Shikha Gaur

Arginine Starvation Impairs Mitochondrial Respiratory Function in ASS1-Deficient Breast Cancer Cells

Tumors that cannot make arginine undergo mitochondrial dysfunction in the absence of extracellular arginine and die by autophagy. Starved to Death by Lack of Arginine Some breast tumors have low abundance of argininosuccinate synthetase 1 (ASS1), an enzyme involved in the synthesis of the amino acid arginine. Although ASS1 deficiency enhances the growth of these tumors, these cells are sensitive to depletion of external arginine. ADI-PEG20 (pegylated arginine deiminase), a modified form of a microbial enzyme, metabolizes arginine. Qiu et al. found that in various breast cancer cell lines that were deficient in ASS1, addition of ADI-PEG20 to the medium induced widespread mitochondrial dysfunction, which in turn triggered autophagy, an adaptive catabolic process that can lead to cell death if unchecked. In mice, ADI-PEG20 treatment slowed the growth of tumors formed from ASS1-deficient breast cancer cells only if these cells could undergo autophagy. Low ASS1 abundance in tumor samples from patients with breast cancer correlated with low survival rates, suggesting that treatments that induce arginine starvation may be beneficial in patients with ASS1-deficient breast cancers. Autophagy is the principal catabolic response to nutrient starvation and is necessary to clear dysfunctional or damaged organelles, but excessive autophagy can be cytotoxic or cytostatic and contributes to cell death. Depending on the abundance of enzymes involved in molecule biosynthesis, cells can be dependent on uptake of exogenous nutrients to provide these molecules. Argininosuccinate synthetase 1 (ASS1) is a key enzyme in arginine biosynthesis, and its abundance is reduced in many solid tumors, making them sensitive to external arginine depletion. We demonstrated that prolonged arginine starvation by exposure to ADI-PEG20 (pegylated arginine deiminase) induced autophagy-dependent death of ASS1-deficient breast cancer cells, because these cells are arginine auxotrophs (dependent on uptake of extracellular arginine). Indeed, these breast cancer cells died in culture when exposed to ADI-PEG20 or cultured in the absence of arginine. Arginine starvation induced mitochondrial oxidative stress, which impaired mitochondrial bioenergetics and integrity. Furthermore, arginine starvation killed breast cancer cells in vivo and in vitro only if they were autophagy-competent. Thus, a key mechanism underlying the lethality induced by prolonged arginine starvation was the cytotoxic autophagy that occurred in response to mitochondrial damage. Last, ASS1 was either low in abundance or absent in more than 60% of 149 random breast cancer biosamples, suggesting that patients with such tumors could be candidates for arginine starvation therapy.

Pharmacodynamic and pharmacogenomic study of the nanoparticle conjugate of camptothecin CRLX101 for the treatment of cancer

CRLX101 is a nanopharmaceutical consisting of cyclodextrin-based polymer molecule and camptothecin. The CRLX101 nanoparticle is designed to concentrate and slowly release camptothecin in tumors over an extended period of time. Tumor biopsy and blood samples collected from patients with advanced solid malignancies before and after CRLX101 treatment are subjected to immunohistochemistry and pharmacogenomics. The expression of Topoisomerase-1, Ki-67, CaIX, CD31 and VEGF decreased after CRLX101 treatment. The expressions of these proteins are inversely proportional with survival duration of the patients. The Drug Metabolism Enzymes and Transporters (DMET) array shows an allele frequency in patients similar to global populations with none of the SNPs associated with toxicity. The results suggest that the observed lower toxicity is not likely to be due to different genotypes in SNPs. CRLX101 demonstrates a promising anti-tumor activity in heavily pre-treated or treatment-refractory solid tumor malignancies presumably by inhibition of proliferation and angiogenesis correlating with tumor growth inhibition. From the clinical editor: In this cancer treatment study clinical samples collected from patients were subjected to immunohistochemistry and pharmacogenomics. The expressions of key proteins that are inversely proportional with survival duration of the patients decreased after treatment with CRLX101, a camptothecin slow-release nanoparticle conjugate. This anti-tumor activity in heavily pre-treated and treatment resistant solid tumors, promises a novel therapeutic approach.

