Septins are an evolutionarily conserved group of GTP-binding and filament-forming proteins that belong to the large superclass of P-loop GTPases. While originally discovered in yeast as cell division cycle mutants with cytokinesis defects, they are now known to have diverse cellular roles which include polarity determination, cytoskeletal reorganization, membrane dynamics, vesicle trafficking, and exocytosis. Septin proteins form homo-and heterooligomeric polymers which can assemble into higher-order filaments. They are also known to interact with components of the cytoskeleton, ie actin and tubulin. The precise role of GTP binding is not clear but a current model suggests that it is associated with conformational changes which alter binding to other proteins. There are at least 12 human septin genes, and although information on expression patterns is limited, most undergo complex alternative splicing with some degree of tissue specificity. Nevertheless, an increasing body of data implicates the septin family in the pathogenesis of diverse disease states including neoplasia, neurodegenerative conditions, and infections. Here the known biochemical properties of mammalian septins are reviewed in the light of the data from yeast and other model organisms. The data implicating septins in human disease are considered and a model linking these data is proposed. It is posited that septins can act as regulatable scaffolds where the stoichiometry of septin associations, modifications, GTP status, and the interactions with other proteins allow the regulation of key cellular processes including polarity determination. Derangements of such septin scaffolds thus explain the role of septins in disease states.

The septins are an evolutionarily conserved family of GTP-binding proteins involved in diverse processes including vesicle trafficking, apoptosis, remodelling of the cytoskeleton, infection, neurodegeneration, and neoplasia. The present paper reports a comprehensive study of septin gene expression by DNA microarray methods in 10 360 samples of normal, diseased, and tumour tissues. A novel septin, SEPT13, has been identified and is shown to be related to SEPT7. It is shown that SEPT13 and the other known human septins are expressed in all tissue types but some show high expression in lymphoid (SEPT1, 6, 9, and 12) or brain tissues (SEPT2, 3, 4, 5, 7, 8, and 11). For a given septin, some isoforms are highly expressed in the brain and others are not. For example, SEPT8_v2 and v1, 1* and 3 are highly expressed in the brain and cluster with SEPT2, 3, 4, 5, 7, and 11. However, a probe set specific for SEPT8_v1 with low brain expression clusters away from this set. Similarly, SEPT4 has lymphoid and non-lymphoid forms; SEPT2 has lymphoid and central nervous system (CNS) forms; and SEPT6 and SEPT9 are elevated in lymphoid tissues but both have forms that cluster away from the lymphoid forms. Perturbation of septin expression was widespread in disease and tumours of the various tissues examined, particularly for conditions of the CNS, where alterations in all 13 septin genes were identified. This analysis provides a comprehensive catalogue of the septin family in health and disease. It is a key step in understanding the role of septins in physiological and pathological states and provides insight into the complexity of septin biology.

The septin family of genes has been implicated in a variety of cellular processes including cytokinesis, membrane transport and fusion, exocytosis, and apoptosis. One member of the septin family maps to chromosome 17q25.3, a region commonly deleted in sporadic ovarian and breast tumours, and has also been identified as a fusion partner of MLL in acute myeloid leukaemias. The present study demonstrates that the pattern of expression of multiple splice variants of this septin gene is altered in ovarian tumours and cell lines. In particular, expression of the zeta transcript is detectable in the majority of tumours and cell lines, but not in a range of non-malignant adult and fetal tissues. Zeta expression is accompanied by loss of the ubiquitous beta transcript. Somatic mutations of the gene were not detected in ovarian tumours, but it was demonstrated that beta expression in tumour cell lines can be reactivated by 5-azacytidine treatment, suggesting a role for methylation in the control of expression of this gene.

The septins are a family of GTP-binding proteins, evolutionarily conserved from yeast through to mammals, with roles in multiple core cellular functions. Here we provide an overview of our current knowledge of septin structure and function and focus mainly on mammalian septins, but gain much insight by drawing on knowledge of septins in other organisms. We describe their genomic and transcriptional complexity: a complexity manifest also in the diversity of scaffold structures that septins can form. Septin complexes can act to localize interacting proteins at specific intracellular locales and can also define membrane compartments by defining diffusion barriers. By such activities, septins can contribute to the definition of spatial asymmetry and cell polarity and we suggest a potential role in stem cell biology. Finally, we review the evidence that septins contribute to various disease states and argue that it is a breakdown in the tight regulation of their expression (particularly of individual isoforms), and also their inherent ability to oligomerize, which is pathogenic. Study of the perturbation of septin complex formation in disease will provide valuable insights into septin biology and will be a fertile ground for study.

Several lines of evidence indicate that altered expression of SEPT9 is seen in human neoplasia. In particular there is evidence of altered expression of the SEPT9_v4 isoform. The functional consequences of this remain unclear. We have studied the expression of wild-type- and GTP-binding mutants (G144V and S148N) of the SEPT9_v4 isoform in the MCF7 cell line as a model for its deregulation in neoplasia. We find that SEPT9_v4 expression induces dramatic actin cytoskeletal reorganization with the formation of processes around the cell periphery. Expression of the SEPT9_v4 isoform and a G144V mutant cause delocalization of endogenous SEPT9 from filamentous structures but the S148N mutant does not have this effect. In addition SEPT9_v4 isoform expression enhances cell motility and is associated with perturbation of directional movement. Expression of SEPT9_v4 GTP binding mutants also has potent effects on morphology and motility and causes loss of normal polarity, as judged by Golgi reorientation assays. The phenotypes induced by expression of the SEPT9_v4 isoform and the GTP mutants provide an insight into possible mechanisms of SEPT9_v4 function and suggest that the GTPase functions have both ras- and rab-like features. We propose a model in which overexpression of the SEPT9_v4 isoform in neoplasia is associated with perturbation of SEPT9 complexes, leading to phenotypes associated with neoplasia.

