bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2218: Radiation-induced hypomethylation triggers urokinase plasminogen activator (uPA) transcription in meningioma cells
Authors: Velpula Kiran Kumar , Gogineni Venkateswara Rao , Nalla Arun Kumar , Dinh Dzung H. , Rao Jasti S.
Published: August 2012

Our previous studies have shown the role of radiation-induced urokinase plasminogen activator (uPA) expression in meningioma tumor progression. In the present study, we investigated whether modulation of DNA methylation profiles could regulate uPA expression. Radiation treatment induced hypomethylation in meningioma cells with a decrease in DNMT1 and MBD expression. However, oxidative damage by H2O2 or pretreatment of irradiated cells with NAC did not show any influence on these proteins, thereby indicating a radiation-specific change in the methylation patterns among meningioma cells. Further, we identified that hypomethylation is coupled to an increase in uPA expression in these cells. Azacytidine treatment induced a dose-dependent surge of uPA expression while pretreatment with Sodium butyrate inhibited the radiation-induced uPA expression; these results complement our prior findings. Methylation-specific PCR on bisulfite treated genomic DNA revealed a diminished methylation of uPA promoter in irradiated cells, which supports the role of epigenetics in uPA gene regulation. Transfection with shRNA expressing plasmids targeting CpG islands of the uPA promoter showed a marked decline in uPA expression with subsequent decreases in invasion and proliferation of meningioma cells. Further, radiation treatment is coupled to the recruitment of SP1 transcription into the nuclear extracts, which was abrogated by shRNA treatment. Our experiments to study the signaling events demonstrated the activation of MEK-ERK in radiation treated cells while U0126 (MEK/ERK inhibitor) blocked hypomethylation, recruitment of SP1, and uPA expression. In agreement with our in vitro data, low DNMT1 levels and high uPA were found in intracranial tumors given radiation treatment as compared to untreated tumors. In conclusion, our data suggest radiation-mediated hypomethylation triggers uPA expression in meningioma cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2218. doi:1538-7445.AM2012-2218
Abstract 2221: Dimerization and DNA binding specificities unravelled by the structure of MITF
Authors: Pogenberg Vivian , Ogmundsdottir Margret H. , Bergsteinsdottir Kristin , Milewski Morlin , Deineko Viktor , Phung Bengt , Schepsky Alexander , Hoek Keith , Wilmanns Matthias , Steingrimsson Eirikur
Published: August 2012

The regulatory protein MITF (microphthalmia associated transcription factor) is essential for melanocyte development and acts as a master regulator of this cell type. Mice carrying loss-of-function mutations in Mitf lack melanocytes altogether and in humans, MITF mutations result in the pigmentation and deafness syndromes Waardenburg and Tietz. In melanoma, MITF plays a critical role as a lineage-survival oncogene and may act as a molecular switch downstream of the Braf pathway, determining whether the cells proliferate or form metastasis. Here we describe the crystal structure of MITF in presence and absence of DNA and use complementary functional experiments to show that Isoleucine 212 of the basic region is critical for discriminating between target sequences. The structures also reveal a kink between the second helix and the Leucine Zipper of MITF which has not been described in other related proteins. This kink is due to a shift in the heptad repeats of the Zipper region and is important for selection of dimerization partners. Our results provide new and important insights into the mechamisms of MITF mediated transcription regulation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2221. doi:1538-7445.AM2012-2221
Abstract 2222: Oncogene-mediated alterations in chromatin conformation
Authors: Rickman David S. , Soong T. David , Moss Benjamin , Mosquera Juan Miguel , Dlabal Jan , Terry Stephane , MacDonald Theresa , Bunting Karen , Demichelis Francesca , Melnick Ari , Elemento Olivier , Rubin Mark A.
Published: August 2012

Emerging evidence suggests that chromatin adopts a non-random three dimensional (3D) topology and that the organization of genes into structural hubs and domains affects their transcriptional status. How chromatin conformation changes in diseases such as cancer is poorly understood. Moreover, how oncogenic transcription factors, which bind to thousands of sites across the genome, influence gene regulation by globally altering the topology of chromatin requires further elucidation. To address these questions, we performed unbiased high-resolution mapping of intra- and inter-chromosome interactions upon over-expression of ERG, an oncogenic transcription factor frequently over-expressed in prostate cancer as a result of a gene fusion. By integrating data from genome-wide chromosome conformation capture (Hi-C), ERG binding and gene expression, we demonstrate for the first time that an oncogenic transcription factor can induce global, reproducible and functionally coherent changes in chromatin organization. We also show using spectral karyotyping that ERG over-expression is associated with a specific translocation involving chromosomes 13 and 15 at loci that are enriched in ERG binding. The results presented here have broader implications, as many driving genetic lesions in other cancer types involve altered levels of transcription factors due to genomic alterations (e.g. EWS-FLI1, c-Myc, n-Myc, PML-RARα).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2222. doi:1538-7445.AM2012-2222
Abstract 2228: mTORC2 is a promising therapeutic target in epithelial ovarian cancer
Authors: Hisamatsu Takeshi , Mabuchi Seiji , Hashimoto Kae , Isobe Aki , Sawada Kenjiro , Kimura Tadashi
Published: August 2012

