Survival Of Carcinoma Cells Research Articles

Expression and effects of JMJD2A histone demethylase in endometrial carcinoma.

Previous studies have demonstrated that JMJD2A is a potential oncogene and is overexpressed in human tumors. However, its role in the endometrial carcinoma remains largely unknown. In this study, we discovered that JMJD2A was overexpressed in endometrial carcinoma, using immunohistochemistry, quantitative real- time polymerase chain reaction, and western blotting. Downregulation of JMJD2A led to reduced endometrial carcinoma RL95-2 and ISK cell proliferation, invasion and metastasis as asessed with cell counting kit-8, cell migration and invasive assays. Collectively, our results support that JMJD2A is a promoter of endometrial carcinoma cell proliferation and survival, and is a potential novel drug target.

Expression of the lysyl oxidase propeptide in hepatocellular carcinoma and its clinical relevance

Lysyl oxidase is an important extracellular matrix remodeling enzyme and plays critical roles in tumor progression and development. Its tumor-suppressor activity has been shown to depend on the propeptide region. Previous studies have reported that the expression levels of lysyl oxidase propeptide (LOX-PP) are associated with cancer of the breast, pancreas, lung, prostate and gastrointestinal system. However, to date, the exact effects and molecular mechanisms of LOX-PP in hepatocellular carcinoma progression are still unclear. The present study aimed to investigate the expression and clinical significance of LOX-PP in human hepatocellular carcinoma. First, 42 cases of hepatocellular carcinoma and corresponding adjacent non-cancerous tissues (ANCTs) were collected, and the expression of LOX-PP in these samples was assessed by immunohistochemistry (IHC). The clinicopathological characteristics of all patients were recorded. Next, in in vitro studies, recombinant adenovirus LOX (ad-LOX-PP) was used to infect hepatocellular carcinoma cell lines to determine the function of LOX-PP. To determine whether ad-LOX-PP affects hepatocellular carcinoma cell survival, cell viability was examined by CCK-8 assay, and cell cycle progression was assessed by flow cytometry. We also investigated the effects of LOX-PP on the expression of cell cycle regulators (cyclin D1 and cyclin E) by western blot analysis. The migration and invasion capacities of hepatocellular carcinoma cells were observed by wound-healing and tranwell invasion assays. To further investigate how LOX-PP affects migration levels of matrix metallopeptidase (MMP)-2 and MMP-9 were assessed by western blot analysis. Additionally, markers of the PI3K and MAPK signaling pathway were detected to further confirm the mechanisms of LOX-PP. As a result, reduced expression of LOX-PP was found in hepatocellular carcinoma tissues, when compared with that in the ANCTs (15 vs. 83%, P<0.01), and its expression was associated with tumor stage and distant metastasis (each P<0.05). Proliferation in hepatocellular carcinoma cells was significantly decreased in the ad-LOX-PP group as indicated by CCK-8 assay. LOX-PP significantly reduced the expression of Ki-67, while prominent increases in the rate of apoptosis and cell cycle arrest were observed. Similarly, cell migration was significantly inhibited in the ad-LOX-PP group as evidenced by transwell invasion and wound-healing assays. The expression levels of MMP-2 and MMP-9 were attenuated in the ad-LOX-PP group, suggesting that LOX-PP inhibits hepatocellular carcinoma cell migration via down-regulation of MMPs expression. When LOX-PP expression was potentiated by an adenovirus containing LOX-PP, the expression of p-ERK was significantly downregulated, indicating that LOX-PP inhibits hepatocellular carcinoma cell proliferation and induces its apoptosis probably through downregulation of the MAPK/ERK pathway.

Gramicidin A Induces Metabolic Dysfunction and Energy Depletion Leading to Cell Death in Renal Cell Carcinoma Cells

Ionophores are lipid-soluble organic molecules that disrupt cellular transmembrane potential by rendering biologic membranes permeable to specific ions. They include mobile-carriers that complex with metal cations and channel-formers that insert into the membrane to form hydrophilic pores. Although mobile-carriers possess anticancer properties, investigations on channel-formers are limited. Here, we used the channel-forming ionophore gramicidin A to study its effects on the growth and survival of renal cell carcinoma (RCC) cells. RCC is a histologically heterogeneous malignancy that is highly resistant to conventional treatments. We found that gramicidin A reduced the in vitro viability of several RCC cell lines at submicromolar concentrations (all IC50 < 1.0 μmol/L). Gramicidin A exhibited similar toxicity in RCC cells regardless of histologic subtype or the expression of either the von Hippel-Lindau tumor suppressor gene or its downstream target, hypoxia-inducible factor-1α. Gramicidin A decreased cell viability equal to or greater than the mobile-carrier monensin depending on the cell line. Mechanistic examination revealed that gramicidin A blocks ATP generation by inhibiting oxidative phosphorylation and glycolysis, leading to cellular energy depletion and nonapoptotic cell death. Finally, gramicidin A effectively reduced the growth of RCC tumor xenografts in vivo. These results show a novel application of gramicidin A as a potential therapeutic agent for RCC therapy.

