Vineshkumar Thidil Puliyappadamba

Mechanistic evaluation of the signaling events regulating curcumin-mediated chemosensitization of breast cancer cells to 5-fluorouracil

5-Fluorouracil (5-FU) is the first rationally designed antimetabolite, which achieves its therapeutic efficacy through inhibition of the enzyme thymidylate synthase (TS), which is essential for the synthesis and repair of DNA. However, prolonged exposure to 5-FU induces TS overexpression, which leads to 5-FU resistance in cancer cells. Several studies have identified curcumin as a potent chemosensitizer against chemoresistance induced by various chemotherapeutic drugs. In this study, we report for the first time, with mechanism-based evidences, that curcumin can effectively chemosensitize breast cancer cells to 5-FU, thereby reducing the toxicity and drug resistance. We found that 10 μM 5-FU and 10 μM curcumin induces a synergistic cytotoxic effect in different breast cancer cells, independent of their receptor status, through the enhancement of apoptosis. Curcumin was found to sensitize the breast cancer cells to 5-FU through TS-dependent downregulation of nuclear factor-κB (NF-κB), and this observation was confirmed by silencing TS and inactivating NF-κB, both of which reduced the chemosensitizing efficacy of curcumin. Silencing of TS suppressed 5-FU-induced NF-κB activation, whereas inactivation of NF-κB did not affect 5-FU-induced TS upregulation, confirming that TS is upstream of NF-κB and regulates the activation of NF-κB in 5-FU-induced signaling pathway. Although Akt/PI3kinase and mitogen-activated protein kinase pathways are activated by 5-FU and downregulated by curcumin, they do not have any role in regulating the synergism. As curcumin is a pharmacologically safe and cost-effective compound, its use in combination with 5-FU may improve the therapeutic index of 5-FU, if corroborated by in vivo studies and clinical trials.

Constitutive and ligand-induced EGFR signalling triggers distinct and mutually exclusive downstream signalling networks

EGFR overexpression plays an important oncogenic role in cancer. Regular EGFR protein levels are increased in cancer cells and the receptor then becomes constitutively active. However, downstream signals generated by constitutively activated EGFR are unknown. Here we report that the overexpressed EGFR oscillates between two distinct and mutually exclusive modes of signaling. Constitutive or non-canonical EGFR signaling activates the transcription factor IRF3 leading to expression of IFI27, IFIT1, and TRAIL. Ligand-mediated activation of EGFR switches off IRF3 dependent transcription, activates canonical ERK and Akt signals, and confers sensitivity to chemotherapy and virus-induced cell death. Mechanistically, the distinct downstream signals result from a switch of EGFR associated proteins. EGFR constitutively complexes with IRF3 and TBK1 leading to TBK1 and IRF3 phosphorylation. Addition of EGF dissociates TBK1, IRF3, and EGFR leading to a loss of IRF3 activity, Shc-EGFR association and ERK activation. Finally, we provide evidence for non-canonical EGFR signaling in glioblastoma.

Targeted proteasome inhibition by Velcade induces apoptosis in human mesothelioma and breast cancer cell lines

IntroductionThoracic malignancies and human breast cancer (HBC) continue to be aggressive solid tumors that are poor responders to the existing conventional standard chemotherapeutic approaches. Malignant pleural mesothelioma (MPM) is an asbestos-related tumor of the thoracic pleura that lacks effective treatment options. Altered ubiquitin proteasome pathway is frequently encountered in many malignancies including HBC and MPM and thus serves as an important target for therapeutic intervention strategies. Although proteasome inhibitor Velcade (Bortezomib) has been under clinical investigation for a number of cancers, limited preclinical studies with this agent have thus far been conducted in HBC and MPM malignancies.PurposeTo study the biological and molecular responses of MPM and HBC cells to Velcade treatments, and to identify mechanisms involved in transducing growth inhibitory effects of this agent.MethodsFlow-cytometric analyses coupled with western immunoblotting and gene-array methodologies were utilized to determine mechanisms of Velcade-dependent growth suppression of five MPM (H2595, H2373, H2452, H2461, and H2714) and two breast cancer (MDA MB-468, SKBR-3) cell lines.ResultsOur data revealed significant reduction in cell growth properties that were dose and time dependent. Velcade treatment resulted in G2M phase arrest, increased expression of cyclin-dependent kinase inhibitor p21 and pro-apoptotic protein Bax. Pretreatment of mesothelioma cells with Velcade showed synergistic effect with cisplatin combination regimens. High-throughput gene expression profiling among Velcade treated and untreated mesothelioma cell lines resulted in identification of novel transducers of apoptosis such as CARP-1, XAF1, and Troy proteins.ConclusionsVelcade targets cell cycle and apoptosis signaling to suppress MPM and HBC growth in part by activating novel transducers of apoptosis. This pilot study has paved way for further in-depth analysis of the downstream target molecules associated with presensitization of mesothelioma cells in finding effective therapeutic treatment options for both mesothelioma and recalcitrant breast cancers.

