bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2921: NAD(P)H quinone oxidoreductase 1 regulates pro-inflammatory mediators associated with prostate tumorigenesis.
Authors: Thapa Dinesh , Kumar Addanki P. , Ghosh Rita
Published: August 2014

Oxidative stress and inflammation are both important in the neoplastic transformation of the prostate. NAD(P)H Quinone Oxidoreductase 1 (NQO1) is a key antioxidant enzyme that protects against quinone-derived reactive intermediates and maintains a cellular pool of antioxidants. Various studies conducted using NQO1 knockout mice suggested its potential role in carcinogenesis. However the role of NQO1 has not been fully explored in prostate cancer. Here we show that NQO1 silencing by a specific shRNA (shNQO1) increased cell survival, proliferation, colony formation, migration and hormone-independent survival in hormone-responsive human prostate cancer cells LNCaP. Genome wide array revealed that NQO1 blockade significantly upregulated pro-inflammatory mediators (e.g., IL-32, CCL2, IL-8, IL-17C, IL-10RA, CXCR2, CXCR7, NOS3) associated with prostate tumorigenesis. Cytokine profiling further confirmed the increased IL-8 production in conditioned-media from NQO1 knockdown cells compared to the parental cells. However, enforced expression of NQO1 in these cells did not alter IL-8 and other inflammatory mediators. Our data indicate that inflammatory mediators produced by NQO1 knockdown LNCaP cells may be responsible for the hormone-independent survival. Since these inflammatory mediators influence prostate cancer development and castration resistance, strategies modulating cellular antioxidants to regulate NQO1 and other important players in NQO1-signaling may provide multifaceted opportunities for prostate cancer prevention. Supported by a postdoctoral fellowship from CPRIT (DT) and RO1 CA 149516 (RG).

Citation Format: Dinesh Thapa, Addanki P. Kumar, Rita Ghosh. NAD(P)H quinone oxidoreductase 1 regulates pro-inflammatory mediators associated with prostate tumorigenesis. [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 2921. doi:10.1158/1538-7445.AM2013-2921
Abstract 2922: Stoichiometric depletion of glutathione by anticancer agent JS-K.
Authors: Holland Ryan J. , Maciag Anna E. , Cheng Robert Y.-S. , Rodriguez Luis G. , Saavedra Joseph E. , Anderson Lucy M. , Keefer Larry K.
Published: August 2014

JS-K is a nitric oxide (NO)-releasing drug of the O2-arylated diazeniumdiolate family that has demonstrated pronounced cytotoxicity and antitumor properties in a variety of cancer models. It is activated for NO release by reaction with glutathione (GSH), a step that consumes an equivalent of GSH, effectively irreversibly. The two equivalents of NO thus released can be oxidized intracellularly, leading to S-nitrosylation and S-glutathionylation of GSH in a cascade of further reactions that consume additional equivalents of GSH and generate GSH disulfide (GSSG). The GSH/GSSG redox couple is the major redox buffer of the cell, helping maintain a reducing environment under basal conditions. Here we show that depletion of GSH on treating U937 leukemia cells with JS-K markedly raises the reduction potential of the cell, initiating MAPK stress signaling pathways, and inducing apoptosis. Microarray analysis confirmed signaling gene changes at the transcriptional level and revealed alteration in the expression of several genes crucial for maintenance of cellular redox homeostasis, as well as cell proliferation and survival, including MYC. The data indicate that multiplicative depletion of reduced GSH and deregulation of intracellular redox balance are important steps in the mechanism of JS-K's cytotoxic action.

An additional GSH-consuming pathway was found to occur with Double JS-K, an aryl bis(diazeniumdiolate) created with the purpose of doubling the payload of NO. Double JS-K caused pronounced protein glutathionylation, in contrast to its monovalent counterpart. Serving as a bivalent electrophile, Double JS-K irreversibly crosslinked GSH to protein thiols through its aromatic linker. The results provide a previously unrecognized component of the multifaceted mechanism of action of bivalent electrophiles of the arylated diazeniumdiolate class.

