Alex D. Chacko

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

Dynamical systems analysis of mitochondrial BAK activation kinetics predicts resistance to BH3 domains.

Introduction The molecular mechanism underlying mitochondrial BAK activation during apoptosis remains highly controversial. Two seemingly conflicting models have been proposed. In one, BAK requires so-called activating BH3 only proteins (aBH3) to initiate its conformation change. In the other, displacement from inhibitory pro-survival BCL-2 proteins (PBPs) and monomerization of BAK by PBP selective dissociator BH3-only proteins (dBH3) is sufficient. Methodology/Principal Findings To better understand the kinetic implications of these conflicting but highly evidence-based models, we have conducted a deterministic, dynamical systems analysis to explore the kinetics underlying the first step of BAK activation, as a non-linear reaction system. We show that dBH3 induced BAK activation is efficient, even in the absence of aBH3s, provided constitutive interaction of PBPs with open conformation BAK occurs in an adenoviral E1B 19K-like manner. The pattern of PBP expression robustly predicts the efficacy of dBH3s. Conclusion Our findings accommodate the prevailing BAK activation models as potentially coexisting mechanisms capable of initiating BAK activation, and supports a model based approach for predicting resistance to therapeutically relevant small molecule BH3 mimetics.

Voltage dependent anion channel-1 regulates death receptor mediated apoptosis by enabling cleavage of caspase-8.

BackgroundActivation of the extrinsic apoptosis pathway by tumour necrosis factor related apoptosis inducing ligand (TRAIL) is a novel therapeutic strategy for treating cancer that is currently under clinical evaluation. Identification of molecular biomarkers of resistance is likely to play an important role in predicting clinical anti tumour activity. The involvement of the mitochondrial type 1 voltage dependent anion channel (VDAC1) in regulating apoptosis has been highly debated. To date, a functional role in regulating the extrinsic apoptosis pathway has not been formally excluded.MethodsWe carried out stable and transient RNAi knockdowns of VDAC1 in non-small cell lung cancer cells, and stimulated the extrinsic apoptotic pathway principally by incubating cells with the death ligand TRAIL. We used in-vitro apoptotic and cell viability assays, as well as western blot for markers of apoptosis, to demonstrate that TRAIL-induced toxicity is VDAC1 dependant. Confocal microscopy and mitochondrial fractionation were used to determine the importance of mitochondria for caspase-8 activation.ResultsHere we show that either stable or transient knockdown of VDAC1 is sufficient to antagonize TRAIL mediated apoptosis in non-small cell lung cancer (NSCLC) cells. Specifically, VDAC1 is required for processing of procaspase-8 to its fully active p18 form at the mitochondria. Loss of VDAC1 does not alter mitochondrial sensitivity to exogenous caspase-8-cleaved BID induced mitochondrial depolarization, even though VDAC1 expression is essential for TRAIL dependent activation of the intrinsic apoptosis pathway. Furthermore, expression of exogenous VDAC1 restores the apoptotic response to TRAIL in cells in which endogenous VDAC1 has been selectively silenced.ConclusionsExpression of VDAC1 is required for full processing and activation of caspase-8 and supports a role for mitochondria in regulating apoptosis signaling via the death receptor pathway.

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

BackgroundThe 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.MethodsWe have examined the effect of over-expression of SEPT9_i4 on the dynamics of microtubule polymer mass in cultured cells.ResultsWe 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.ConclusionsGiven 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.

BAK and NOXA Are Critical Determinants of Mitochondrial Apoptosis Induced by Bortezomib in Mesothelioma

Based on promising preclinical efficacy associated with the 20S proteasome inhibitor bortezomib in malignant pleural mesothelioma (MPM), two phase II clinical trials have been initiated (EORTC 08052 and ICORG 05–10). However, the potential mechanisms underlying resistance to this targeted drug in MPM are still unknown. Functional genetic analyses were conducted to determine the key mitochondrial apoptotic regulators required for bortezomib sensitivity and to establish how their dysregulation may confer resistance. The multidomain proapoptotic protein BAK, but not its orthologue BAX, was found to be essential for bortezomib-induced apoptosis in MPM cell lines. Immunohistochemistry was performed on tissues from the ICORG-05 phase II trial and a TMA of archived mesotheliomas. Loss of BAK was found in 39% of specimens and loss of both BAX/BAK in 37% of samples. However, MPM tissues from patients who failed to respond to bortezomib and MPM cell lines selected for resistance to bortezomib conserved BAK expression. In contrast, c-Myc dependent transactivation of NOXA was abrogated in the resistant cell lines. In summary, the block of mitochondrial apoptosis is a limiting factor for achieving efficacy of bortezomib in MPM, and the observed loss of BAK expression or NOXA transactivation may be relevant mechanisms of resistance in the clinic.

Abstract 4445: Pegylated arginine deiminase induces mitochondrial apoptosis and synergizes with cisplatin in ASS1-negative malignant pleural mesothelioma