EGFR wild type antagonizes EGFRvIII-mediated activation of Met in glioblastoma

Epidermal growth factor receptor (EGFR)vIII is the most common EGFR mutant found in glioblastoma (GBM). EGFRvIII does not bind ligand, is highly oncogenic and is usually coexpressed with EGFR wild type (EGFRwt). EGFRvIII activates Met, and Met contributes to EGFRvIII-mediated oncogenicity and resistance to treatment. Here, we report that addition of EGF results in a rapid loss of EGFRvIII-driven Met phosphorylation in glioma cells. Met is associated with EGFRvIII in a physical complex. Addition of EGF results in a dissociation of the EGFRvIII–Met complex with a concomitant loss of Met phosphorylation. Consistent with the abrogation of Met activation, addition of EGF results in the inhibition of EGFRvIII-mediated resistance to chemotherapy. Thus, our study suggests that ligand in the milieu of EGFRvIII-expressing GBM cells is likely to influence the EGFRvIII–Met interaction and resistance to treatment, and highlights a novel antagonistic interaction between EGFRwt and EGFRvIII in glioma cells.

Antagonists of Anaphase-promoting Complex (APC)-2-Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 Interaction Are Novel Regulators of Cell Growth and Apoptosis

CARP-1/CCAR1, a perinuclear phosphoprotein, is a regulator of cell growth and apoptosis signaling. Although CARP-1 is a regulator of chemotherapy-dependent apoptosis, it is also a part of the NF-κB proteome and a co-activator of steroid/thyroid nuclear receptors as well as β-catenin signaling. Our yeast two-hybrid screen revealed CARP-1 binding with the anaphase-promoting complex/cyclosome E3 ubiquitin ligase component APC-2 protein. CARP-1 also binds with anaphase-promoting complex/cyclosome co-activators Cdc20 and Cdh1. Following mapping of the minimal epitopes involved in CARP-1 binding with APC-2, a fluorescence polarization assay was established that indicated a dissociation constant (Kd) of 480 nm for CARP-1/APC-2 binding. Fluorescence polarization assay-based high throughput screening of a chemical library yielded several small molecule antagonists of CARP-1/APC-2 binding, termed CARP-1 functional mimetics. CFM-4 (1(2-chlorobenzyl)-5′-phenyl-3′H-spiro[indoline-3,2′-[1,3,4]thiadiazol]-2-one), a lead compound, binds with and stimulates CARP-1 expression. CFM-4 prevents CARP-1 binding with APC-2, causes G2M cell cycle arrest, and induces apoptosis with an IC50 range of 10–15 μm. Apoptosis signaling by CFM-4 involves activation of caspase-8 and -9 and caspase-mediated ubiquitin-proteasome pathway-independent loss of cyclin B1 and Cdc20 proteins. Depletion of CARP-1, however, interferes with CFM-4-dependent cell growth inhibition, activation of caspases, and apoptosis. Because CFM-4 also suppresses growth of drug-resistant human breast cancer cells without affecting the growth of human breast epithelial MCF-10A cells, elevating CARP-1 by CFM-4 and consequent apoptosis could in principle be exploited to further elucidate, and perhaps effectively target, often deregulated cell cycle pathways in pathological conditions, including cancer.

An EGFR wild type-EGFRvIII-HB-EGF feed-forward loop regulates the activation of EGFRvIII.