Citation Format: Ryan J. Holland, Anna E. Maciag, Robert Y.-S. Cheng, Luis G. Rodriguez, Joseph E. Saavedra, Lucy M. Anderson, Larry K. Keefer. Stoichiometric depletion of glutathione by anticancer agent JS-K. [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 2922. doi:10.1158/1538-7445.AM2013-2922
Abstract 2924: The role of autophagy in telomere damage-induced replicative senescence.
Authors: Charles Florie , Debnath Jayanta , Stohr Bradley
Published: August 2014

Recent studies have implicated autophagy in multiple aspects of cancer biology, including oncogene-induced senescence (OIS) and genome instability. Autophagy is thought to control the transition to OIS through its ability to regulate the Senescence Associated Secretory Phenotype (SASP), which is necessary and sufficient for OIS. In addition, autophagy can suppress genome instability by limiting p62 accumulation in an apoptosis-deficient setting. In this study, we explored the role of autophagy in replicative senescence, an important barrier to tumorigenesis that is driven by telomere dysfunction and that is very distinct from OIS. We modeled replicative senescence by expressing mutant telomerase in human diploid fibroblasts, which rapidly induces telomere dysfunction and senescence. We observed strong induction of autophagic flux in response to telomere dysfunction prior to and during senescence. To determine the role of autophagy in the cellular response to telomere damage, we examined multiple components of the dysfunctional telomere response: transition and maintenance of senescence, SASP, DNA damage response, genome instability, and heterochromatin foci. In cells depleted of key autophagy mediators (ATGs), we did not observe a clear defect in the transition to senescence, as was previously seen with OIS. However, we did observe an outgrowth population of non-senescent cells among the senescent autophagy-deficient population, suggesting a possible defect in the maintenance of senescence. We also found that telomere dysfunction induced secretion of IL6 in an autophagy-dependent manner. Lastly, we observed an increase in polyploid cells in Atg7-deficient checkpoint-proficient cells, suggesting a role for autophagy in maintaining genome stability during the dysfunctional telomere response. Our results suggest that autophagy controls several aspects of the cellular response to telomere dysfunction in normal human cells.

Citation Format: Florie Charles, Jayanta Debnath, Bradley Stohr. The role of autophagy in telomere damage-induced replicative senescence. [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 2924. doi:10.1158/1538-7445.AM2013-2924
Abstract 2928: Interleukin-1 receptor-associated kinase-2 (IRAK2) is a critical mediator of endoplasmic reticulum (ER) stress.
Authors: Palaniyandi Ravanan , Benosman Samir , Hou Ying-Chen , Correa Ricardo G. , Yu Minjia , Gulen Muhammet Fatih , Li Xiaoxia , Thomas James , Matsuzawa Yasuko , Matsuzawa Shu-Ichi , Reed John C.
Published: August 2014

Endoplasmic reticulum (ER) stress occurs when unfolded proteins accumulate in the lumen of the organelle, triggering signal transduction events that contribute either to cellular adaptation and recovery or alternatively to cellular dysfunction and death. ER stress has been implicated in numerous diseases, including cancer where harsh tumor microenvironments induce ER stress and create pressure for evolution of malignant clones that have acquired mechanisms for successful adaptation to stressful environments. To identify novel modulators of ER stress, we undertook a siRNA library screen of the kinome, revealing Interleukin-1 Receptor-Associated Kinase-2 (IRAK2) as a contributor to unfolded protein response (UPR) signaling and ER stress-induced cell death. Knocking down expression of IRAK2 (but not IRAK1) in cultured mammalian cells suppresses ER stress-induced expression of the pro-apoptotic transcription factor CHOP and activation of stress kinases. Similarly, RNAi-mediated silencing of the IRAK family member Tube (but not Pelle) suppresses activation of stress kinase signaling induced by ER stress in Drosophila cells. The action of IRAK2 maps to the IRE1 pathway, rather than the PERK or ATF6 components of the UPR. Interestingly, ER stress also induces IRAK2 gene expression in an IRE1/XBP1-dependent manner, suggesting a mutually supporting amplification loop involving IRAK2 and IRE1. In vivo, ER stress induces Irak2 expression in mice. Moreover, Irak2 gene knockout mice display defects in ER stress-induced CHOP expression and IRE1 pathway signaling. These findings demonstrate an unexpected linkage of the innate immunity machinery to UPR signaling, revealing IRAK2 as a novel amplifier of the IRE1 pathway involved in ER stress signaling. Future analysis of the role of IRAK2 in animal models of cancer will aid in assessing whether IRAK2 provides a novel targets for potential cancer therapeutics development.

