SEPT9_v4 expression induces morphological change, increased motility and disturbed polarity

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. Copyright (c) 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Properties of SEPT9 Isoforms and the requirement for GTP binding

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. Copyright © 2004 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Expression of the SEPT9_i4 isoform confers resistance to microtubule-interacting drugs

BACKGROUND: The evolutionarily conserved septin family of genes encode GTP binding proteins involved in a variety of cellular functions including cytokinesis, apoptosis, membrane dynamics and vesicle trafficking. Septin proteins can form hetero-oligomeric complexes and interact with other proteins including actin and tubulin. The human SEPT9 gene on chromosome 17q25.3 has a complex genomic architecture with 18 different transcripts that can encode 15 distinct polypeptides. Two distinct transcripts with unique 5' ends (SEPT9_v4 and SEPT9_v4*) encode the same protein. In tumours the ratio of these transcripts changes with elevated levels of SEPT9_v4* mRNA, a transcript that is translated with enhanced efficiency leading to increased SEPT9_i4 protein.

METHODS: We have examined the effect of over-expression of SEPT9_i4 on the dynamics of microtubule polymer mass in cultured cells.

RESULTS: We show that the microtubule network in SEPT9_i4 over-expressing cells resists disruption by paclitaxel or cold incubation but also repolymerises tubulin more slowly after microtubule depolymerisation. Finally we show that SEPT9_i4 over-expressing cells have enhanced survival in the presence of clinically relevant microtubule acting drugs but not after treatment with DNAinteracting agents.

CONCLUSIONS: Given that SEPT9 over-expression is seen in diverse tumours and in particular ovarian and breast cancer, such data indicate that SEPT9_v4 expression may be clinically relevant and contribute to some forms of drug resistance.

Truncated estrogen receptor alpha 46-kDa isoform in human endothelial cells: relationship to acute activation of nitric oxide synthase.

Background Estrogen acutely activates endothelial nitric oxide synthase (eNOS). However, the identity of the receptors involved in this rapid response remains unclear. Methods and Results We detected an estrogen receptor (ER) transcript in human endothelial cells that encodes a truncated 46-kDa ER (1a-hER-46). A corresponding 46-kDa ER protein was identified in endothelial cell lysates. Transfection of cDNAs encoding the full-length ER (ER-66) and 1a-hER-46 resulted in appropriately sized recombinant proteins identified by anti-ER antibodies. Confocal microscopy revealed that a proportion of both ER-66 and hER-46 was localized outside the nucleus and mediated specific cell-surface binding of estrogen as assessed by FITC-conjugated, BSA-estrogen binding studies. Both ER isoforms colocalized with eNOS and mediated acute activation of eNOS in response to estrogen stimulation. However, estrogen-stimulated transcriptional activation mediated by 1a-hER-46 was much less than with ER-66. Furthermore, 1a-hER-46 inhibited classical hER-66 mediated transcriptional activation in a dominant-negative fashion. Conclusions These findings suggest that expression of an alternatively spliced, truncated ER isoform in human endothelial cells confers a unique ability to mediate acute but not transcriptional responses to estrogen.

Glucocorticoid receptor β and histone deacetylase 1 and 2 expression in the airways of severe asthma.

Rationale Upregulation of glucocorticoid receptor ß (GRß) has been implicated in steroid resistance in severe asthma, although previous studies are conflicting. GRß has been proposed as a dominant negative isoform of glucocorticoid receptor a (GRa) but it has also been suggested that GRß can cause steroid resistance via reduced expression of histone deacetylase 2 (HDAC2), a key regulator of steroid responsiveness in the airway.


Objectives To examine GRß, GRa, HDAC1 and HDAC2 expression at transcript and protein levels in bronchial biopsies from a large series of patients with severe asthma, and to compare the findings with those of patients with mild to moderate asthma and healthy volunteers.


Methods Bronchoscopic study in two UK centres with real-time PCR and immunohistochemistry performed on biopsies, western blotting of bronchial epithelial cells and immunoprecipitation with anti-GRß antibody.


Measurements and main results Protein and mRNA expression for GRa and HDAC2 did not differ between groups. GRß mRNA was detected in only 13 of 73 samples (seven patients with severe asthma), however immunohistochemistry showed widespread epithelial staining in all groups. Western blotting of bronchial epithelial cells with GRß antibody detected an additional ‘cross-reacting’ protein, identified as clathrin. HDAC1 expression was increased in patients with severe asthma compared with healthy volunteers.


Conclusions GRß mRNA is expressed at low levels in a minority of patients with severe asthma. HDAC1 and HDAC2 expression was not downregulated in severe asthma. These data do not support upregulated GRß and resultant reduced HDAC expression as the principal mechanism of steroid resistance in severe asthma. Conflicting GRß literature may be explained in part by clathrin cross-reactivity with commercial antibodies.