Abstract B043: Role of IRS2-microtubule interactions in breast carcinoma cell survival

Abstract The biggest obstacle for the eradication of breast cancer is the metastatic spread of tumor cells beyond the primary cancer site. Insulin-like Growth Factor-1 receptor (IGF-1R) signaling has been implicated in mammary tumor progression from localized to metastatic disease. Signal transduction by the IGF-1R requires the Insulin Receptor Substrate (IRS) adaptor proteins IRS1 and IRS2. These scaffold proteins lack intrinsic kinase activity but organize signaling downstream of the receptor. Despite their homology, these proteins have distinct functions, with IRS1 regulating tumor cell proliferation and IRS2 regulating metabolism, survival and invasion. In vivo studies performed by our group demonstrated that Irs2-/- tumors are poorly metastatic, whereas Irs1-/- tumors that express elevated Irs2 are highly metastatic, when compared with wild type tumors. These studies suggest that IRS2 is a positive regulator of mammary tumor metastasis. Importantly, the mechanism(s) responsible for the divergent roles of these homologous proteins in tumor biology remains unknown. Recent studies from our group and others suggest that the subcellular localization of IRS1 and IRS2 may play a role in their distinct functions. Specifically, in human breast tumors, IRS1 is expressed either in the nucleus or cytoplasm and IRS2 is expressed either at the cell membrane or in the cytoplasm. Of note, patients with breast tumors that have IRS2 localized at the cell membrane have decreased overall survival. We hypothesized that trafficking of the IRS proteins to distinct subcellular compartments is essential for their differential functions in breast cancer and that the cytoskeleton would play an important role in this localization. Our preliminary studies using MDA-MB-231 and SUM-159 breast carcinoma cells revealed that IRS2-mediated signaling, but not IRS1-mediated signaling, requires an intact microtubule cytoskeleton. Treatment of breast carcinoma cells with microtubule disrupting agents inhibited IGF1 stimulated activation of PI3K/Akt signaling in cells that predominantly signal through IRS2. In contrast, cells that signal predominantly through IRS1 retained the ability to activate Akt after disruption of the microtubule cytoskeleton. Suppression of IRS2 expression abrogated the effect of microtubule disruption on Akt activation, whereas restoration of Irs2 in Irs2-/- mouse mammary tumor cells sensitized these cells to the effects of microtubule disrupting agents. Likewise, overexpression of IRS1 in MDA-MB-435 breast carcinoma cells that express predominantly IRS2 caused resistance to the effects of microtubule disruption on IGF1-stimulated Akt activation. Given the importance of Akt for tumor cell survival, we assessed the impact of IRS2 signaling on the survival of breast carcinoma cells after microtubule disruption. Cells that signal predominantly through IRS2 had diminished cell survival after forty-eight hours of treatment with Nocodazole and Vinblastine. Moreover, Irs2-/- cells are more resistant to these agents, and restoration of Irs2 expression resulted in a significant increase in cell death. Taken together our data suggest that IRS2 may interact with the microtubule cytoskeleton and that this interaction plays a role in the ability of IRS2 to activate Akt signaling and promote breast carcinoma cell survival. More importantly, our data implicates IRS2 as a biomarker of tumor sensitivity to microtubule disrupting agents. Understanding the mechanism by which IRS2 interacts with microtubules and how this influences its function may facilitate the development of new therapeutic approaches for the effective treatment of breast cancer metastasis. Citation Format: Jose R. Mercado-Matos, Jennifer Clark, Andrew J. Piper, Jenny Janusis, Leslie M. Shaw. Role of IRS2-microtubule interactions in breast carcinoma cell survival. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B043.

Quinoline 3-sulfonamides inhibit lactate dehydrogenase A and reverse aerobic glycolysis in cancer cells

BackgroundMost normal cells in the presence of oxygen utilize glucose for mitochondrial oxidative phosphorylation. In contrast, many cancer cells rapidly convert glucose to lactate in the cytosol, a process termed aerobic glycolysis. This glycolytic phenotype is enabled by lactate dehydrogenase (LDH), which catalyzes the inter-conversion of pyruvate and lactate. The purpose of this study was to identify and characterize potent and selective inhibitors of LDHA.MethodsHigh throughput screening and lead optimization were used to generate inhibitors of LDHA enzymatic activity. Effects of these inhibitors on metabolism were evaluated using cell-based lactate production, oxygen consumption, and 13C NMR spectroscopy assays. Changes in comprehensive metabolic profile, cell proliferation, and apoptosis were assessed upon compound treatment.Results3-((3-carbamoyl-7-(3,5-dimethylisoxazol-4-yl)-6-methoxyquinolin-4-yl) amino) benzoic acid was identified as an NADH-competitive LDHA inhibitor. Lead optimization yielded molecules with LDHA inhibitory potencies as low as 2 nM and 10 to 80-fold selectivity over LDHB. Molecules in this family rapidly and profoundly inhibited lactate production rates in multiple cancer cell lines including hepatocellular and breast carcinomas. Consistent with selective inhibition of LDHA, the most sensitive breast cancer cell lines to lactate inhibition in hypoxic conditions were cells with low expression of LDHB. Our inhibitors increased rates of oxygen consumption in hepatocellular carcinoma cells at doses up to 3 microM, while higher concentrations directly inhibited mitochondrial function. Analysis of more than 500 metabolites upon LDHA inhibition in Snu398 cells revealed that intracellular concentrations of glycolysis and citric acid cycle intermediates were increased, consistent with enhanced Krebs cycle activity and blockage of cytosolic glycolysis. Treatment with these compounds also potentiated PKM2 activity and promoted apoptosis in Snu398 cells.ConclusionsRapid chemical inhibition of LDHA by these quinoline 3-sulfonamids led to profound metabolic alterations and impaired cell survival in carcinoma cells making it a compelling strategy for treating solid tumors that rely on aerobic glycolysis for survival.