(Supported by NIH grant AG-15393).

Citation Format: Ravanan Palaniyandi, Samir Benosman, Ying-Chen Hou, Ricardo G. Correa, Minjia Yu, Muhammet Fatih Gulen, Xiaoxia Li, James Thomas, Yasuko Matsuzawa, Shu-Ichi Matsuzawa, John C. Reed. Interleukin-1 receptor-associated kinase-2 (IRAK2) is a critical mediator of endoplasmic reticulum (ER) stress. [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 2928. doi:10.1158/1538-7445.AM2013-2928

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 293: TGF-b regulates Par-4 expression through XIAP in ovarian and endometrial cancers.
Authors: Fabi Francois , Parent Sophie , Leblanc Valérie , Asselin Eric
Published: August 2014

The lethality of gynecological malignancies can be explained by their propensity to develop chemioresistance and metastases. The loss of apoptotic pathways is one of the major underlying mechanisms by which tumors become resistant to treatments. This loss can be explained by the silencing of pro-apoptotic proteins that would normally contrive the cell into suicide. Par-4 is a tumor suppresor protein that is widely expressed but only functionally silenced in most endometrial cancer. This protein presents the unique ability to induce apoptosis selectively in cancer cells. We have recently demonstrated that Par-4 is a substrate of caspase-3 and the resulting cleaved fragment retains its apoptotic-inducing capabilities. We have also evidenced that TGF-β, a known instigator of EMT, regulates the invasiveness of endometrial cancer cells and Par-4 expression. TGF-β has also been shown to increase the expression of XIAP, an important promoter of cell survival and invasiveness in endometrial cancer. In this study, we have investigated the effect of TGF-ß and XIAP on Par-4 expression in ovarian and endometrial cancer.

We have performed rescue experiment in which MEFs cells from XIAP KO mice were cultured and transfected with either an empty plasmid, a wild-type XIAP plasmid or a XIAP mutated RING domain plasmid. We have then submitted these cells to cisplatin treatment. In the empty vector transfectants, we have found that the absence of XIAP results in Par-4 cleavage, increased cleaved caspase-3 and cleaved PARP. The rescue of XIAP inhibited the apoptotic machinery and Par-4 cleavage thus resulting in increased chemoresistance. This effect was reversed using the mutated XIAP plasmid. We have also treated endometrial cancer cells (HeLa, KLE) with TGF-ß3 and observed an higher expression of EMT markers such as Snail, Vimentin and N-Cadherin; Par-4 was also upregulated by TGF-ß3. Finally, we have overexpressed ovarian A2780 cancer cells with Par-4 plasmid, which resulted in the upregulation of Snail and Vimentin. We confirmed these results using Par-4 siRNA and found that Snail and Vimentin expression were reduced.

Altogether, results of the present study shed new light in the underlying mechanisms of Par-4 regulation and apoptosis induction. The assembled data also suggests that TGF-β plays a cardinal role in the regulation of Par-4 expression, which regulates EMT in gynecological cancers.