Members of the evolutionarily conserved septin family of genes are emerging as key components of several cellular processes including membrane trafficking, cytokinesis, and cell-cycle control events. SEPT9 has been shown to have a complex genomic architecture, such that up to 15 different isoforms are possible by the shuffling of five alternate amino termini and three alternate carboxy termini. Genomic and transcriptional alterations of SEPT9 have been associated with neoplasia. The present study has used a Sept9-specific antibody to determine the pattern of isoform expression in a range of tumour cell lines. Western blot analysis indicated considerable variation in the relative amounts and isoform content of Sept9. Immunofluorescence studies showed a range of patterns of cytoplasmic localization ranging from mainly particulate to mainly filamentous. Expression constructs were also generated for each amino terminal isoform to investigate the patterns of localization of individual isoforms and the effects on cells of ectopic expression. The present study shows that the epsilon isoform appears filamentous in this overexpression system while the remaining isoforms are particulate and cytoplasmic. Transient transfection of individual constructs into tumour cell lines results in cell-cycle perturbation with a G2/M arrest and dramatic growth suppression, which was greatest in cell lines with the lowest amounts of endogenous Sept9. Similar phenotypic observations were made with GTP-binding mutants of all five N-terminal variants of Sept9. However, dramatic differences were observed in the kinetics of accumulation of wild-type versus mutant septin protein in transfected cells. In conclusion, the present study shows that the expression patterns of Sept9 protein are very varied in a panel of tumour cell lines and the functional studies are consistent with a model of septin function as a component of a molecular scaffold that contributes to diverse cellular functions. Alterations in the levels of Sept9 protein by overexpression of individual isoforms can clearly perturb cellular behaviour and may thus provide a mechanistic explanation for observations of deranged septin expression in neoplasia.

Key tenets of modern biology are the central place of protein in cell regulation and the flow of genetic information from DNA to RNA to protein. However, it is becoming increasingly apparent that genomes are much more complex than hitherto thought with remarkably complex regulatory systems. The notion that the fraction of the genome involved in coding protein is all that matters is increasingly being questioned as the roles of non-coding RNA (ncRNA) in cellular systems becomes recognised. The RNA world, including microRNA (miRNA), small inhibitory RNA (siRNA) and other RNA species, are now recognised as being crucial for the regulation of chromatin structure, gene expression, mRNA processing and splicing, mRNA stability and translational control. Furthermore such ncRNA systems may be perturbed in disease states and most notably in neoplasia, including in haematological malignancies. Here the burgeoning evidence for a role of miRNA in neoplasia is reviewed and the importance of understanding the RNA world emphasised.

The septins are a family of 14 genes, the products of which are thought to function primarily as scaffolds which recruit other proteins to particular cellular locations. This gene family is highly conserved and encodes proteins involved in a variety of cellular functions including cytokinesis, apoptosis and vesicle trafficking. Septin colocalisation with actin and tubulin has also been reported. Previous work from this laboratory mapped the SEPT9 gene to a region of allelic imbalance on chromosome 17q in sporadic epithelial ovarian tumours. SEPT9 undergoes extensive alternate splicing at both the 5’ (v1, v2 v3, v4, v4* and v5) and 3’ (a, b and c) ends, with SEPT9_v4 and _v4* differing only in their 5’UTRs, therefore encoding the same polypeptide. We have shown perturbed expression of SEPT9 transcripts in neoplasia, specifically up-regulation of SEPT9_v1 and _v4* transcripts. In a range of normal tissues we noted three alternatively spliced 3’ transcripts (a, b & c) were expressed. However, in ovarian tumour development expression of 2 of these (b & c) is decreased. This suggests that expression of the various SEPT9 transcripts is independently regulated. We therefore sought to gain some insight into control of expression of these various splice variants and why specific patterns of deregulation might contribute to neoplasia. With respect to the cell cycle, we observed by immunofluoresence that SEPT9 protein is visible around the cell membrane during anaphase and into telophase. During cytokinesis the SEPT9 protein localises to the cleavage furrow and is part of the ‘ring’ structure of dividing cells. We also used a double thymidine block to synchronise HeLa cells but there was no alteration in levels of transcripts with specific 5’ or 3’ ends in these cells. Next we looked at expression of the SEPT9 5’ and 3’ ends in response to cellular stress, in particular DNA damage following doxorubicin treatment. We observe an up-regulation of the 5’ v4* transcript and 3’ c variant after doxorubicin treatment in a p53 wild type background but not in p53 null cells. We then used a model of ovarian surface epithelial cells immortalised with telomerase and a temperature sensitive SV40 large T antigen which proliferate at 330C but senesce when shifted to the non-permissive temperature (370C). We see that SEPT9_v2 and v4 mRNA levels increase significantly as cells undergo senescence. Finally, we note that the b and c variants share the same short 3’UTR (57 bases) while the a variant has a long 3’UTR (1.938kb). Bioinformatics of both 3’UTRs identified several putative microRNA binding sites present in the 3’UTR of the a variant but only one in that of b and c. We show that the 3’UTR of a drives a reporter gene when cloned upstream of luciferase and in a sequence dependant manner. In summary, our data supports the hypothesis that the alternatively spliced transcripts of SEPT9 play individual roles within the cell and that the 3’UTRs appear to be important in the regulation of SEPT9. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 260.