Carboxypeptidase E promotes cancer cell survival, but inhibits migration and invasion

Carboxypeptidase E (CPE), a prohormone processing enzyme is highly expressed and secreted from (neuro)endocrine tumors and gliomas, and has been implicated in cancer progression by promoting tumor growth. Our study demonstrates that secreted or exogenously applied CPE promotes survival of pheochromocytoma (PC12) and hepatocellular carcinoma (MHCC97H) cells under nutrient starvation and hypoxic conditions, but had no effect on their proliferation. CPE also reduced migration and invasion of fibrosarcoma (HT1080) cells. We show that CPE treatment mediates survival of MHCC97H cells during metabolic stress by up-regulating the expression of anti-apoptotic protein BCL-2, and other pro-survival genes, via activation of the ERK1/2 pathway.

Notice of Retraction

Cellular fibronectin (cFN) is one of the main components of tissue extracellular matrices and is involved in multiple physiologic and pathologic processes such as embryogenesis, wound healing, inflammation and tumor progression. The function of fibronectin in regulating normal cell adhesion and migration is well documented, but its function in cancer progression is only partially unraveled. We have reported previously that fibronectin stimulates the proliferation and survival of non-small lung carcinoma cells through upregulation of pro-oncogenic signals related to cyclooxygenase-2/phosphatidylinositol-3-kinase/protein kinase B (COX-2/PI3-K/AKT)/mammalian target of rapamycin triggered by activation of the integrin α5β1. Here, we extend these studies by showing that fibronectin promotes epithelial-mesenchymal transition (EMT) in lung cancer cells. We found that cFN, but not plasma fibronectin or type 1 collagen, induces lung carcinoma cell scattering in vitro, promotes cell migration and invasion of Matrigel and stimulates the expression of the mesenchymal marker α-smooth muscle actin while decreasing the expression of the epithelial marker E-cadherin through PI3-K and Erk pathways. Interestingly, the extra domain A (EDA) within cFN was found to be crucial for this process, as confirmed by testing cells overexpressing EDA or cells exposed to EDA-containing matrices. We found that the integrin α9, but not α5, mediated cFN-induced EMT as silencing integrin α9 neutralized cFN-induced EMT. Overall, our findings show that the EDA domain within cFN induces EMT in lung carcinoma cells through integrin α9-mediated activation of PI3-K and Erk.

Effects of the monoamine oxidase inhibitors pargyline and tranylcypromine on cellular proliferation in human prostate cancer cells

Chemotherapy is one of the therapeutic strategies that has been used for the inhibition of cancer cell proliferation in several types of cancer, including prostate cancer. Although monoamine oxidase (MAO) inhibitors, phytoestrogen and antioxidants used in chemotherapy have been systematically studied, their effects on cancer cell growth remain to be fully understood. The purpose of this study was to investigate the effects of the MAO inhibitors, pargyline and tranylcypromine on cell survival in human prostate carcinoma (LNCaP-LN3) cells. After treating LNCaP-LN3 cells with pargyline or tranylcypromine, we examined cell proliferation, cell cycle pattern, apoptosis and the expression levels of apoptosis-related genes. The proliferation of cells exposed to pargyline decreased in a dose- and time-dependent manner, while tranylcypromine-treated cells showed the opposite results. Treatment with pargyline significantly induced cell cycle arrest at the G1 phase compared to the control and tranylcypromine-treated cells. In addition, pargyline induced an increase in the cell death rate by promoting apoptosis; however, tranylcypromine had no effect on LNCaP-LN3 cells. Based on our results, we suggest that pargyline is more powerful than tranylcypromine for the treatment of human prostate cancer.