Citation Format: Francois Fabi, Sophie Parent, Valérie Leblanc, Eric Asselin. TGF-b regulates Par-4 expression through XIAP in ovarian and endometrial cancers. [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 293. doi:10.1158/1538-7445.AM2013-293
Abstract 2938: Altered caspase-8 protein expression and activity in TRAIL-resistant normal human fibroblasts.
Authors: Crowder Roslyn N. , Dicker David T. , El-Deiry Wafik S.
Published: August 2014

TNF-related apoptosis-inducing ligand (TRAIL)-mediated cell death is currently being utilized in newly developed cancer therapies. The molecular basis of normal cell resistance to TRAIL-mediated apoptosis, and thus therapeutic index, remains largely unexplored. We investigated TRAIL sensitivity in normal fibroblasts (HFF, WI-38, MRC-5) and found that normal fibroblasts have decreased protein expression of initiator caspase-8 that is required for complete TRAIL signaling. TRAIL treatment caused caspase-8 cleavage in TRAIL-sensitive cancer cells. Yet, no caspase-8 cleavage was observed in normal fibroblasts after TRAIL treatment. TRAIL treated fibroblasts had modest increase in caspase-8 activity compared to TRAIL-susceptible cancer cells.

Previous studies have found caspase-8 polyubiquitination necessary for caspase-8 aggregation and complete activation. Additionally, reports show deubiquitination of caspase-8 limits caspase-8 activity. Therefore, we examined caspase-8 ubiquitination in normal fibroblasts. Caspase-8 ubiquitination, analyzed by Western blotting, in normal fibroblasts was found to be decreased compared to TRAIL-sensitive cancer cells. TRAIL sensitivity in normal fibroblasts after treatment with deubiquitinase inhibitor PR-619 was also assessed. Normal fibroblasts had markedly decreased cell viability after co-treatment with TRAIL and PR-619 compared to TRAIL-only treated fibroblasts. Cancer cell viability after co-treatment with TRAIL and PR-619 was similar to cancer cells merely treated with TRAIL. Normal fibroblasts co-treated with TRAIL and PR-619 had increased caspase-8 cleavage, PARP cleavage, and caspase-8 activity. The increased cell death seen in normal fibroblasts with TRAIL and PR-619 co-treatment was greatly inhibited when caspase inhibitor ZVAD was added, demonstrating the cell death with TRAIL and PR-619 was caspase-mediated. Our data suggest that caspase-8 deubiquitination may be a contributing factor in normal fibroblast resistance to TRAIL-mediated apoptosis. Our current studies are focused on investigating the role of deubiquitinase A20, previously found to deubiquitinate caspase-8, in normal fibroblast caspase-8 deubiquitination.

Citation Format: Roslyn N. Crowder, David T. Dicker, Wafik S. El-Deiry. Altered caspase-8 protein expression and activity in TRAIL-resistant normal human fibroblasts. [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 2938. doi:10.1158/1538-7445.AM2013-2938
Abstract 2941: The role of lysine acetylation in the tumor cell response to stress.
Authors: Heppler Lisa , Andersen Joshua L.
Published: August 2014

Solid breast tumors contain heterogenous microenvironments where tumor cells are often exposed to metabolic stress (e.g., hypoxia due to poor blood supply). Such environments select for tumor cells that can adapt metabolically to survive, while other cells fail to adapt and undergo cell death. The survival of cells through periods of hypoxia can promote chemoresistance and metastasis (1). Thus, it is critical that we develop therapeutic strategies to enhance metabolic-stress-induced tumor cell death. One promising strategy is the modulation of lysine acetylation pathways by HDAC inhibitors that potently promote cell death in response to various stimuli, including hypoxia/glucose withdrawal. Given the relatively non-specific nature of chemical HDAC inhibitors, the precise acetylation-regulating enzymes and pathways that govern cell death in these settings have yet to be fully elucidated. Our goal is to identify the cellular factors that link acetylation to cell death in response to hypoxia and other metabolic stresses, with the hope that such factors could be exploited therapeutically in cancer.