Purpose: The mammalian target of rapamycin (mTOR) is frequently activated in epithelial ovarian cancer, and is regarded as an attractive therapeutic target for therapy. Preclinical investigations using rapamycin and its analogs have demonstrated significant growth-inhibitory effects on the growth of ovarian cancer, but those researches were focused only on mTOR Complex 1 (mTORC1), not on mTOR Complex 2 (mTORC2). We investigated the activity of mTORC2 and its role in epithelial ovarian cancer. Experimental design: Using human ovarian cancer samples and cell lines, (serous adenocarcinoma and clear cell carcinoma), we first examined the expression and activity of mTORC2. We next investigated the role of mTORC2 on the proliferation and invasiveness of overian cancer cells by knocking down the expression of rictor. Moreover, using 2 pairs of everolimus-sensitive parental and everolimus-resistant sublines, we examined whether mTORC2 is involved in the mechanism of acquired resistance to everolimus. Results: mTORC2 is frequently activated in human ovarian cancer, especially in clear cell carcinoma of the ovary. mTORC2 stimulated the proliferation and invasiveness of ovarian cancer cells in vitro. mTORC2 was hyperactivated in everolimus-resistant sublines compared to their parental cell lines. Inhibition of mTORC2 activity by siRNA sensitize the everolimus-resistant ovarian cancer cells to everolimus. Conclusion: mTORC2 is frequently activated in epithelial ovarian cancer, and is a promising therapeutic target.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2228. doi:1538-7445.AM2012-2228
Abstract 2240: Pre-clinical potency of INK128, a highly potent TORC1/2 kinase inhibitor in the HER2 amplified breast cancer model
Authors: De Pradip KR. , Sun Yuliang , Wu Hui , Rommel Christian , Yi Liu , Dey Nandini , Leyland-Jones Brian
Published: August 2012

Background: The PI3K-AKT-mTOR pathway is commonly activated in breast cancers due to the amplification of receptor tyrosine kinase (HER2), the mutation of PIK3CA or the loss of expression/function of PTEN. About 20% of HER2+ breast cancer patients have PIK3CA mutation, which has resulted in a trend toward shorter progression-free survival with trastuzumab-based therapy. Resistance (intrinsic or acquired) to trastuzumab is a significant clinical challenge, partly due to the overriding activation of PI3K-AKT-mTOR pathway. The PI3K pathway activation leads to stimulation of the key growth and proliferation regulatory kinase, mTOR which can be blocked by allosteric inhibitors (rapalogous) or by ATP-comptetive mTOR kinase inhibitor (TORC1/2). Purpose: We investigated the effects of INK128; a potent and selective TORC1/2 kinase inhibitor alone or in conjunction with trastuzumab in HER2 amplified breast tumor cell lines. We also sought to identify the molecular response determinants of mTOR kinase inhibitor predicting potent activity in HER2+/trastuzumab-resistant cell lines. Experimental Design: Here, we used the HER2 amplified trastuzumab-sensitive breast tumor cell line (BT474), the trastuzumab-resistant breast tumor cell line (BT474HR) and HER2 amplified/PIK3CA mutated breast tumor cell lines (HCC1954, UACC893). Results: We demonstrate that 1) HER2+ cells are sensitive to INK128 (IC50 ∼40nM), 2) INK128 is also effective in trastuzumab-resistant and PIK3CA mutaed/HER2+ breast tumor cells (IC50 ∼20 and 20-30nM respectively), 3) INK128 is mechanistically distinct from rapalogous by a more potent and comprehensive inhibition of PI3K-AKT-mTOR signaling network: a) avoids S6K inhibition-mediated feedback activation of PI3K-AKT and b) abrogates 4EBP1 phosphorylation; 4) ligand-induced HIF1α accumulation is inhibited by prior treatment of INK128, and 5) the combination of trastuzumab plus INK128 more effectively blocked PI3K-AKT-mTOR signaling and HIF1α accumulation both in parental and trastuzumab-resistant cells. Conclusion: Our results suggest that i) therapeutic targeting of PI3K-AKT-mTOR signaling should be effective in abrogating resistance to trastuzumab therapy in HER2+ breast cancer, ii) combination of trastuzumab and INK128 is a promising treatment approach for HER2 overexpressed as well as trastuzumab-resistant breast cancer patients and iii) dual TORC1/2 inhibitor also mitigates the feedback activation of AKT caused by selective inhibition of mTORC1, and because of this, INK128 may yield greater therapeutic benefit in breast cancer patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2240. doi:1538-7445.AM2012-2240
Abstract 2242: The MAPK and PI3K signaling pathways in breast cancer: Crosstalk mechanisms and the effect on cell proliferation
Authors: Monlish Darlene A. , Cavanaugh Jane E.
Published: August 2012