Autocrine human growth hormone increases sensitivity of mammary carcinoma cell to arsenic trioxide-induced apoptosis

Human growth hormone (hGH) has been increasingly implicated in a variety of cancers; its up-regulation is observed in breast cancer and correlates with a poor outcome. Autocrine hGH promotes mammary carcinoma cell survival, proliferation, immortalization; it confers an invasive phenotype as a result of an epithelial–mesenchymal transition and contributes to chemoresistance and radioresistance. Arsenic trioxide (ATO) is being successfully used as a first and second line therapy for the treatment of patients with acute promyelocytic leukemia. It also inhibits tumor cell growth and induces apoptosis in a broad range of solid tumors. In the present study, we investigated the effect of hGH on sensitivity of a mammary adenocarcinoma cell to ATO, using a stable hGH-transfectant MCF-7 cell line, MCF7-hGH. Our results demonstrated for the first time that the overexpression of hGH increased sensitivity of the breast cancer cell line MCF-7 to ATO through apoptotic and anti-proliferative mechanisms. The effect of ATO on the transcriptional level of genes involved in survival (Bcl-2, Bax and Survivin), self-sufficiency in growth signals (c-Myc, ARF, Cdc25A, p53 and Bax), immortalization (hTERT) and invasion and metastasis (MMP-2 and MMP-9, uPA and uPAR and E-cadherin) was more pronounced in MCF7-hGH compared with its parental MCF-7 line. Our study may highlight the potential application of ATO for the treatment of patients with breast cancer, especially in those who have metastatic and chemoresistant tumor phenotype possibly due to the over expression of hGH.

Cytotoxicity of hepatocellular carcinoma cells to hyperthermic and ablative temperature exposures: In vitro studies and mathematical modelling

Purpose: Image-guided ablative therapies use temperatures greater than 45 °C to kill abnormal cells. There is limited published data of cell survival after ablative temperature exposures, which is of importance to predict ablation zone dimensions. The objective of this study was to determine and mathematically model survival of hepatocellular carcinoma cells following ablative temperature exposures (45–60 °C).Materials and methods: Hepatocellular carcinoma (HCC) cell lines were plated in 96-well plates, and heated between 45 and 60 °C for 0–32 min. Heating was applied by a rapid media exchange with heated Hank’s balanced salt solution (HBSS) in a temperature-controlled water bath. Cell viability was determined by MTS assay. Survival data was modelled by the Arrhenius model, and the thermal isoeffective dose (TID) model where kinetic parameters were determined via non-linear optimisation.Results: Results suggest that the thermal dose based on cumulative equivalent minutes and parameters as used for hyperthermia exposures (<43 °C) is not applicable for ablative exposures. We found R = 0.72 for temperatures between 45–60°C for the TID model. The Arrhenius parameters were frequency factor A = 3.25E43 1/s, and activation energy Ea = 281 kJ/mol. These parameters correlate well with a prior study in the same cell line, and with threshold temperatures for necrosis from in vivo studies.Conclusions: Our results suggest that standard TID model kinetic parameters based on hyperthermia studies, often also used at ablation temperatures, are not applicable at these higher temperatures for HCC cells.

Correction: S100A8 and S100A9 Are Associated with Colorectal Carcinoma Progression and Contribute to Colorectal Carcinoma Cell Survival and Migration via Wnt/β-Catenin Pathway

Correction: S100A8 and S100A9 Are Associated with Colorectal Carcinoma Progression and Contribute to Colorectal Carcinoma Cell Survival and Migration via Wnt/β-Catenin Pathway

S100A8 and S100A9 Are Associated with Colorectal Carcinoma Progression and Contribute to Colorectal Carcinoma Cell Survival and Migration via Wnt/β-Catenin Pathway

Background and ObjectiveS100A8 and S100A9, two members of the S100 protein family, have been reported in association with the tumor cell differentiation and tumor progression. Previous study has showed that their expression in stromal cells of colorectal carcinoma (CRC) is associated with tumor size. Here, we investigated the clinical significances of S100A8 and S100A9 in tumor cells of CRC and their underlying molecular mechanisms.MethodsExpression of S100A8 and S100A9 in colorectal carcinoma and matching distal normal tissues were measured by reverse transcriptase polymerase chain reaction (RT-PCR), immunohistochemistry and western blot. CRC cell lines treated with the recombinant S100A8 and S100A9 proteins were used to analyze the roles and molecular mechanisms of the two proteins in CRC in vitro.ResultsS100A8 and S100A9 were elevated in more than 50% of CRC tissues and their expression in tumor cells was associated with differentiation, Dukes stage and lymph node metastasis. The CRC cell lines treatment with recombinant S100A8 and S100A9 proteins promoted the viability and migration of CRC cells. Furthermore, the two recombinant proteins also resulted in the increased levels of β-catenin and its target genes c-myc and MMP7. β-catenin over-expression in CRC cells by Adβ-catenin increased cell viability and migration. β-catenin knock-down by Adsiβ-catenin reduced cell viability and migration. Furthermore, β-catenin knockdown also partially abolished the promotive effects of recombinant S100A8 and S100A9 proteins on the viability and migration of CRC cells.ConclusionsOur work demonstrated that S100A8 and S100A9 are linked to the CRC progression, and one of the underlying molecular mechanisms is that extracellular S100A8 and S100A9 proteins contribute to colorectal carcinoma cell survival and migration via Wnt/β-catenin pathway.

Abstract 845: Sirtuin1, a longevity gene, is over-expressed and enhances proliferation and survival of endometrial carcinoma cells via deacetylation of p53.