Mechanistic Control of Carcinoembryonic Antigen-related Cell Adhesion Molecule-1 (CEACAM1) Splice Isoforms by the Heterogeneous Nuclear Ribonuclear Proteins hnRNP L, hnRNP A1, and hnRNP M

Carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) is expressed in a variety of cell types and is implicated in carcinogenesis. Alternative splicing of CEACAM1 pre-mRNA generates two cytoplasmic domain splice variants characterized by the inclusion (L-isoform) or exclusion (S-isoform) of exon 7. Here we show that the alternative splicing of CEACAM1 pre-mRNA is regulated by novel cis elements residing in exon 7. We report the presence of three exon regulatory elements that lead to the inclusion or exclusion of exon 7 CEACAM1 mRNA in ZR75 breast cancer cells. Heterologous splicing reporter assays demonstrated that the maintenance of authentic alternative splicing mechanisms were independent of the CEACAM1 intron sequence context. We show that forced expression of these exon regulatory elements could alter CEACAM1 splicing in HEK-293 cells. Using RNA affinity chromatography, three members of the heterogeneous nuclear ribonucleoprotein family (hnRNP L, hnRNP A1, and hnRNP M) were identified. RNA immunoprecipitation of hnRNP L and hnRNP A1 revealed a binding motif located central and 3′ to exon 7, respectively. Depletion of hnRNP A1 or L by RNAi in HEK-293 cells promoted exon 7 inclusion, whereas overexpression led to exclusion of the variable exon. By contrast, overexpression of hnRNP M showed exon 7 inclusion and production of CEACAM1-L mRNA. Finally, stress-induced cytoplasmic accumulation of hnRNP A1 in MDA-MB-468 cells dynamically alters the CEACAM1-S:CEACAM1:L ratio in favor of the l-isoform. Thus, we have elucidated the molecular factors that control the mechanism of splice-site recognition in the alternative splicing regulation of CEACAM1.

A Small-Molecule Blocking Ribonucleotide Reductase Holoenzyme Formation Inhibits Cancer Cell Growth and Overcomes Drug Resistance

Ribonucleotide reductase (RNR) is an attractive target for anticancer agents given its central function in DNA synthesis, growth, metastasis, and drug resistance of cancer cells. The current clinically established RNR inhibitors have the shortcomings of short half-life, drug resistance, and iron chelation. Here, we report the development of a novel class of effective RNR inhibitors addressing these issues. A novel ligand-binding pocket on the RNR small subunit (RRM2) near the C-terminal tail was proposed by computer modeling and verified by site-directed mutagenesis and nuclear magnetic resonance (NMR) techniques. A compound targeting this pocket was identified by virtual screening of the National Cancer Institute (NCI) diverse small-molecule database. By lead optimization, we developed the novel RNR inhibitor COH29 that acted as a potent inhibitor of both recombinant and cellular human RNR enzymes. COH29 overcame hydroxyurea and gemcitabine resistance in cancer cells. It effectively inhibited proliferation of most cell lines in the NCI 60 human cancer panel, most notably ovarian cancer and leukemia, but exerted little effect on normal fibroblasts or endothelial cells. In mouse xenograft models of human cancer, COH29 treatment reduced tumor growth compared with vehicle. Site-directed mutagenesis, NMR, and surface plasmon resonance biosensor studies confirmed COH29 binding to the proposed ligand-binding pocket and offered evidence for assembly blockade of the RRM1-RRM2 quaternary structure. Our findings offer preclinical validation of COH29 as a promising new class of RNR inhibitors with a new mechanism of inhibition, with broad potential for improved treatment of human cancer.

Inhibitors of mTOR overcome drug resistance from topoisomerase II inhibitors in solid tumors

The present study was performed to investigate the possible role of mTOR inhibitors in restoring chemosensitivity to adriamycin/cisplatin and elucidate the underlying mechanism. Combining adriamycin/cisplatin with torisel synergistically inhibited the cell proliferation in human oropharyngeal carcinoma cell line KB and its multidrug-resistant subclone KB/7D. Combining adriamycin and torisel inhibited the phosphorylation of 4EBP-1 and p70S6K, the proteins involved in mTOR pathway, increased expression of γH2AX indicative of DNA damage, triggered cell cycle arrest at G2/M and apoptosis. We conclude that chromatin decondensation by DNA damage provided an easy access for torisel to block the translation of proteins essential for DNA repair thereby restoring the chemosensitivity.