The purpose of the current study is to assess crosstalk between the mitogen-activated protein kinase (MAPK) and the phosphatidylinositol 3-kinase (PI3K) pathways in breast cancer. More specifically, the goal is to investigate the interaction between the extracellular signal regulated kinases 1/2 and 5 (ERK1/2 and ERK5) and Akt and how these kinases act in concert to promote sustained proliferation in a breast cancer model. The MAPK and PI3K pathways mediate numerous cellular processes, including differentiation, growth, proliferation, and survival. To date, most related studies have focused on assessment of the crosstalk mechanisms between ERK1/2 and Akt, while the interplay of ERK5 has remained largely unstudied. Breast cancer patients with increased levels of ERK5 have been found to have a poorer prognosis than patients with lower levels, warranting further investigation of this kinase. The interaction between ERK5 and ERK1/2, as well as between ERK5 and Akt, and how these signaling mechanisms act in conjunction to promote cell proliferation in breast cancer cells remains to be elucidated. The BT-474 (ER+/PR+/HER2+) and MDA-MB-231 (ER-/PR-/HER2-) human breast cancer cell lines were utilized to examine ERK1/2, ERK5 and Akt activation following treatment with ERK and Akt inhibitors or siRNA for these kinases, respectively. Western blot analysis was performed to determine expression and activation of the kinases. An MTT assay was used to assess changes in cell proliferation following kinase inhibition for 12, 24, 48, 72 and 96 hours. In vivo studies utilizing the transgenic MMTV-neu mouse model were also performed. In the BT-474 cell line, inhibition of Akt resulted in increased ERK1/2 and ERK5 expression and activation. Interestingly, the increases in both total and phosphorylated ERK1/2 and ERK5 following Akt-inhibition were dose-dependent, meriting further study. Excised mammary tumors from our animal model demonstrated an age-dependent increase in both ERK1/2 and ERK5. Notably, an age-dependent decrease in Akt was also observed, supporting the hypothesis that crosstalk between these pathways may exist. Future studies involving dual kinase inhibition are planned to further assess crosstalk and proliferation. The data obtained from these studies indicate the presence of crosstalk between the ERK1/2, ERK5 and Akt pathways. A better understanding of this crosstalk may lead to patient-specific drug regimens to decrease the cytotoxic effects of chemotherapeutics and improve patient mortality.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2242. doi:1538-7445.AM2012-2242
Abstract 2246: Combined grape polyphenols regulate mammalian target of rapamycin (mTOR) signaling via a dual mechanism of Akt inhibition and AMPK activation
Authors: Rivera-Rivera Amilcar , Castillo-Pichardo Linnette , Dharmawardhane Suranganie
Published: August 2012

Breast Cancer (BC) is the major prevalent cancer in women in the United States and Puerto Rico with metastatic breast cancer (MBC), often resulting in death. Recent studies have shown that the Akt/mammalian target of rapamycin (mTOR) pathway is a critical signaling cascade that drives MBC. We recently reported that a combined diet of grape polyphenols (resveratrol, quercetin and catechin: RQC) at equimolar concentration is effective at inhibition of mammary tumor growth and metastasis to lung and bones in nude mice. The objective of this study is to investigate a role for RQC in prevention of MBC via inhibition of the Akt/AMPK/mTOR pathway. We found that RQC at 5 μM each inhibits Akt and p70S6 kinase (downstream effector of mTOR) activities in a metastatic variant of the MDA-MB-435 cell line and MDA-MB-231 human BC cells. RQC at 5 μM demonstrated a 2-fold activation of adenosine monophophate-activated protein kinase (AMPK), an endogenous inhibitor of mTOR that acts opposite to Akt. To determine the contribution of individual resveratrol, quercetin, and catechin to the effect of combined RQC on mTOR signaling, MDA-MB-231 cells were treated with RQC at 1, 3 and 9 μM individually and in combination. Results show that at 1 μM, individual or combined RQC did not increase AMPK activity, while at 3 μM, only Quercetin increased AMPK activity. Individual compounds at 1 μM did not affect Akt activity, while combined RQC decreased Akt activity by 20%. At 3 μM, Akt activity is downregulated by all compounds individually and in combination with combined RQC exerting a higher inhibitory effect on Akt activity than individual compounds. In general, the combination of RQC was more efficient at inhibition of the Akt/mTOR pathway than individual compounds. Thus, RQC has the potential to be used alone or in combination with other cancer therapeutics to impede progressive BC. This study was sponsored by the Department of Defense/Breast Cancer Research Program award W81XWH-07-1-0330 to SD and the U54 grant No. CA-096297 to UPR Comprehensive Cancer Center.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2246. doi:1538-7445.AM2012-2246
Abstract 2249: Mutations to the GSK3β regulatory region of CTNNB1 increase TCF transcription activity and are resistant to degradation by GSK3β
Authors: Manring Heather R. , Antinozzi Peter A.
Published: August 2012