V3 versican isoform alters the behavior of human melanoma cells by interfering with CD44/ErbB-dependent signaling

Versican is a hyaluronan-binding, extracellular chondroitin sulfate proteoglycan produced by several tumor types, including malignant melanoma, which exists as four different splice variants. The short V3 isoform contains the G1 and G3 terminal domains of versican that may potentially interact directly or indirectly with the hyaluronan receptor CD44 and the EGFR, respectively. We have previously described that overexpression of V3 in MeWo human melanoma cells markedly reduces tumor cell growth in vitro and in vivo. In this study we have investigated the signaling mechanism of V3 by silencing the expression of CD44 in control and V3-expressing melanoma cells. Suppression of CD44 had the same effects on cell proliferation and cell migration than those provoked by V3 expression, suggesting that V3 acts through a CD44-mediated mechanism. Furthermore, CD44-dependent hyaluronan internalization was blocked by V3 expression and CD44 silencing, leading to an accumulation of this glycosaminoglycan in the pericellular matrix and to changes in cell migration on hyaluronan. Furthermore, ERK1/2 and p38 activation after EGF treatment were decreased in V3-expressing cells suggesting that V3 may also interact with the EGFR through its G3 domain. The existence of a EGFR/ErbB2 receptor complex able to interact with CD44 was identified in MeWo melanoma cells. V3 overexpression resulted in a reduced interaction between EGFR/ErbB2 and CD44 in response to EGF treatment. Our results indicate that the V3 isoform of versican interferes with CD44 and the CD44-EGFR/ErbB2 interaction, altering the signaling pathways, such as ERK1/2 and p38 MAPK, that regulate cell proliferation and migration.

The androgen receptor controls expression of the cancer-associated sTn antigen and cell adhesion through induction of ST6GalNAc1 in prostate cancer

Patterns of glycosylation are important in cancer, but the molecular mechanisms that drive changes are often poorly understood. The androgen receptor drives prostate cancer (PCa) development and progression to lethal metastatic castration-resistant disease. Here we used RNA-Seq coupled with bioinformatic analyses of androgen-receptor (AR) binding sites and clinical PCa expression array data to identify ST6GalNAc1 as a direct and rapidly activated target gene of the AR in PCa cells. ST6GalNAc1 encodes a sialytransferase that catalyses formation of the cancer-associated sialyl-Tn antigen (sTn), which we find is also induced by androgen exposure. Androgens induce expression of a novel splice variant of the ST6GalNAc1 protein in PCa cells. This splice variant encodes a shorter protein isoform that is still fully functional as a sialyltransferase and able to induce expression of the sTn-antigen. Surprisingly, given its high expression in tumours, stable expression of ST6GalNAc1 in PCa cells reduced formation of stable tumours in mice, reduced cell adhesion and induced a switch towards a more mesenchymal-like cell phenotype in vitro. ST6GalNAc1 has a dynamic expression pattern in clinical datasets, beingsignificantly up-regulated in primary prostate carcinoma but relatively down-regulated in established metastatic tissue. ST6GalNAc1 is frequently upregulated concurrently with another important glycosylation enzyme GCNT1 previously associated with prostate cancer progression and implicated in Sialyl Lewis X antigen synthesis. Together our data establishes an androgen-dependent mechanism for sTn antigen expression in PCa, and are consistent with a general role for the androgen receptor in driving important coordinate changes to the glycoproteome during PCa progression.

Tumor suppressor Ras-association domain family 1 isoform A is a novel regulator of cardiac hypertrophy

BACKGROUND: Ras signaling regulates a number of important processes in the heart, including cell growth and hypertrophy. Although it is known that defective Ras signaling is associated with Noonan, Costello, and other syndromes that are characterized by tumor formation and cardiac hypertrophy, little is known about factors that may control it. Here we investigate the role of Ras effector Ras-association domain family 1 isoform A (RASSF1A) in regulating myocardial hypertrophy.

METHODS AND RESULTS: A significant downregulation of RASSF1A expression was observed in hypertrophic mouse hearts, as well as in failing human hearts. To further investigate the role of RASSF1A in cardiac (patho)physiology, we used RASSF1A knock-out (RASSF1A(-)(/)(-)) mice and neonatal rat cardiomyocytes with adenoviral overexpression of RASSF1A. Ablation of RASSF1A in mice significantly enhanced the hypertrophic response to transverse aortic constriction (64.2% increase in heart weight/body weight ratio in RASSF1A(-)(/)(-) mice compared with 32.4% in wild type). Consistent with the in vivo data, overexpression of RASSF1A in cardiomyocytes markedly reduced the cellular hypertrophic response to phenylephrine stimulation. Analysis of molecular signaling events in isolated cardiomyocytes indicated that RASSF1A inhibited extracellular regulated kinase 1/2 activation, likely by blocking the binding of Raf1 to active Ras.

CONCLUSIONS: Our data establish RASSF1A as a novel inhibitor of cardiac hypertrophy by modulating the extracellular regulated kinase 1/2 pathway.