EGFRvIII is a key oncogene in glioblastoma (GBM). EGFRvIII results from an in-frame deletion in the extracellular domain of EGFR, does not bind ligand and is thought to be constitutively active. Although EGFRvIII dimerization is known to activate EGFRvIII, the factors that drive EGFRvIII dimerization and activation are not well understood. Here we present a new model of EGFRvIII activation and propose that oncogenic activation of EGFRvIII in glioma cells is driven by co-expressed activated EGFR wild type (EGFRwt). Increasing EGFRwt leads to a striking increase in EGFRvIII tyrosine phosphorylation and activation while silencing EGFRwt inhibits EGFRvIII activation. Both the dimerization arm and the kinase activity of EGFRwt are required for EGFRvIII activation. EGFRwt activates EGFRvIII by facilitating EGFRvIII dimerization. We have previously identified HB-EGF, a ligand for EGFRwt, as a gene induced specifically by EGFRvIII. In this study, we show that HB-EGF is induced by EGFRvIII only when EGFRwt is present. Remarkably, altering HB-EGF recapitulates the effect of EGFRwt on EGFRvIII activation. Thus, increasing HB-EGF leads to a striking increase in EGFRvIII tyrosine phosphorylation while silencing HB-EGF attenuates EGFRvIII phosphorylation, suggesting that an EGFRvIII–HB-EGF–EGFRwt feed-forward loop regulates EGFRvIII activation. Silencing EGFRwt or HB-EGF leads to a striking inhibition of EGFRvIII-induced tumorigenicity, while increasing EGFRwt or HB-EGF levels resulted in accelerated EGFRvIII-mediated oncogenicity in an orthotopic mouse model. Furthermore, we demonstrate the existence of this loop in human GBM. Thus, our data demonstrate that oncogenic activation of EGFRvIII in GBM is likely maintained by a continuous EGFRwt–EGFRvIII–HB-EGF loop, potentially an attractive target for therapeutic intervention.

A novel mechanism of cell growth regulation by Cell Cycle and Apoptosis Regulatory Protein (CARP)-1

Background CARP-1/CCAR1, a perinuclear phospho-protein, regulates signaling by adriamycin, steroids, or growth factors. However, intracellular events that regulate CARP-1-dependent cell growth are not fully understood. Results Here we investigated whether CARP-1 is involved in signaling induced by the protein kinase A inhibitor H89. Treatments of human breast cancer cells with H89 resulted in apoptosis that involved enhanced CARP-1 threonine phosphorylation and expression. Depletion of CARP-1, on the other hand, abrogates apoptosis induced by H89. CARP-1 binds with signal transducer TAZ and over-expression of TAZ inhibits apoptosis by CARP-1. CARP-1 (651-759) interacts with a novel, N-terminal epitope of TAZ. H89 treatment stimulates threonine phosphorylation of CARP-1 (651-759), while substitution of threonine667 to alanine interferes with its binding with TAZ and apoptosis by H89. In addition, expression of wild type or CARP-1 (651-759) causes loss of c-myc expression due, in part, to suppression of c-myc transcription. Conclusions CARP-1 threonine667 regulates H89-dependent signaling by a novel pathway that involves modulation of CARP-1 interaction with TAZ and transcriptional down-regulation of c-myc.

The role of NF-κB in the pathogenesis of glioma

Activation of NF-κB affects multiple aspects of cancer biology including cell survival and resistance to treatment. Glioblastoma (GBM) is the most common primary malignant tumor of the brain in adults and is resistant to treatment. Recent studies have reported that NF-κB activation in GBM is widespread and have elucidated the underlying regulatory mechanisms. EGFR gene amplification and mutation are among the key genetic alterations in GBM, and aberrant EGFR signaling is a key activator of NF-κB in GBM. In this review we discuss the evidence for activation of NF-κB in GBM and the key signaling pathways involved. Substantial evidence suggests a role for NF-κB in the pathogenesis of GBM and its resistance to treatment, indicating that NF-κB pathways may be useful targets for treatment.

Curcumin inhibits B[a]PDE‐induced procarcinogenic signals in lung cancer cells, and curbs B[a]P‐induced mutagenesis and lung carcinogenesis

Benzo[a]pyrene is a procarcinogen present in environment and cigarette smoke, which could be bio-transformed in vivo to B[a]PDE, a potent carcinogen known to form DNA adducts and induce mutations. We observed that curcumin, a known chemopreventive, could significantly inhibit the survival of lung cancer cells exposed to B[a]PDE. It also downregulates B[a]PDE-induced nuclear translocation of NF-κB as assessed by Electrophoretic Mobility Shift Assay (EMSA) and NF-κB-dependent reporter gene assay. Ames assay demonstrated its ability to revert the mutagenic property of benzo[a]pyrene. These observations prompted us to evaluate the efficacy of curcumin in preventing B[a]P-induced lung carcinogenesis in vivo and to explore the molecular mechanism associated with it. The average number of tumor nodules present in the lungs of the Swiss albino mice, which received benzo[a]pyrene, was significantly high compared to that received curcumin as 2% diet along with B[a]P. Curcumin treatment significantly reverted histopathological deviations in the lung tissues due to benzo[a]pyrene ingestion. Moreover, curcumin diet reduced benzo[a]pyrene-induced activation of NF-κB and MAPK signaling and Cox-2 transcription in lung tissues of mice. Taken together, this study illustrates multifaceted efficacy of curcumin in preventing lung cancer.