Abstract Background: Sirtuin1 (SIRT1) is one of the NAD-dependent histone deacetylases, known as a longevity gene induced by caloric restriction or various stresses. SIRT1 also contributes to cell survival under stress conditions through deacetylation of apoptosis or cell cycle-related proteins. The overexpression of SIRT1 in several cancers was reported, however, the expression and function of SIRT1 in endometrial cancer remained undetermined. In this study, we examined the role of SIRT1 on the carcinogenesis and survival of endometrial carcinoma cells. Methods: The expressions of SIRT1 protein was examined by immunostaining using formalin-fixed tissue specimens of 110 endometrial carcinomas, 72 normal endometria and 24 endometrial hyperplasias. Immunoreactivity was evaluated according to the ratio of positive cells in 500 cells and described as a positivity index (PI, full score 100). All of surgical specimens were used after obtaining the consents. The mRNA and protein levels of SIRT1 in endometrial carcinoma cell lines, Ishikawa and HEC1B, were examined using real-time RT-PCR and Western blotting under stresses such as ultraviolet, hypoxia and cisplatin (CDDP) treatment. The viability of those cells under CDDP treatment was examined using WST-1 assay with/without suppression of SIRT1 by SIRT1 inhibitor (EX527) or siRNA. The effect of SIRT1 inhibition on the acetylation of p53 and the p53-related protein expression was in Ishikawa and HEC1B cells were examined using Western blotting under CDDP treatment. Results: Imunoreactivity of SIRT1 protein of carcinoma (PI=37.0±25.6) was significantly higher than that of normal endometrium (PI=25.3±26.5, P= 0.006). The expression of SIRT1 was significantly higher in grade 3 tumors (P= 0.006) and those with lympho-vascular space invasion (P= 0.044). There is a weak correlation between the expression of SIRT1 and growth potential indicated by Ki67 (P=0.051). The expression of SIRT1 protein was inversely correlated with that of p53 in carcinoma tissues and cell lines. In addition, the suppression of SIRT1 significantly reduced cell proliferation activity (P=0.01) and resistance against CDDP (P=0.004) in Ishikawa and HEC1B cells. The suppression of SIRT1 enhanced the acetylation of p53 protein and its downstream proteins. Conclusions: SIRT1 is over-expressed in endometrial carcinoma and may be involved in the carcinogenesis and development. SIRT1 may enhance the survival of endometrial carcinoma cells against various stresses, especially CDDP exposure, via inhibition of p53 by deacetylation. These findings suggest that SIRT1 can be a potent therapeutic target of endometrial carcinoma. Citation Format: Ryoichi Asaka, Tsutomu Miyamoto, Kaori Ishikawa, Yasushi Yamada, Hisanori Kobara, Akihisa Suzuki, Shun'ichiro Taniguchi, Tanri Shiozawa. Sirtuin1, a longevity gene, is over-expressed and enhances proliferation and survival of endometrial carcinoma cells via deacetylation of p53. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 845. doi:10.1158/1538-7445.AM2013-845

Abstract 5418: Rapid LDH5 inhibition reverses malignant metabolic phenotype and impairs survival of hepatocellular carcinoma cells .

Abstract Many cancer cells generate energy by rapidly converting glucose to lactate in the cytosol, a process termed aerobic glycolysis. This metabolic phenotype is recognized as one of the hallmarks of cancer and is enabled by lactate dehydrogenase (LDH), which catalyzes pyruvate to lactate inter-conversion. We find that hepatocellular carcinoma cells express micromolar quantities of LDH5 and that LDH5 protein down-regulation takes about 5 days allowing time for the cells to adapt their metabolism. Since metabolic processes happen in minutes, addressing consequences of LDH5 inhibition by protein down-regulation is inadequate. We screened the GSK compound library and identified a series of quinoline acids as NADH-competitive LDH5 inhibitors. Subsequent lead optimization yielded molecules with LDH5 inhibitory potencies as low as 2-3 nM and selectivity over LDH1 on the order of 10-100-fold. These molecules were cell-permeable and did not have any appreciable activity against a panel of approximately fifty common enzymes, receptors and ion channels, making them the most potent and selective LDH5 inhibitors identified to date. Using these tool inhibitors, we find that rapid chemical inhibition of LDH5 in Snu398 hepatocellular carcinoma cells results in profound inhibition of lactate production and increase in pyruvate as measured by mass spectrometric analysis. Real-time analysis by NMR spectroscopy of live Snu398 cells fed with 13C-labeled glucose demonstrated that chemical LDH5 inhibition led to a rapid decrease in glucose uptake and concomitant slow-down of lactate production. Comprehensive analysis of more than 500 metabolites upon LDH5 inhibition in Snu398 cells revealed that the cytosolic glycolysis pathway was significantly impeded with some up-stream intermediates increasing as much as 40-fold. As the cell lost its ability for cytosolic glucose processing, the TCA cycle activity increased indicating that pyruvate entered the mitochondria and restored their activity resulting in increased oxygen consumption upon LDH5 inhibition. Several pathways that rely on glycolytic and TCA intermediates were also upregulated, including fatty acid metabolism and pentose phosphate pathway. LDH5 inhibition also strongly potentiated PKM2 activity. These profound metabolic alterations greatly impaired cell survival and induced cell death in Snu398 cells. In summary, we have shown that rapid chemical inhibition of LDH5 leads to profound metabolic alterations and impairs cell survival in hepatocellular carcinoma cells making it a compelling strategy for treating solid tumors relying on aerobic glycolysis. Citation Format: Julia Billiard, Roland Annan, Jennifer Ariazi, Jacques Briand, Kristin Brown, Nino Campobasso, Subhas Chakravorty, Deping Chai, Mariela Colón, Elizabeth Davenport, Christopher Dodson, Nathan Gaul, Seth Gilbert, Anthony Jurewicz, Hong Lu, Dean McNulty, Jeanelle McSurdy-Freed, Lisa Miller, Kelvin Nurse, Paru Rao Nuthulaganti, Chad Quinn, Jessica Schneck, Gilbert Scott, Tony Shaw, Christian Sherk, Angela Smallwood, Sharon Sweitzer, James Villa, Gregory Waitt, Richard Wooster, Kevin Duffy. Rapid LDH5 inhibition reverses malignant metabolic phenotype and impairs survival of hepatocellular carcinoma cells . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5418. doi:10.1158/1538-7445.AM2013-5418