Previous studies have implicated protein lysine acetylation in the coordination of cellular metabolism to the available nutrient supply (2). In line with this idea, our preliminary data suggest that lysine acetylation pathways dictate whether breast tumor cells survive (through metabolic adaptation) or die in response to hypoxia and glucose deprivation. Moreover, we have observed that general increases in protein lysine acetylation precede the activation of pro-apoptotic caspases in response to these stresses. In addition, our proteomics efforts have shown that breast tumors that are sensitive to hypoxia/glucose withdrawal exhibit significant increases in acetylation across the proteome, whereas resistant cells show very little change. Together, our data suggest that lysine acetylation pathways play a role in metabolic adaption and survival under conditions of hypoxia/glucose withdrawal. We are currently using an RNAi approach to target all known deacetylases, acetyl-transferases, and metabolic enzymes that modulate acetylation (e.g., acetyl-CoA synthetase) in order to identify the specific acetylation-regulating factors that govern tumor cell susceptibility to metabolic stress.

Citation Format: Lisa Heppler, Joshua L. Andersen. The role of lysine acetylation in the tumor cell response to stress. [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 2941. doi:10.1158/1538-7445.AM2013-2941
Abstract 2942: Acetylation of intracellular protein induces apoptotic cell death in Aspirin-treated human breast cancer cells.
Authors: Lee Yu Ran , Jeon Byeong Hwa , Choi Sunga
Published: August 2014

Acetylation is one of the dynamic post-translational modification of lysine residues on protein determining the structure, function and intracellular localization that plays an important role in the signal transduction pathways related to diverse cell functions - proliferation, cell death, and differentiation as well as transcriptional regulation. Recently, it has been reported that acetylating agents including chemical histone deacetylase inhibitors and plant polyphenols lead to the increased acetylation of histone and non-histone proteins, which induces to rapid cancer cell death. In this present study, we are focused on molecular mechanism of cell death in two Aspirin-treated human breast cancer cells, MCF-7 and MDA-MB-231. Trichostatin A (TSA) treatment was combined for maintaining acetylation of intracellular proteins at 0.1 μM concentration, which was not caused cell death itself. It was observed that Aspirin/TSA treatment caused the significant inhibition of cell growth in a dose- and time-dependent manner in both MDA-MB-231 and MCF-7 cells. The induced cell death in MDA-MB-231 by Aspirin/TSA treatment was associated with several specific features of apoptosis, which were proved by biochemical assays containing DNA fragmentation, Annexin V staining and cell cycle analysis. On the other hand, the exposure of MCF-7 to Aspirin/TSA caused necrotic rather than apoptotic mechanism judged by DNA fragmentation assay. These results suggest that different cell death pathway in two breast cancer cell lines is implicated in acetylated protein mediated signals. Previous reports suggest sporadic tumor cell necrosis is associated with extracellular release of cellular protein such as the high mobility group box 1 and may activate innate immune system. Interestingly, we confirmed secretory protein in cell-culture supernatant by immunoprecipitation using anti-acetyllysin antibody. Secretion of the acetylated protein was elevated dose- and time- dependent manner after Asprin treatment. Although extracellular role of the secretory protein in response to acetylation has not been clearly identified yet, this acetylated protein may be involved in initiation of apoptotic cell death signals and/or activating innate immune response. Taken together, theses results indicated that acetylated protein plays an essential role in anti-tumorigenesis in response to acetylating agent such as Aspirin.

Citation Format: Yu Ran Lee, Byeong Hwa Jeon, Sunga Choi. Acetylation of intracellular protein induces apoptotic cell death in Aspirin-treated human breast 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 2942. doi:10.1158/1538-7445.AM2013-2942

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 2945: Anti-tumor effect of insulin-like growth factor-1 receptor inhibition in head and neck squamous cell carcinoma.
Authors: Khalil Ashraf A. , Axelrod Mark J. , Hubbard Matthew A. , Maxwell Anne K. , Taniguchi Linnea E. , Mendez Rolando E. , Jameson Mark J.
Published: August 2014

Back ground: Insulin-like growth factor-1 receptor (IGF1R) activity is upregulated in a variety of human cancers and IGF1R signaling has been implicated as a mechanism of resistance to various cancer therapies, including radiotherapy, cytotoxic chemotherapy, and targeted molecular therapy. We have demonstrated that IGF1R activation can induce resistance to targeted therapy of head and neck squamous cell carcinomas (HNSCCs) using epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). Targeting the IGF1R by selective small molecule kinase inhibition may thus hold promise as an adjuvant treatment for HNSCC.