Effects of iodonium-class flavin dehydrogenase inhibitors on growth, reactive oxygen production, cell cycle progression, NADPH oxidase 1 levels, and gene expression in human colon cancer cells and xenografts

Iodonium-class flavoprotein dehydrogenase inhibitors have been demonstrated to possess antiproliferative potential and to inhibit reactive oxygen production in human tumor cells, although the mechanism(s) that explains the relationship between altered cell growth and the generation of reactive oxygen species (ROS) remains an area of active investigation. Because of the ability of these compounds to inhibit the activity of flavoprotein-containing epithelial NADPH oxidases, we chose to examine the effects of several iodonium-class flavoprotein inhibitors on human colon cancer cell lines that express high, functional levels of a single such oxidase (NADPH oxidase 1, or Nox1). We found that diphenyleneiodonium (DPI), di-2-thienyliodonium (DTI), and iodonium diphenyl inhibited the growth of Caco2, HT-29, and LS-174T colon cancer cells at concentrations (10-250nM for DPI, 0.5-2.5μM for DTI, and 155nM to 10μM for iodonium diphenyl) substantially lower than needed for DU145 human prostate cancer cells, which do not possess functional NADPH oxidase activity. Drug treatment was associated with decreased H2O2 production and diminished intracellular ROS levels, lasting up to 24h, after short-term (1-h) exposure to the iodonium analogs. Decreased tumor cell proliferation was caused, in part, by a profound block in cell cycle progression at the G1/S interface in both LS-174T and HT-29 cells exposed to either DPI or DTI; and the G1 block was produced, for LS-174T cells, by upregulation of p27 and a drug concentration-related decrease in the expression of cyclins D1, A, and E that was partially prevented by exogenous H2O2. Not only did DPI and DTI decrease intracellular ROS, they both also significantly decreased the mRNA expression levels of Nox1, potentially contributing to the prolonged reduction in tumor cell reactive oxygen levels. We also found that DPI and DTI significantly decreased the growth of both HT-29 and LS-174T human tumor xenografts, at dose levels that produced peak plasma concentrations similar to those utilized for our in vitro experiments. These findings suggest that iodonium analogs have therapeutic potential for NADPH oxidase-containing human colon cancers in vivo and that at least part of their antineoplastic mechanism of action may be related to targeting Nox1.

Mutants TP53 p.R273H and p.R273C but not p.R273G enhance cancer cell malignancy.

Mutation of the tumor suppressor TP53 gene occurs in greater than half of all human cancers. In addition to loss of tumor suppressor function of wild‐type TP53, gain‐of‐function mutations endow cancer cells with more malignant properties. R273 is a mutation hotspot with the p.R273H, p.R273C, and p.R273G variants occurring most commonly in patient samples. To better understand the consequences of these R273 mutations, we constructed cancer cell lines expressing TP53 p.R273H, p.R273C, or p.R273G and explored their characteristics. We found that p.R273H and p.R273C, but not p.R273G, enhanced proliferation, invasion, and drug resistance in vitro. Furthermore, breast cancer susceptibility protein 1 was upregulated by mutant TP53 p.R273H and p.R273C in response to DNA damage and repair. Transcriptional analysis of the TP53‐R273 mutants by RNA‐seq confirmed that the apoptosis pathway was less active in p.R273H and p.R273C, compared with R273G. Molecular dynamics simulation further revealed that TP53‐R273G binds more tightly to DNA than TP53‐R273H or TP53‐R273C. These findings indicate that mutation of TP53 at a single codon has different effects, and likely clinical implications. p.R273H and p.R273C lead to a more aggressive phenotype than p.R273G. These findings may contribute to future diagnosis and therapy in TP53 mutant cancers.

Small interfering RNAs targeting cyclin D1 and cyclin D2 enhance the cytotoxicity of chemotherapeutic agents in mantle cell lymphoma cell lines.

Abstract Cyclin D1 (CCND1) is a known cell cycle regulator whose overexpression is a hallmark of mantle cell lymphoma (MCL). Although molecular techniques have unified the diagnostic approach to MCL, no therapeutic advances have been made to target this particular pathway. The significance of CCND1 in the pathogenesis and treatment of MCL has yet to be defined. We have taken advantage of RNA interference (RNAi) to down-regulate CCND1 expression in two MCL cell lines (Granta-519 and Jeko-1) to investigate the cytotoxic effect of combining RNAi with conventional chemotherapeutic agents. We designed four small interfering RNAs (siRNAs) specific to CCND1, one specific to CCND2, and one dual-targeting siRNA that simultaneously down-regulates CCND1 and CCND2. Etoposide and doxorubicin were used as chemotherapeutics in combination with the siRNAs. The transfected siRNAs in MCL cell lines triggered 40–60% reduction in target mRNA and protein levels. Importantly, the siRNA-mediated reduction in cyclins resulted in decreased IC50 (50% inhibitory concentration) values for both doxorubicin and etoposide. The combination of siRNA-mediated inhibition of the cyclins along with chemotherapeutic agents could potentially be used to lower the effective doses of the chemotherapeutic agents and reduce drug-related toxicities.