Abstract 3451: Gramicidin-based nanopores induce cellular energy depletion and cell death in renal cell carcinoma.

Abstract The plasma membrane physically separates the intracellular components of a cell from the extracellular environment and its integrity is absolutely essential to sustain cellular functions. Ionophores are lipid soluble organic molecules that disrupt cellular transmembrane potential by rendering biological membranes permeable to specific ions. They are classified on the basis of their mechanism of action; mobile carriers complex with metal cations to shield their hydrophilic charge from the interior of the lipid bilayer and channel-formers insert into the membrane to form hydrophilic pores that permit the rapid passage of selected ions through membranes. Research over the past decade has described an new role for mobile carrier ionophores as novel anticancer therapeutics yet the potential anticancer attributes of channel-forming ionophores have not been explored. In this study we utilized the channel-forming ionophore gramicidin A (GA) as a model nanopore to study its effects upon the growth and survival of renal cell carcinoma (RCC) cells. RCC is a histologically heterogeneous malignancy that is highly resistant to conventional chemotherapy. We found that disruption of plasma membrane integrity with GA reduced the in vitro viability of several RCC cell lines when treated at sub-micromolar concentrations (all IC50 &amp;lt; 1.0μM). Comparison of GA with the mobile carrier ionophore monensin revealed that cell viability was decreased by GA to a degree that was equal to or greater than monensin depending on the cell line. Mechanistic examination determined that GA induced cell death that was associated with cellular energy depletion. Importantly, GA exhibited similar toxicity in RCC cells regardless of histological subtype or the expression of either the von Hippel-Lindau (VHL) tumor suppressor or its downstream target hypoxia-inducible factor (HIF) -1α. Taken together, these results demonstrate the utility of nanopore-forming drugs as a new strategy for cancer therapy. Citation Format: Justin M. David, Ayyappan K. Rajasekaran. Gramicidin-based nanopores induce cellular energy depletion and cell death in renal cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3451. doi:10.1158/1538-7445.AM2013-3451

Abstract 905: Design and synthesis of regorafenib analogues against signal transducers and activators of transcription 3 in hepatocellular carcinoma cells.

Abstract Background: Regorafenib has been approved for the treatment of metastatic colorectal cancer (m-RCC) by FDA. The major targets of regorafenib are membrane-bound and intracellular kinases including RET, VEGFR1, VEGFR2, VEGFR3, KIT, PDGFR-alpha, PDGFR-beta, FGFR1, FGFR2, TIE2, DDR2, Trk2A, Eph2A, RAF-1, BRAF, BRAFV600E, SAPK2, PTK5, and Abl pathways. In HCC, signal transducers and activators of transcription 3 (STAT3) is a key transcription factor involving in cell proliferation and survival in hepatocellular carcinoma cells, Targeting STAT3 by small molecule is a promising strategy in HCC therapy. Methods: We have designed and synthesized a series of regorafenib analogues without kinase inhibitory activity. These compounds are treated with and analyzed in terms of STAT3 activity in HCC cell lines. Also, Effects of regorafenib analogues induced cell death are evaluated. Results: We demonstrate that regorafenib and its analogues repress STAT3 phosphorylation, subsequently abrogate STAT3-regulated protein, Mcl-1 and cyclin D1 and further induce apoptosis in HCC cells. Overexpression of STAT3 in PLC-5 cells attenuates apoptotic effect with regorafenib and its analogues treatment, suggesting STAT3, at least, is a major target. Interestingly, the new analugues of regorafenib are devoid of kinase inhibitory activity which implies the involvement of other regulation factors. The activity of negative regulator of STAT3, such as protein tyrosine phosphatases, is under investigation. In summary, the new analogues provide a lead for the design of a new class of agents that inhibit STAT3 along and synergize with clinical drugs in HCC.(Supported by NSC 100-2325-B-010-007) Citation Format: Jung-Chen Su, Kuen-Feng Chen, Wen-Tien Tai, Jui-Wen Huang, Chung-Wai Shiau. Design and synthesis of regorafenib analogues against signal transducers and activators of transcription 3 in hepatocellular carcinoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 905. doi:10.1158/1538-7445.AM2013-905

Abstract 3046: Lipocalin2, an iron transporter, functions as a novel oncogene and enhances the proliferation and survival of endometrial carcinoma cell.

