Esther A. Peterson

High SEPT9_v1 Expression in Human Breast Cancer Cells Is Associated with Oncogenic Phenotypes

Altered expression of the human septin gene, SEPT9, and its murine homologue, Sept9, has been implicated in neoplasia. However, their role(s) in oncogenesis remains poorly understood. We found amplification of SEPT9 in 67% of breast cancer cells (BCC) when compared with immortalized human mammary epithelial cells (IHMEC) as well as high levels of SEPT9 expression in the majority (61%) of the BCCs studied, unlike IHMECs. Expression profiling of variant SEPT9 transcripts and translated products revealed that high expression of the variant, SEPT9_v1, in contrast to other variants, was widespread in BCCs (55% of the BCCs) but not in IHMECs. High expression of SEPT9_v1 was also observed in primary breast cancer samples by immunohistochemical studies. We subsequently examined the phenotypic consequences of SEPT9_v1 expression in human breast cells. Retroviral expression of SEPT9_v1 in IHMEC cell culture models showed that SEPT9_v1 accelerated growth kinetics, stimulated cell motility, promoted invasion in Matrigel Transwell assays, increased genomic instability with the development of aneuploidy, and stimulated morphologic changes. Significant cytokinesis defects and disruption of tubulin microfilaments were also observed by immunofluorescence when SEPT9_v1 was ectopically expressed in IHMECs. Furthermore, SEPT9_v1 markedly enhanced neoplastic transformation in Hs578T cells, a BCC with no endogenous expression of the SEPT9_v1 isoform. Small interfering RNA-mediated and short hairpin RNA-mediated inhibition of SEPT9_v1 expression in two BCCs with high levels of endogenous SEPT9_v1 expression inhibited neoplastic growth properties of the cells. Taken together, our findings suggest that increased SEPT9_v1 expression contributes to the malignant pathogenesis of some breast tumors.

Conquering the complex world of human septins: implications for health and disease

Peterson EA and Petty EM. Conquering the complex world of human septins: implications for health and disease.

Characterization of a SEPT9 interacting protein, SEPT14, a novel testis-specific septin

Septins are a highly conserved family of GTP-binding cytoskeletal proteins implicated in multiple cellular functions, including membrane transport, apoptosis, cell polarity, cell cycle regulation, cytokinesis, and oncogenesis. Here we describe the characterization of a novel interacting partner of the septin family, initially cloned from a human testis expression library following yeast two-hybrid isolation to identify SEPT9 binding partners. Upon further genomic characterization and bioinformatics analyses it was determined that this novel septin-interacting partner was also a new member of the mammalian septin family, named SEPT14. SEPT14 maps to 7p11.2 in humans and includes a conserved GTPase domain and a predicted carboxy-terminus coiled-coil domain characteristic of other septins. Three potential translational start methionines were identified by 5′ RACE-PCR encoding proteins of 432-, 427-, and 425-residue peptides, respectively. SEPT14 shares closest homology to SEPT10, a human dendritic septin, and limited homology to SEPT9 isoforms. SEPT14 colocalized with SEPT9 when coexpressed in cell lines, and epitope-tagged forms of these proteins coimmunoprecipitated. Moreover, SEPT14 was coimmunoprecipitated from rat testes using SEPT9 antibodies, and yeast two-hybrid analysis suggested SEPT14 interactions with nine additional septins. Multitissue Northern blotting showed testis-specific expression of a single 5.0-kb SEPT14 transcript. RT-PCR analysis revealed that SEPT14 was not detectable in normal or cancerous ovarian, breast, prostate, bladder, or kidney cell lines and was only faintly detected in fetal liver, tonsil, and thymus samples. Interestingly, SEPT14 was expressed in testis but not testicular cancer cell lines by RT-PCR, suggesting that further investigation of SEPT14 as a testis-specific tumor suppressor is necessary.

Up-regulation of SEPT9_v1 stabilizes c-Jun-N-terminal kinase and contributes to its pro-proliferative activity in mammary epithelial cells.

SEPT9_v1, the largest transcript of the septin gene family member, SEPT9, encodes a septin isoform implicated in the tumorigenic transformation of mammary epithelial cells. High levels of SEPT9_v1 expression also have been observed in both breast cancer cell lines, primary breast cancers as well as other solid tumor malignancies. We found a novel interaction between SEPT9_v1 and the c-Jun-N-terminal kinase (JNK), a mitogen-activated protein kinase important in cellular stress responses, cell proliferation, and cell survival. We found that up-regulation of SEPT9_v1 stabilizes JNK by delaying its degradation, thereby activating the JNK transcriptome. C-jun kinase assays in mammary epithelial cells expressing SEPT9_v1, compared to controls, exhibited increased JNK/c-Jun transcriptional activity. This increase was associated with increased levels of cyclin D1, a critical component of the proliferative response required for progression through G(1) of the cell cycle in many cell types. These findings demonstrate the first link between a septin protein and the JNK signaling pathway. Importantly, it suggests a novel functional role of SEPT9_v1 in driving cellular proliferation of mammary epithelial cells, a hallmark feature of oncogenesis that is directly relevant to breast cancer.

Duplication within the SEPT9 gene associated with a founder effect in North American families with hereditary neuralgic amyotrophy

Hereditary neuralgic amyotrophy (HNA) is an autosomal dominant disorder associated with recurrent episodes of focal neuropathy primarily affecting the brachial plexus. Point mutations in the SEPT9 gene have been previously identified as the molecular basis of HNA in some pedigrees. However in many families, including those from North America demonstrating a genetic founder haplotype, no sequence mutations have been detected. We report an intragenic 38 Kb SEPT9 duplication that is linked to HNA in 12 North American families that share the common founder haplotype. Analysis of the breakpoints showed that the duplication is identical in all pedigrees, and molecular analysis revealed that the duplication includes the 645 bp exon in which previous HNA mutations were found. The SEPT9 transcript variants that span this duplication contain two in-frame repeats of this exon, and immunoblotting demonstrates larger molecular weight SEPT9 protein isoforms. This exon also encodes for a majority of the SEPT9 N-terminal proline rich region suggesting that this region plays a role in the pathogenesis of HNA.

Mutant GATA3 Actively Promotes the Growth of Normal and Malignant Mammary Cells

Background/Aim: GATA3, a transcription factor expressed in luminal breast epithelial cells, is required for mammary gland development. Heterozygous GATA3 mutations occur in up to 15% of estrogen receptor (ER)-positive breast tumors and have been proposed to be null alleles resulting in haploinsufficiency; however, the mutation spectrum of GATA3 in breast cancer is in sharp contrast to that found in HDR syndrome, a true GATA3 haploinsufficiency disease. Materials and Methods: Transgenic mice, 3D cultures and xenografts were used to examine the effect of mutant GATA3 expression on mammary cell proliferation. Results: Mutant GATA3 accelerated tumor growth of ZR751 cell xenografts and promoted precocious lobuloalveolar development in transgenic mouse mammary glands. Conclusion: GATA3 mutations, recently observed in breast cancer, encode active transcription factors, which elicit proliferative phenotypes in normal mammary epithelium and promote the growth of ER-positive breast cancer cell lines.