Hepatocellular carcinoma (HCC) is currently the third most common cause of cancer related mortality worldwide. As with many cancers, aberrant activation of Wnt/β-catenin signaling plays a critical pathogenic role in the disease. Somatic mutations in genes of the Wnt pathway are widely observed in tumor tissue, where it has been estimated that 15-20% of tumors possess activating mutations in beta-catenin (CTNNB1). The turnover of CTNNB1 protein is tightly regulated in cells by the degradation complex which includes the CK1α and GSK3β kinases in a complex with Axin and APC. Mutations within the CTNNB1 gene at the CK1α phosphorylation site (Ser45) and GSK3β phosphorylation sites (Ser33, Ser37, Thr41) are observed clinically and result in CTNNB1 protein accumulation and enhanced transcription of Wnt target genes. The goal of this study is to characterize additional clinically observed CTNNB1 mutations in HCC. It is hypothesized that mutations within the phosphorylation domain in exon 3 will impact the phosphorylation of these key regulatory sites of CTNNB1. To assess the impact of three observed CTNNB1 mutations (H36P, I35S and S23R) on Wnt signaling activity, mutant CTNNB1 constructs were first created and their impact on Wnt signaling was measured using a luciferase-based Wnt reporter assay in transfected HEK293T cells. Compared to the wild type CTNNB1 control, Wnt transcription activity in the presence of H36P, I35S, and S23R mutants were increased by 6.55 ± 2.08, 6.76 ± 2.44, and 2.36 ± 1.01 fold, respectively. This compared to a 5.91 ± 1.38 fold increase observed with the phosphorylation mutant S45P. To evaluate if these mutations had a measurable impact on GSK3β regulation of CTNNB1 levels, each mutant was coexpressed with a GSK3β expression vector and evaluated using the same Wnt reporter system. As anticipated, GSK3β coexpression reduced Wnt reporter activity of the CTNNB1 control by 3.6 fold ± .33 where the S45P mutant was not affected. Coexpression of GSK3β did not attenuate increased Wnt activity observed with the mutants H36P and I35S, however it did decrease activity observed with the S23R by a similar magnitude of the wild type CTNNB1 control (2.27 fold ± .93). These results describe the activating nature of H36P, S45P, and I35S which likely mediate their effect via impaired CTNNB1 phosphorylation. To a lesser extent, S23R activated Wnt activity, however, is subject to attenuation by GSK3β phosphorylation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2249. doi:1538-7445.AM2012-2249
Abstract 225: Protein tyrosine kinase 6 directly phosphorylates p130 CRK-associated substrate to promote peripheral adhesion complex formation and cell migration
Authors: Zheng Yu , Tyner Angela L.
Published: August 2012

Protein tyrosine kinase 6 (PTK6) is a nonmyristoylated intracellular tyrosine kinase evolutionarily related to Src kinases. Aberrant PTK6 expression and intracellular localization have been detected in human prostate tumors. Although the majority of total PTK6 is localized within the cytoplasm of PC3 cells, a human prostate cancer cell line, we found that the activated pool of PTK6 phosphorylated on tyrosine residue 342 is localized at the membrane. Ectopic expression of membrane targeted PTK6 leads to enhanced activation of PTK6 at the membrane and the formation of 6 to 12 striking focal adhesion-like complexes localized at the periphery of each cell, termed peripheral adhesion complexes. Using confocal microscopy, we show that p130 CRK-Associated Substrate (p130CAS), FAK, Paxillin and Vinculin, as well as increased levels of tyrosine phosphorylated proteins and active PTK6 are localized within the induced peripheral adhesion complexes. We determined that peripheral adhesion complex formation is dependent upon PTK6 tyrosine kinase activity. We demonstrate that p130CAS is a novel direct substrate of PTK6 that works as a crucial adapter protein in PTK6 mediated formation of peripheral adhesion complexes. We found that activation of ERK5 signaling downstream of p130CAS is indispensible for this process. Knockdown of p130CAS attenuates oncogenic signaling induced by membrane targeted PTK6, including ERK5 and AKT activation. We show that formation of peripheral adhesion complexes is associated with enhanced cell migration. PC3 cells expressing membrane targeted PTK6 exhibit increased migratory ability, which can be impaired by knockdown of p130CAS or ERK5. In contrast, knockdown of endogenous PTK6 in PC3 cells reduces cell migration, which is accompanied with decreased phosphorylation of p130CAS and ERK5. Our study reveals a novel function for PTK6 at the plasma membrane, and suggests that a PTK6-p130CAS-ERK5 signaling cascade plays an important role in cancer cell migration and invasion.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 225. doi:1538-7445.AM2012-225
Abstract 2253: New targets of mTORC1 pathway in ER-positive cells
Authors: Sedletcaia Anna , Unger Helen A. , Plotzker Naamah , Holz Marina K.
Published: August 2012

mTOR complex 1 (mTORC1) regulates protein translation, autophagy, and cell growth. These cellular functions are often dysregulated in cancer, therefore identification of mTORC1 targets in control of these processes is of great interest for development of targeted cancer therapies. While rapamycin shown an effect in early clinical trials, the effectiveness of rapamycin in treatment of breast cancer has been variable. Estrogen receptor (ER) ≤ mediates the proliferative effects of estrogen and represents an important clinical target in treatment of ER-positive cancers. Tamoxifen is an anti-estrogen that has become the standard agent for the treatment of ER-positive breast cancer, where it acts as an antagonist. However, resistance to tamoxifen, and other endocrine or anti-estrogen therapies develops in many cases. One mechanism by which resistance develops is through phosphorylation of ERα, allowing it to act in estrogen-independent manner. Phosphorylation of Ser118 and Ser167 is important for transcriptional activity of ERα. The regulation of these sites is complex, involving inputs from the mTOR and MAPK signaling pathways. Importantly, ERα phosphorylation correlates with resistance to tamoxifen, and is a prognostic marker for disease progression and survival. Therefore, the goal of our study is to identify new targets of mTORC1 pathway important for growth and proliferation of ER-positive cells. Thus, inhibition of downstream components of the mTORC1 pathway together in combination with anti-estrogen therapy may prove to be an effective strategy in treatment of ER-positive cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2253. doi:1538-7445.AM2012-2253
Abstract 2261: Heat-shock and autophagy in cancer: Novel role for DGK iota
Authors: Samarasinghe Buddhini N. , Wales Christina T. , Jacobs Aaron T.
Published: August 2012