Abstract Background: The mechanism of carcinogenesis and progression of endometrial carcinoma has not been fully understood. To elucidate the molecular machinery, we performed microarray analysis using mRNA extracted from normal endometria, endometrial hyperplasia and carcinoma and found lipocalin2 (LCN2), an iron transporter, to be markedly up-regulated along with disease progression. Furthermore, the over-expression of LCN2 and its receptor, SLC22A17, was associated with poor outcome of the patients with endometrial carcinoma. In the present study, we further analyzed the molecular functions of LCN2 using endometrial carcinoma cells. Methods: An endometrial carcinoma cell line HHUA over-expressing both LCN2 and SLC22A17 was used. We established LCN2-silenced HHUA (HHUAsh) and control-HHUA (HHUAc) by transfection of LCN2-shRNA and scrambled shRNA producing vector, respectively. The growth and migration were examined by WST-1 assay and wound healing assay. Cell viability under stress conditions such as ultraviolet (UV) irradiation or cisplatin (CDDP) treatment, with or without iron-chelation, was measured by WST-1. The intensity of apoptosis was analyzed by TUNEL staining and Apostrand assay. The expression of phosphorylated Akt was examined by Western blotting. In vivo tumor formation was observed by the subcutaneous injection of those HHUA cells to nude mice. The colony formation of these HHUA cells in soft agar was evaluated. In addition, the LCN2 cDNA containing retrovirus vector (LCN2-RET) was transfected to NIH3T3 to observe the transformation ability. Results: The cell migration of HHUAsh was decreased compared with HHUAc. After UV irradiation and CDDP treatment, the residual cell viability of HHUAsh (25.4% and 37.1%, respectively) was significantly lower than that of HHUAc (51.2% and 77.6%, p&amp;lt;0.05). Under these stresses, the enhanced apoptosis and down-regulation of phosphorylated Akt was observed in HHUAsh. Interestingly, these differences were abolished by iron-chelation. The growth of HHUAsh showed no difference with that of HHUAc in vitro. However, LCN2-silencing significantly reduced the tumor growth in nude mice (84% reduction). Moreover, the down-regulation of phospho-Akt expression was observed in tumors of LCN2-silenced HHUA. The colony formation in soft agar of HHUAsh was significantly reduced (66.3% reduction) from that of HHUAc, and this difference was rescued to 12% by addition of recombinant LCN2. In addition, NIH3T3 transfected by LCN2-RET formed focuses. Conclusions: The present study revealed that LCN2 is involved in the genesis and survival of endometrial carcinoma cells under stress conditions and in the tumor growth in vivo, via p-Akt signaling in an iron-dependent manner, suggesting LCN2 to be a novel oncogene. Citation Format: Tsutomu Miyamoto, Ryoichi Asaka, Yasushi Yamada, Kaori Ishikawa, Hisanori Kobara, Akihisa Suzuki, Tanri Shiozawa. Lipocalin2, an iron transporter, functions as a novel oncogene and enhances the proliferation and survival of endometrial carcinoma cell. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3046. doi:10.1158/1538-7445.AM2013-3046

Abstract 4095: Paradoxical activation of the RAF-MEK-ERK pathway in response to nilotinib induces synthetic lethality with MEK inhibition in head and neck cancer cells.

Abstract Cancers of the head and neck are difficult to treat because both the therapy and the tumor impact essential functions such as speech and swallowing and they can significantly alter facial appearance. The RAS/RAF/MEK/ERK cascade is one of the major signaling pathways that leads to head and neck squamous cell carcinoma (HNSCC) cell survival. There are three RAS genes (HRAS, KRAS, and NRAS) and together, they are mutated in about 30% of human cancers. RAS can also be activated by upstream signaling. Paradoxically RAF inhibition induces RAF-dimerization and causes subsequent activation of MEK/ERK pathway in presence of activated RAS in melanoma. Similarly the ABL inhibitor nilotinib acts as weak inhibitor of RAF kinase and also induces RAF-dimerization and subsequent activation of MEK/ERK in the presence of activated RAS in leukemia and pancreatic cancer cells. Nilotinib synergizes with MEK inhibitition to kill nilotinib-resistant leukemia cells (Abl T351I). We hypothesized that nilotinib and the MEK inhibitor MEK162 would be synergistic in HNSCC due to the frequent activation of RAS via upstream growth factor receptors. RAS mutations are relatively uncommon in HNSCC. We screened 60 validated HNSCC cell lines through sequenome analysis and we found three HNSCC cell lines with activating HRAS mutations (G12D and Q61L) [∼5%]. We found that nilotinib induces significant RAF dimerization and subsequent activation of MEK-ERK pathway irrespective of RAS mutational status. We next investigated how paradoxical MEK/ERK pathway activation affected the growth of HNSCC cells. In this context we treated a panel of HNSCC cell lines harboring WT and mutated HRAS with nilotinib and MEK162. Nilotinib alone had nearly no effect on cell viability and MEK162 had a moderate effect on some HNSCC lines. The combination of nilotinib and MEK162 synergized to inhibit the growth of HNSCC cells independent of RAS mutational status. The drug combination led to an accumulation of cells in G1 and a significant reduction in cell proliferation as measured with BrDU incorporation and colony formation. HNSCC cells with mutant HRAS undergo apoptosis in presence of both the drugs. In contrast none of these drugs induce senescence in HNSCC cell lines. Here we show that in HNSCC, nilotinib as single agent drives the paradoxical activation of RAF-MEK-ERK pathway in RAS mutation-independent manner and we uncovered a synthetic lethal pathway that can be used treat HNSCC. Animal studies are planned. This work was supported by University of Texas SPORE in Head and Neck Cancer P50 CA097007. Citation Format: Banibrata Sen, Tuhina Mazumdar, Shaohua Peng, Katherine S. Hale, Bonnie S. Glisson, Faye M. Johnson. Paradoxical activation of the RAF-MEK-ERK pathway in response to nilotinib induces synthetic lethality with MEK inhibition in head and neck cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4095. doi:10.1158/1538-7445.AM2013-4095