Aims: (1) To assess the anti-tumor effects of two IGF1R/insulin receptor TKIs, BMS-754807 and OSI-906, in multiple HNSCC cell lines in vitro. (2) To evaluate the effect of combined IGF1R and EGFR inhibition in HNSCC cell lines in vitro.

Methods: The effect of IGF1R inhibition on cell proliferation, viability, survival, and apoptosis was assessed using alamarBlue, trypan blue exclusion, clonogenic, and flow cytometric assays in 9 HNSCC cell lines. Alterations in downstream IGF1R signaling were assessed by standard immunoblot. The impact of combining an EGFR-TKI with an IGF1R-TKI was measured.

Result: Both BMS-754807 and OSI-906 inhibited IGF1R phosphorylation at 200 nM. Complete inhibition of the phosphorylation of IRS-1 and Akt required higher concentrations. Both inhibitors slowed proliferation, decreased cell viability, and decreased cell survival in all 9 cell lines. The EC50 to inhibit proliferation was in the range of X to X microM for BMS-754807 and X to X microM for OSI-906. Both drugs induced apoptosis in HNSCC tumor cell lines based on flow cytometric analysis of caspase-3 cleavage and PARP cleavage. In combination with gefitinib and lapatinib, BMS-754807 and OSI-906 were capable of synergistic growth inhibition.

Conclusion: Inhibition of IGF1R activity using the selective TKIs BMS-754807 and OSI906 has an antitumor effect on HNSCC cells in vitro. These drugs are synergistic with EGFR-TKIs and co-targeting the two receptors may represent an approach to overcoming clinical resistance to targeted anti-growth factor therapy in HNSCC.

Citation Format: Ashraf A. Khalil, Mark J. Axelrod, Matthew A. Hubbard, Anne K. Maxwell, Linnea E. Taniguchi, Rolando E. Mendez, Mark J. Jameson. Anti-tumor effect of insulin-like growth factor-1 receptor inhibition in head and neck squamous 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 2945. doi:10.1158/1538-7445.AM2013-2945
Abstract 2947: PARP-1 regulates death receptor 5-mediated apoptosis in pancreatic cancer.
Authors: Yuan Kaiyu , Sun Yong , Zhou Tong , McDonald Jay , Chen Yabing
Published: August 2014

Pancreatic cancer is a highly malignant tumor with limited therapy and poor prognosis. Activation of death receptors (DR)-induced apoptotic pathways using antibodies or agonists for the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapy. However, most pancreatic tumors are resistant to TRAIL therapy. The present studies aimed to identify combination therapies that enhance the efficacy of TRAIL therapy by sensitizing resistant pancreatic cancer cells to TRAIL-induced apoptosis, and to investigate the underlying mechanisms.

Using TRA-8, a monoclonal agonistic antibody specific for DR5, we demonstrated that TRA-8 induced apoptosis of 60% and 83% of BxPc-3 and MiaPaCa-2 pancreatic cancer cells in 24 hours. By contrast, only 20% and 24% of PANC-1 and Suit-2 cells underwent apoptosis in response to TRA-8 (n=3), despite the fact that they express high levels of the DR5 receptor. The expression of poly(ADP-ribose) polymerase-1 (PARP-1) was higher in the PANC-1 and Suit-2 cells than that BxPc-3 and MiaPaCa-2 cells, which may contribute to their resistance to TRA-8-induced apoptosis. Inhibition of PARP-1 with a pharmacologic inhibitor (PJ-34) sensitized PANC-1 to TRA-8 induced apoptosis in a dose-dependent manner. Furthermore, small interfering RNAs specifically knocking down PARP-1 in PANC-1 cells enhanced TRA-8-induced apoptosis in vitro by 2.5 fold, as well as the efficacy of TRA-8 therapy on tumorigenesis of PANC-1 cells in a mouse xenograft model by 3.8 fold (n=8, p<0.01). PARP-1 knockdown increased TRA-8-induced activation of caspase-8 in the death-induced signaling complex (DISC). Immuoprecipitation with DR5 antibody identified the recruitment of PARP-1 and PARP1-mediated protein poly-ADP-ribosylation(pADPr) modification in the DR5-associated DISC, which was associated with caspase 8 activation in the DISC. Our studies determined a novel function of PARP-1, independent of its known function in DNA repair, in regulating caspase 8 activation in the DISC. These results not only provide novel molecular insights into the function of PARP-1 in regulating the extrinsic apoptosis machinery, but also support the intervention of combining PARP inhibitors with death receptor agonists to enhance the efficacy of pancreatic cancer therapy.