Autophagy is a cellular process by which damaged proteins, protein aggregates, and organelles are sequestered by lysosome-like compartments and degraded. Autophagy contributes to quality control, cell death, and senescence. Defects in autophagy cause the accumulation of cellular damage, inhibit cell death processes, and promote an inflammatory state that facilitates tumor development. In contrast, the heat shock response, which is mediated primarily by the transcription factor, HSF1 is frequently enhanced in cancer. Despite this inverse relationship between autophagy and heat-shock in cancer, the direct influence of heat-shock and HSF1-mediated gene expression on autophagy has not been investigated. Our study suggests that heat-shock mediated gene expression suppresses autophagy in a human colorectal cancer cell line. Our data show that diacylglycerol kinase iota (DGK iota) expression is regulated by HSF1. DGK iota phosphorylates diacylglycerol, generating phosphatidic acid (PA), which is a potent suppressor of autophagy through its activation of the serine/threonine kinase mTOR (mammalian target of rapamycin). The aim of our study was to evaluate the influence of HSF-1-mediated gene expression on autophagy, and to examine the specific contribution of DGK iota in this effect. We utilized RKO colorectal cancer cells for this investigation and assayed autophagy using complimentary techniques, including: conversion of LC3-I to LC3-II, which was monitored by Western blot; and the formation of autophagic vesicles (autophagosomes), which were visualized by high-content screening confocal microscopy of cells harboring an LC3-GFP expression construct. The influence of basal and heat-shock (42°C)-simulated HSF1 activity on autophagy was analyzed using siRNA. Silencing of HSF1 led to a profound increase in autophagic markers, suggesting a role for HSF1-driven gene expression in the suppression of autophagy. In HSF1-deficient cells we also observed increased phosphorylation of p70S6K, which is a surrogate marker for mTOR activation. We confirmed that DGK iota mRNA and protein expression is mediated by HSF1. Using biochemical inhibitors of DGK activity, we observed a significant increase in autophagy, supporting our hypothesis that DGK iota participates in suppressing autophagy. Additional siRNA experiments of DGK iota are being performed to confirm this effect, along with measurements of cellular PA levels. Taken together, these results indicate an important role for HSF1 in the suppression of autophagy, and provides mechanistic insight into a potential, tumor-promoting role for DGK iota.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2261. doi:1538-7445.AM2012-2261
Abstract 2265: Embelin induces autophagy in ovarian cancer cells
Authors: Jehan Zeenath , Al-obaisi Khadija Abdul Rahman , Uddin Shahab , Al-Kuraya Khawla
Published: August 2012

Embelin is an active ingredient of traditional herbal medicine that exhibits cytotoxic effect and inhibits cell proliferation in various cancer cell types. It is a small molecular inhibitor of XIAP undergoing preclinical testing for its anticancer effects in many different cancer types. In this study we have investigated its role as a chemotherapeutic agent in a panel of ovarian cancer cell lines. Utilising the MTT assay and annexin staining we demonstrate that embelin exhibits a potent, dose dependent cytotoxic effect in human ovarian cancer cells. Surprisingly in contrast to the previously reported cytotoxicity of embelin in many other cancer cell types, treatment of ovarian cancer cells with embelin promoted autophagy and not apoptosis as evidenced by the death without caspase 3 activation and induces cellular swelling and cytoplasmic vacuolization with evident features of necrotic cell death. Treatment of ovarian cancer cells with embelin demonsrnstrated autophagic hallmarks, including an increased number of autophagosomes were observed through acridine orange staining. Embelin also induced cell autophagy, as demonstrated by the punctuate distribution of GFP-LC3, as well as the LC3 cleavage and beclin1 activation. Red green fluorescence in acridine orange stained cells by flowcyometry further confirmed the autophagy action by embelin treatment. Our findings demonstrate a new death pathway underlying the cytotoxic effect of embelin, but also suggest that targeting autophagy could augment the anticancer activity, providing the framework for further development of embelin as a new chemotherapeutic agent for ovarian cancer treatment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2265. doi:1538-7445.AM2012-2265
Abstract 2268: Suicidal tendencies: The promotion of cell death by a prolactin antagonist through autophagy in ovarian cancer
Authors: Wen Yunfei , Zand Behrouz , Yu Kang , Bottsford-Miller Justin , Stone Rebbeca L. , Carroll Amy , Pecot Chad V. , Pradeep Sunila , Wu Sherry , Hu Wei , Ozpolat Bulent , Coleman Robert , Sood Anil K.
Published: August 2012