Coxsackie and adenovirus receptor is a critical regulator for the survival and growth of oral squamous carcinoma cells

Coxsackie and adenovirus receptor (CAR) is essential for adenovirus infection to target cells, and its constitutive expression in various cancerous and normal tissues has been reported. Recently, the biological role of CAR in human cancers of several different origins has been investigated with respect to tumor progression, metastasis and tumorigenesis. However, its biological function in tumor cells remains controversial. Here we report the critical role of CAR in growth regulation of oral squamous cell carcinomas (SCCs) in vitro and in vivo via the specific interaction with Rho-associated protein kinase (ROCK). Loss of endogenous CAR expression by knockdown using specific small interfering RNA (siRNA) against CAR facilitates growth suppression of SCC cells due to cell dissociation, followed by apoptosis. The consequent morphological reaction was reminiscent of anoikis, rather than epithelial-mesenchymal transition, and the dissociation of oral SCC cells was triggered not by lack of contact with extracellular matrix, but by loss of cell-to-cell contact caused by abnormal translocation of E-cadherin from surface membrane to cytoplasm. Immunoprecipitation assays of the CAR-transfected oral SCC cell line, HSC-2, with or without ROCK inhibitor (Y-27632) revealed that CAR directly associates with ROCKI and ROCKII, which results in inhibition of ROCK activity and contributes to maintenance of cell-to-cell adhesion for their growth and survival. Based on these findings, in vivo behavior of CAR-downregulated HSC-2 cells from siRNA knockdown was compared with that of normally CAR-expressing cells in intraperitoneally xenografted mouse models. The mice engrafted with CAR siRNA-pretreated HSC-2 cells showed poor formation of metastatic foci in contrast to those implanted with the control siRNA-pretreated cells. Thus, CAR substantially has an impact on growth and survival of oral SCC cells as a negative regulator of ROCK in vitro and in vivo.

Mitochondria-Targeted Nitroxide, Mito-CP, Suppresses Medullary Thyroid Carcinoma Cell Survival In Vitro and In Vivo

Medullary thyroid carcinoma (MTC) is a neuroendocrine tumor mainly caused by mutations in the RET proto-oncogene. For MTC therapy, the U.S. Food and Drug Administration recently approved vandetanib and cabozantinib, multikinase inhibitors targeting RET and other tyrosine kinase receptors of vascular endothelial growth factor, epidermal growth factor, or hepatocyte growth factor. Nevertheless, not all patients with the progressive MTC respond to these drugs, requiring the development of additional therapeutic modalities that have distinct activity. We aimed to evaluate mitochondria-targeted carboxy-proxyl (Mito-CP), a mitochondria-targeted redox-sensitive agent, for its tumor-suppressive efficacy against MTC. In vitro cultures of 2 human MTC cell lines, TT and MZ-CRC-1, and TT xenografts in mice were treated with Mito-CP in comparison with vandetanib. The effects on cell survival/death, RET expression, mitochondrial integrity, and oxidative stress were determined. Contrary to vandetanib, Mito-CP induced RET downregulation and strong cytotoxic effects in both cell lines in vitro, including caspase-dependent apoptosis. These effects were accompanied by mitochondrial membrane depolarization, decreased oxygen consumption, and increased oxidative stress in cells. Intriguingly, Mito-CP-induced cell death, but not RET downregulation, was partially inhibited by the reactive oxygen species scavenger, N-acetyl-cysteine, indicating that Mito-CP mediates tumor-suppressive effects via redox-dependent as well as redox-independent mechanisms. Orally administered Mito-CP effectively suppressed TT xenografts in mice, with an efficacy comparable to vandetanib and relatively low toxicity to animals. Our results suggest that Mito-CP can effectively suppress MTC cell growth/survival via a mechanism distinct from vandetanib effects. Mitochondrial targeting may be a potential strategy for MTC therapy.