bottom panel: α-tubulin). (TIFF 514 KB)
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Authors’ original file for figure 1
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Authors’ original file for figure 2
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Authors’ original file for figure 3
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Authors’ original file for figure 4
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Authors’ original file for figure 5
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Authors’ original file for figure 6
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