As a multifunctional pituitary hormone and cytokine, prolactin (PRL) is not only essential for normal reproduction and maintenance of pregnancy, but is involved in over 300 biological activities. There is increasing evidence that PRL plays a role in a variety of cancers including ovarian cancer. Elevated levels of PRL have been reported in patients with ovarian cancer. Furthermore, increased expression of PRL receptors (PRLr) have been shown in ovarian cancers, signifying the importance of PRL-mediated signaling in ovarian carcinogenesis. Abnormal activities of PRLr-mediated signaling pathways such as MAPK and JAK2/STAT5 lead to tumorigenicity and recurrence. Therefore, inhibition of PRL signaling is implicated as a target in cancer therapy; however, no viable strategies have been identified for blocking PRL during tumor progression. Herein, we report a novel strategy to counteract the activity of PRLr-mediated cellular survival in malignant ovarian cancer cells with autophagic cell death. Prolanta (G129R), an analog of human PRL with a single amino-acid substitution at position 129 Glycine with bulky Arginine, induces adaptive autophagy through antagonism of the PRLr expressed on human ovarian cancer cells (HeyA8 and SKOV3). The mouse ovarian cancer cell IG10, which does not express PRLr, had no induction of autophagy upon Prolanta treatment. We also identified that Prolanta competitively binds to PRLr and partially inhibits the oncogenic signaling pathways mediated by PRLr, including JNK/STAT3, PI-3K/Rac/PAK pathways that can lead to cell proliferation, differentiation and survival. In addition, we observed cell death after Prolanta treatment in 3-D-spheroid models. We explored molecular mechanisms behind this autophagic cell death induced by Prolanta. Through biomarker identification using the reverse phase protein array (RPPA), we revealed a signaling complex regulated by Pea-15, an autophagy activator through JNK-pathway, which was activated by Prolanta. Microarray analysis reported an array of autophagic genes activated by Prolanta. Furthermore, in an orthotopic mouse model, Prolanta treatment with or without a taxane caused significant inhibition of SKOV3 and HeyA8 growth. In summary, Prolanta functions as a molecular antagonist to tumor-associated PRL by inducing adaptive autophagic cell death in human ovarian cancer. These preclinical findings reveal the novelty of Prolanta as an adjuvant therapy for ovarian cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2268. doi:1538-7445.AM2012-2268
Abstract 2269: Lapatinib and obatoclax kill cns tumor cells through blockade of erbb1 / 3 / 4 and through inhibition of bcl-xl and mcl-1
Authors: Cruickshanks Nichola A.
Published: August 2012

Previous studies in breast cancer cells have shown that Lapatinib (Lap) and Obatoclax (GX) interact in a greater than additive manner to cause cell death. The present studies sought to expand our analyses to CNS (Glioblastoma & Medulloblastoma) tumor cells and to further advance our understanding of the mechanisms of drug action. Combinational treatment with Lap / GX resulted in enhanced tumor cell death in multiple CNS tumor types. Moreover, it was discovered that cells lacking PTEN function were resistant to drug combination lethality and re-expression of PTEN in PTEN null cells restored drug sensitivity. Furthermore, PTEN expressing cancer cells, sensitive to Lap / GX treatment, demonstrated drug resistance upon knockdown of PTEN. Additionally, we established that inhibition of ERBB1 / 3 / 4 receptors was most significant for enhancing GX lethality, and that knockdown of BCL-XL and particularly MCL-1, interacted to facilitate Lap lethality. Pre-treatment of tumor cells in vitro and in vivo with GX enhanced the lethality of Lap to a greater extent than concomitant treatment and suppressed tumor growth. Treatment of animals carrying orthotopic CNS tumor isolates and those implanted with anoikis resistant breast tumors in the brain with Lap / GX prolonged survival. Collectively our data argues that combinational Lap / GX therapy could be of use in the treatment of tumors located in the CNS. Further studies must be carried out in order to elucidate whether autophagy or apoptosis plays the most predominant role in cell death in response to Lap / GX treatment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2269. doi:1538-7445.AM2012-2269
Abstract 2271: Autophagy induction by low dose cisplatin: The role of p53 in autophagy
Authors: Yang Sei-Hoon , Lee Kang Kyoo , Moon Sun Rock
Published: August 2012

Cisplatin has been mainly used for lung-cancer. However, cisplatin has many side effects, so the usage of cisplatin has a limitation. Recently, autophagy has become an important mechanism of cell death. The purpose of this study was to determine whether low dose cisplatin treated lung cancer cell induce autophagy and the autophagy inhibition resulted in apoptosis or necrosis. H460 cells were treated with 5 μM or 20 μM cisplatin for 12, 24, or 48 h. To detect the cisplatin-induced autophagy, we examined the autophagic vacuoles, and LC3 localization. To confirm the low-dose of cisplatin could induce autophagy, we detectd acidic vesicle using autophagy specific inhibitors(3-MA). To confirm the result of autophagy inhibition, we had done Annexin-V/PI and cell cycle assay. To find out the cisplatin mediated autophagic mechanisms, we examined the apoptotic regulator, p53. We used p53-/- null cancer cell line, H1299, to prove the role of p53 during autophagy. Low dose of cisplatin(5 μM) induced the autophagy after 24 h treatment in H460. Also low dose of cisplatin showed autophagic vacuoles and cytoplasmic LC3 formation in H460. The induction of autophagy by low dose cisplatin was inhibited by 3-MA, which was proven by reduced acidic vesicles. When the autohpagy inhibited, Annexin-V+/PI- and subG1 was an increased. The inhibition of autophagy resulted in decrease of LC3B-II band. Also cleaved caspase-3 and PARP were increased. Taken together, low dose cisplatin induced autophagy and the inhibition of autophagy resulted in the apoptosis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2271. doi:1538-7445.AM2012-2271
Abstract 2272: Heme oxygenase-1 regulates apoptosis and autophagy for the survival of renal cancer cells
Authors: Banerjee Pallavi , Basu Aninda , Wegiel Barbara , Otterbein Leo E. , Choi Augustine M. K. , Pal Soumitro
Published: August 2012

Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme degradation pathway is classically known for its cytoprotective role during tissue injury. However, this protective function of HO-1, which is essential for maintaining cellular homeostasis under stress conditions, may act as a double-edged sword by promoting survival of cancer cells. HO-1 is overexpressed in different cancers, and cancer cells may utilize the anti-apoptotic effects of HO-1 as a shield against chemotherapeutic agents. We have recently demonstrated that activation of the oncogenic Ras pathway induces HO-1 to promote survival of renal cancer cells. Recent studies show that along with apoptosis, type II cell death or autophagy also plays a vital role in regulating the fate of cancer cells. While some groups suggest that toxic autophagy cooperates with chemotherapeutic agents to facilitate apoptosis of cancer cells, others propose a protective role of autophagy in cancer. Here, we investigated whether HO-1 can control apoptosis- and autophagy-related genes to regulate survival of human renal cancer cells (786-0 and Caki-1). We overexpressed HO-1 in these cells either by CoPP treatment, a known inducer of HO-1, or by transfecting the cells with a human HO-1 overexpression plasmid. We observed that the overexpression of HO-1 increased the level of anti-apoptotic protein Bcl-xL as observed by Western blot analysis; and this was associated with a significant decrease in the expression of autophagic proteins Beclin-1 and LC3B-II. The knockdown of HO-1 using specific siRNA led to down-regulation of Bcl-xL, and a marked increase in LC3B-II expression. Moreover, we found that the treatment of renal cancer cells with either rapamycin or sorafenib (two commonly used drugs for the treatment of renal cancer) induced the protein expression of HO-1; and apoptotic effects of these drugs on cancer cells were markedly enhanced in presence of the HO-1 inhibitor SnPP as observed by FACS analysis using Annexin-V staining. Together, our results indicate that HO-1 is up-regulated in renal cancer cells for their survival against chemotherapeutic agents, and it may protect the cells from both apoptosis- and autophagy-mediated death. Besides, recent studies demonstrated that both rapamycin and sorafenib can induce autophagy. Considering this together with our data, we suggest that application of these drugs along with HO-1 inhibitor may result in more efficient killing of renal cancer cells by reducing cytoprotective effects of HO-1 and by simultaneous induction of both autophagy and apoptosis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2272. doi:1538-7445.AM2012-2272
Abstract 2273: Autophagic cell death induced by curcumin is mediated by p38/Beclin 1 to crosstalk with apoptotic cell death in DU145 cells
Authors: Kang Dongxu , Park Wungki , Kim Joo-Hang , Song Jae Jin
Published: August 2012

Curcumin as an anticancer agent was investigated to study how it can regulate cancer cells’ destination to either apoptosis or autophagy. At higher concentration, curcumin induced ROS production leading to JNK and p38 phosphorylation in DU-145 prostate cancer cell. Of the two MAP kinases, p38 was phosphorylated earlier during curcumin treatment, and responsible for curcumin-induced autophagic cell death by using GFP-LC3 expression. p38-induced autophagy signaling pathway occurred at even 1h, and was enhanced by blocking JNK signaling, and JNK signaling was more involved in apoptosis rather than autophagy. In the case of curcumin treatment. Beclin 1, an essential autophagic protein was mainly activated by dissociation from Bcl2 or Bcl-xL after Beclin 1 phosphorylation induced by p38-DAPK, or from Bcl-2 phosphorylation induced by JNK1. Based on these results, we concluded that curcumin exhibited cytotoxicity in DU-145 cells and this cytotoxicity was mediated by p38 signaling pathway triggering both beclin 1 activation to induce autophagic cell death, which in turn inhibits the apoptotic cell death induced by curcumin-ROS-JNK signaling cascade, leading to a complex crosstalk between autophagy and apoptosis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2273. doi:1538-7445.AM2012-2273
Abstract 2276: Coordinate autophagy and PI3K/Akt/mTOR pathway inhibition enhances cell death in melanoma
Authors: Xie Xiaoqi , Mehnert Janice M. , White Eileen P.
Published: August 2012