Citation Format: Kaiyu Yuan, Yong Sun, Tong Zhou, Jay McDonald, Yabing Chen. PARP-1 regulates death receptor 5-mediated apoptosis in pancreatic cancer. [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 2947. doi:10.1158/1538-7445.AM2013-2947
Abstract 2950: Deletion of TRADD sensitizes oxidative stress-induced necrotic cell death.
Authors: Koo Ki-Bang , Lee Da-gyum , Oh Ji-Yoon , Lee Yun-Sun , Kim You-Sun
Published: August 2014

TNF Receptor-Associated Death Domain (TRADD) is an essential mediator of TNF receptor-1-mediated TNF signaling and is responsible for recruitment of other effector proteins. Recruitment of these adaptor proteins leads to the activation of MAP kinases and NF-kB, as well as cell death. it is well known that aspect of ROS biological effects is their regulatory roles on cell death to function as direct activator cell death or as second messengers in the cell death processes. ROS may initiate cell death processes through affecting various signaling cascade. Several studies on the oxidative stress-induced cell death has shown that some of the key TNF signaling molecules serve as the molecular targets of ROS in cell death. However, there is no report concerning whether TRADD has a function in oxidative stress-induced cell death. The availability of TRADD deficiency mice let us investigate the physiological functions of TRADD on oxidative stress-induce cell death. In this study, we found that TRADD deficiency renders cells more susceptible to oxidative stress-induced cell death through the persistent activation of JNK.

Citation Format: Ki-Bang Koo, Da-gyum Lee, Ji-Yoon Oh, Yun-Sun Lee, You-Sun Kim. Deletion of TRADD sensitizes oxidative stress-induced necrotic cell death. [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 2950. doi:10.1158/1538-7445.AM2013-2950
Abstract 2951: DUSP1 is a novel target for enhancing pancreatic cancer cell sensitivity to gemcitabine.
Authors: Liu Fang , Korc Murray
Published: August 2014

Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer with a poor prognosis that is characterized by marked chemoresistance to a broad spectrum of chemotherapeutic drugs, as a consequence of intrinsic cell-autonomous factors, and extrinsic stroma-associated factors. Dual specificity protein phosphatase 1 (DUSP1) has been reported to be overexpressed in pancreatic cancer cells in PDAC. Moreover, DUSP1 was shown to be essential for efficient in vitro colony formation and in vivo tumorigenicity, indicating that DUSP1 may contribute to the biological aggressiveness of PDAC. However, it is not known whether DUSP1 overexpression contributes to PDAC chemoresistance. Using BxPC3 and COLO-357 human pancreatic cancer cells, we show that gemcitabine activated c-JUN N-terminal kinase (JNK) and p38 mitogen activated protein kinase (p38 MAPK), key kinases in two major stress-activated signaling pathways. Activation of JNK and p38 MAPK was associated with increased apoptosis, as evidenced by increased cleavage of PARP and Caspase 3. However, gemcitabine also enhanced DUSP1 transcription, as demonstrated by increased DUSP1 mRNA levels, and increased RNA polymerase II loading at both DUSP1 promoter and gene body. Moreover, shRNA-mediated inhibition of DUSP1 enhanced JNK and p38 MAPK activation and sensitized both cell lines to gemcitabine. Taken together, these results suggest that gemcitabine-mediated upregulation of DUSP1 contributes to a negative feedback loop that attenuates its beneficial actions on stress pathways and apoptosis, raising the possibility that targeting DUSP1 in PDAC may have the dual advantage of suppressing proliferation while enhancing gemcitabine chemosensitivity.