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC We have explored the role of pegylated arginine deiminase (ADI-PEG20), an arginine catabolizing enzyme, in argininosuccinate synthetase 1 (ASS1)-negative malignant pleural mesothelioma (MPM). MPM is a chemorefractory disease, increasing worldwide, with a median survival of less than 1 year. Novel treatments are urgently required. MPM tumors, lacking ASS1, are auxotrophic for arginine and therefore sensitive to arginine depletion driven by ADI-PEG20. First we assessed the impact of ADI-PEG20 on global gene expression to identify novel interacting pathways in the ASS1-negative MPM cell line, JU77, using the human genome U133 Plus 2.0 Array from Affymetrix combined with bioinformatic analysis. Next, three ASS1-negative MPM cell lines (MSTO-211H, 2591 and JU77) were treated in triplicate with different fixed ratios of ADI-PEG20 and cisplatin. Cellular viability was determined 6 days post-treatment by MTS colorimetric assay. Apoptosis protein induction by ADI-PEG20 and platinum, alone and in combination, was measured by western blot after mitochondrial and cytosolic fractionation. Drug interactions were analyzed using the isobologram method of Chou and Talalay. ADI-PEG20 modulated several thousand genes involved in cell cycle, DNA damage, immune, and inflammatory pathways in the ASS1-negative JU77 MPM cell line by 24hrs of drug treatment. In contrast, stable transfection of ASS1 cDNA in ASS1-negative MPM cell lines resulted in resistance to ADI-PEG20 with minimal evidence of gene modulation by 24hr of drug treatment as assessed in the JU77 MPM cell line. ADI-PEG20 triggered mitochondria-dependent apoptosis as evidenced by Smac release from mitochondria into the cytosol of ASS1-negative MPM cell lines. We confirmed that arginine depletion by ADI-PEG20 altered the mTOR/p70S6K signaling pathway and led to dephosphorylation of the translation regulation proteins S6K and E4-BP1. The expression of cell cycle regulating proteins p21 and cyclin D1 was decreased by arginine depletion, alone and in combination with cisplatin. The enhancement of cisplatin cytotoxicity by ADI-PEG20 with loss of ASS1-negative MPM cell viability as early as 24hr post treatment, supports the demonstrated strong synergy between the two drugs. In summary, the antitumor effect of platinum was potentiated markedly using ADI-PEG20, suggesting that combining arginine depletion with the current standard of care, platinum and antifolates, may offer an improvement over doublet chemotherapy alone. 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 4445.

Machine vision based stochastic analysis of cancer cell mitochondrial dysfunction induced by a BH3 domain

We have developed a versatile and rapid method for the quantitative estimation of cell death kinetics, following direct single-shot activation of the mitochondrial death pathway by a cell permeable BH3 activator peptide (D-R8BH3BID). This approach employs timelapse epifluorescent imaging of live cells and a machine- vision based feature extraction algorithm, to measure unidirectional stochastic transitions associated with mitochondrial inner membrane potential depolarization and/or permeability transition, at single cell resolution. This data is transformed to enable construction of a right step-wise survival function using the product limit estimator, and estimation of a median latency parameter (λ), defined for the entire imaged cell population. Estimates of λ computed for cells exhibiting two-colour fluorescence can be compared statistically using the Mantel-Hansel test. This general method has been applied to measure the kinetics and temporal ordering of BH3 domain induced mitochondrial depolarization and inner membrane permeabilization in cancer cells, and demonstrates the robustness of this technique in resolving temporally distinct intracellular events within individual cells.

The Rab27A effector MYRIP as a regulator of survival in non-small cell lung cancer cells.

e13537 Background: Personalising therapy for non-small cell lung cancer has been validated as an effective treatment paradigm. Somatic gene alterations confer sensitivity to inhibition of growth survival pathways irrespective of underlying resistance to chemotherapeutic agents, and enables efficient activation of BCL-2 family-dependent pro-apoptotic signalling. In platinum resistant cancers this signalling is blocked, however the intrinsic pathway remains sensitive to exogenous BH3 death signals. METHODS Microarray profiling coupled to focused RNAi screening was used to reveal those resistance genes involved in derepression of proapoptotic BCL-2 family proteins. Analysis of the effect of MYRIP expression on survival was generated from studying 6 NSCLC datasets from Gene Expression Omnibus which were paired with survival time and status information. The toxic effect of MYRIP depletion on NSCLC and non-cancerous lung cells was determined by cell viability and clonogenic assays. Apoptotic induction was examined by caspase and PARP cleavage experiments, and fractionation of mitochondrial proteins. FACS analysis was used to examine effects on cell cycle. RESULTS MYRIP/Slac2c, a gene hitherto associated with vesicle trafficking but not apoptosis, was overexpressed in platinum resistant NSCLC cells. In particular, cisplatin resistant cells expressed a heavier protein isoform of MYRIP which appeared to be associated with the mitochondria. Silencing of MYRIP induced BIM and BAX/BAK-dependent mitochondrial apoptosis which was selective for NSCLC, but not non-cancerous lung cells. In addition, loss of MYRIP induced S-phase arrest. CONCLUSIONS Overexpression of MYRIP is an independent, poor prognostic factor suggesting that targeting these genes may be therapeutically relevant. In summary, MYRIP exhibits a previously unknown survival function associated with suppression of pro-apoptotic BCL-2 family proteins and maintenance of cancer cell proliferation and survival.

Deterministic Modelling of BAK Activation Kinetics

The molecular mechanism underlying mitochondrial BAK activation during apoptosis remains highly controversial. Two seemingly conflicting models have been proposed. In the activation model, BAK requires so‐called activating BH3 only proteins (aBH3) to initiate its conformation change. In the other, displacement from inhibitory pro‐survival BCL‐2 proteins (PBPs) and monomerization of BAK by PBP restricted dissociator BH3‐only proteins (dBH3) is sufficient. To better understand the kinetic implications of these models and reconcile these conflicting but highly evidence‐based models, we have employed dynamical systems analysis to explore the kinetics underlying BAK activation as a non‐linear reaction system. Our findings accommodate both pure agonism and dissociation as mutually exclusive mechanisms capable of initiating BAK activation. In addition we find our work supports a modelling based approach for predicting resistance to therapeutically relevant small molecules BH3 mimetics.