NADPH Oxidase 1 Supports Proliferation of Colon Cancer Cells by Modulating Reactive Oxygen Species-Dependent Signal Transduction.

Reactive oxygen species (ROS) play a critical role in cell signaling and proliferation. NADPH oxidase 1 (NOX1), a membrane-bound flavin dehydrogenase that generates O2̇̄, is highly expressed in colon cancer. To investigate the role that NOX1 plays in colon cancer growth, we used shRNA to decrease NOX1 expression stably in HT-29 human colon cancer cells. The 80–90% decrease in NOX1 expression achieved by RNAi produced a significant decline in ROS production and a G1/S block that translated into a 2–3-fold increase in tumor cell doubling time without increased apoptosis. The block at the G1/S checkpoint was associated with a significant decrease in cyclin D1 expression and profound inhibition of mitogen-activated protein kinase (MAPK) signaling. Decreased steady-state MAPK phosphorylation occurred concomitant with a significant increase in protein phosphatase activity for two colon cancer cell lines in which NOX1 expression was knocked down by RNAi. Diminished NOX1 expression also contributed to decreased growth, blood vessel density, and VEGF and hypoxia-inducible factor 1α (HIF-1α) expression in HT-29 xenografts initiated from NOX1 knockdown cells. Microarray analysis, supplemented by real-time PCR and Western blotting, revealed that the expression of critical regulators of cell proliferation and angiogenesis, including c-MYC, c-MYB, and VEGF, were down-regulated in association with a decline in hypoxic HIF-1α protein expression downstream of silenced NOX1 in both colon cancer cell lines and xenografts. These studies suggest a role for NOX1 in maintaining the proliferative phenotype of some colon cancers and the potential of NOX1 as a therapeutic target in this disease.

Abstract 1724: Inhibition of the PI3K and mTOR pathways reverses gemcitabine resistance in pancreatic cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL BACKGROUND: Despite chemotherapy, survival for most patients with pancreatic cancer is less than one year. These dismal statistics reflect the lack of effective therapy. Understanding the molecular pathways that drive cancer growth may help target our therapy. Over 90% of pancreatic cancers are K-ras mutant which activates the PI3K/AKT pathway and signals downstream to mTOR leading to cell growth, proliferation and survival. We explored the combination of gemcitabine and BEZ235, a novel dual PI3K/mTOR inhibitor, in pancreatic cancer cell lines. METHODS: Cytotoxicity assays were performed utilizing the CellTiter-96-Aqueous-One-Solution-Cell-Proliferation-Assay-(MTS) from Promega in K-ras mutant MIA PaCa-2 pancreatic cancer cell lines. We generated gemcitabine resistant cell lines by serial passage of cells with increasing concentrations of drug. We utilized the CalcuSyn program by Biosoft to quantify synergism or antagonism with drug combinations. We also collected cells 6 hours after drug treatment for analysis of phosphorylated proteins. RESULTS: MIA PaCa-2 wild-type and gemcitabine resistant cells were treated with 100, 50 and 25 nM of either gemcitabine, BEZ235 or the combination of both. At the 100 nM dose, cell viability of the wild-type cells was 66%, 55% and 39% respectively. In the gemcitabine resistant cells, cell viability was 84%, 63% and 44%. The combination index was 0.51 in the wild-type cells and 0.46 in the gemcitabine resistant cells indicating synergy in both cell lines. In both cell lines, p-S6K and p-AKT levels increased with gemcitabine treatment which was abrogated by treatment with BEZ-235. Interestingly, in the gemcitabine resistant cells, there appeared to be significantly increased levels of p-MEK1 compared to control. CONCLUSION: Because pancreatic cancer remains resistant to current therapies despite recent advances in the development of molecular targeted therapies, there is an urgency to understand these pathways. We showed that the combination of gemcitabine and BEZ235 is synergistic in both cell lines but cell viability still remains high at 39% and 44% respectively. Combination with a MEK inhibitor may help to increase cell apoptosis and studies are on-going. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1724. doi:10.1158/1538-7445.AM2011-1724