The PI3K/Akt/mTOR pathway is a critical survival pathway in numerous malignancies, including melanoma. Inhibitors of this pathway in cancer cells, in particular mTOR, are commonly cytostatic rather that cytotoxic, which may potentially limit efficacy of these agents. mTOR inhibition also induces the survival pathway of autophagy, a catabolic process whereby cells collect and recycle cellular components to sustain energy homeostasis. Thus we tested the hypothesis that autophagy induction by mTOR inhibitors directly limited their efficacy for the treatment of melanoma. Here we found that melanoma exhibits high basal levels of autophagy, as evidenced by a) detection of autophagosomes using LC3 fluorescence and autophagic flux in multiple melanoma cell lines and b) increased LC3 fluorescence in human primary and metastatic melanomas compared to benign nevi. Knockdown of Atg7, a key element of autophagy machinery, resulted in cell death, indicating that survival of melanoma cells is autophagy dependent. We also showed that the autophagy inhibitor hydroxychloroquine (HCQ) shows synergy when combined with the mTOR inhibitor CCI-779 (temsirolimus), inducing cell death via increased apoptosis in melanoma cell lines and in 3-D spheroid cell cultures. Since autophagy eliminates mitochondria that are a source of toxic reactive oxygen species (ROS) and mediators of apoptosis, we tested the possibility that this contributed to melanoma survival. To test this potential mechanism, we examined the role of the transcription factor Nrf2, a master regulator of antioxidant defense. Treatment with CCI-779 inactivated Nrf2 by blocking its nuclear translocation and creating the requirement for autophagy to eliminate ROS-producing mitochondria. Combination treatment with CCI-779 and HCQ then prevented mitochondrial elimination by autophagy, resulting in increased ROS production compared to either agent alone and increased cell death. Conversely, knockdown of the Nrf2 binding protein and inhibitor Keap1 increased Nrf2 activity, resulting in increased cell culture survival and resistance to treatment with CCI-779 and HCQ. These data suggest that coordinate inhibition of the PI3K/Akt/mTOR and autophagy pathways promotes ROS-mediated apoptosis and is a new therapeutic paradigm for the treatment of melanoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2276. doi:1538-7445.AM2012-2276
Abstract 2281: Signaling from lysosomes to mitochondria sensitizes head and neck cancer cells to photodynamic treatment: Role of mitoferrin 2
Authors: Hung Hsin-I , Lemasters John J. , Nieminen Anna-Liisa
Published: August 2012

Photodynamic therapy (PDT) involves activation by light of a photosensitizer in a tissue, resulting in singlet oxygen formation and cell death. The aim of this study was to investigate the interactions between lysosomes and mitochondria in promoting PDT killing of head and neck cancer cells. Three head and neck cancer cell lines (UMSCC1, UMSCC14 and UMSCC22) were subjected to PDT with Pc 4, a mitochondria-targeted photosensitizer. After comparable Pc 4-PDT, cell killing was greater in UMSCC22 than UMSCC1 or UMSCC14 cells. Pc 4-PDT caused 85% cell killing in UMSCC22 cells compared to 20% killing in UMSCC1 or UMSCC14 cells. In non-erythroid cells, mitoferrin 2 (Mfrn2) is an iron transporter in the mitochondrial inner membrane, and PDT-sensitive UMSCC22 cells expressed >5 times higher Mfrn2 protein levels compared to PDT-resistant UMSCC1 and UMSCC14 cells as determined by Western blotting. Bafilomycin, an inhibitor of the vacuolar proton pump of lysosomes and endosomes enhanced Pc 4-PDTcytoxicity. Mfrn2 protein overexpression in low Mfrn2 expressing cells enhanced sensitivity to Pc 4-PDT plus bafilomycin. Conversely, knocking down (90% knock down) Mfrn2 by siRNA decreased cells’ sensitivity to the Pc 4-PDT plus bafilomycin treatment. Iron chelators (desferrioxamine and starch-desferrioxamine) and the inhibitor of the mitochondrial calcium (and ferrous iron) uniporter, Ru360, protected against PDT plus bafilomycin treatment from Pc 4- mitochondria-mediated cell killing. In conclusion, the data suggest that lysosomal iron release and mitochondrial iron uptake through Mfrn2 act synergistically to induce PDT-induced cell killing. Furthermore, Mfrn2 represents a possible biomarker in predicting efficacy of the PDT treatment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2281. doi:1538-7445.AM2012-2281
Abstract 2287: Inhibition of microRNA-32 leads to decreased expression of the survival-enhancing scaffold protein hKSR2 in human myeloid leukemia cells treated with vitamin D derivatives
Authors: Wang Xuening , Chen-Deutsch Xiangwen , Harrison Jonathan S. , Studzinski George P.
Published: August 2012

1,25-dihydroxyvitamin D3 (1,25D) has potential for treatment and/or prevention of human AML by inducing terminal differentiation of AML cells to monocyte-like phenotype, but the mechanisms underlying this action are not fully understood. We hypothesized that one or more microRNAs (miRNAs) known to control mouse hematopoiesis and lineage commitment may contribute to the known ability of 1,25D to control the malignant phenotype. We found that miR181a and miR32 both participate in the differentiating effects of 1,25D, with effects on cellular levels of cell cycle regulator p27Kip1 and the pro-apoptotic protein Bim, respectively. Here we report that in human AML cells modulation of 1,25D-induced expression of miR32 and miR181a can result in the down-regulation of the abundance of the scaffold protein hKSR2, which is associated with inhibition of differentiation. This supports the concept that hKSR2 is under miRNA control in AML cells. However, ectopic expression of miR32 had little effect on hKSR2 protein levels, suggesting that there is an indirect effect of miR32 on hKSR2 expression, and that intracellular levels of the intermediary regulators are already at saturation levels. Since hKSR2 protein has cell survival-enhancing properties, 1,25D-induced miRNAs and hKSR2 changes may reduce the therapeutic efficacy of 1,25D and analogs in human AML, especially when vitamin D analogs are combined with conventional cytotoxic anti-leukemia agents. This warrants consideration in the design of clinical trials. (This research was supported by NIH grant R01 CA 044722-22 from the National Cancer Institute).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2287. doi:1538-7445.AM2012-2287