Citation Format: Fang Liu, Murray Korc. DUSP1 is a novel target for enhancing pancreatic cancer cell sensitivity to gemcitabine. [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 2951. doi:10.1158/1538-7445.AM2013-2951
Abstract 2952: The PKC-iota inhibitor ICA-1 combined with TRAIL for glioblastoma therapy.
Authors: McCray Andrea N. , Desai Shraddha , Acevedo-Duncan Mildred
Published: August 2014

Glioblastoma multiforme is a very aggressive brain tumor. Temozolimide is the standard clinical therapy following surgery which offers a limited survival advantage. The low survival rates of glioblastoma patients require further investigation into pre-clinical combination therapy options with apoptosis abilities. One such chemotherapeutic approach is the use of the novel PKC-iota inhibitor, ICA-1, combined with rhTRAIL. T98G or U87 glioblastoma cells were treated with ICA-1 for 48h or 72h, respectively, with or without rhTRAIL in a 24-well format. Based on flow cytometric analysis, the overall apoptosis rate for the T98G cells exposed to ICA-1 plus TRAIL was the most significant with no distinction between early and late apoptosis rates. In addition, the immunofluorescence analysis showed higher T98G cell death for the combination therapy. Further flow cytometric analysis showed that ICA-1 plus TRAIL generated significantly more late apoptosis for U87 cells than any other treatment. The mechanism of action for this combinatorial therapy is currently under investigation. However, our in vitro data demonstrated that ICA-1 plus TRAIL induced 60-70% apoptosis in U87 glioblastoma cells and T98G glioblastoma cells which may offer a better survival advantage in pre-clinical animal models.

Citation Format: Andrea N. McCray, Shraddha Desai, Mildred Acevedo-Duncan. The PKC-iota inhibitor ICA-1 combined with TRAIL for glioblastoma therapy. [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 2952. doi:10.1158/1538-7445.AM2013-2952
Abstract 2953: Inhibition of calmodulin enhances TRA-8-induced apoptosis in pancreatic cancer cells .
Authors: Yuan Kaiyu , Zhou Tong , McDonald Jay , Chen Yabing
Published: August 2014

Current chemotherapy and radiotherapy are not curative for pancreatic cancer. Tumor necrosis factor-related apoptosis-inducing ligand or agonist antibodies for death receptor 4/5 have been shown to be a promising cancer therapy. TRA-8, a humanized activating antibody for the death receptor 5 (DR5), is a potent apoptosis-inducing reagent for cancer cells. However, several pancreatic cell lines are resistant to TRA-8-induced apoptosis. We have previously demonstrated that calmodulin (CaM) antagonists enhance the Fas death receptor-mediated apoptosis pathway. Therefore, we investigated whether inhibition of CaM might affect DR5-induced apoptotic signaling in pancreatic cancer cells.

We found that CaM antagonists, trifluoperazine (TFP) and tamoxifen (TMX), dose dependently enhanced TRA-8-induced apoptosis of the TRA-8-resistant PANC-1 pancreatic cells. Consistently, TFP and TMX increased TRA-8-induced activation of caspase 8, caspase 9 and caspase 3. Pretreatment of PANC-1 with Z-IETD-FMK, the caspase-8 inhibitor, decreased TFP-enhanced apoptosis by 2 fold, and markedly decreased caspase 3 activation. Immunoprecipitation analysis of the DR5-induced death inducing signaling complex (DISC) identified the interaction of CaM with DR5. Activation of DR-5 by TRA-8 increased the recruitment of CaM into the DISC, which was inhibited by TFP. In addition, TFP decreased the recruitment of the adaptor protein, FADD, and other DISC proteins that regulate cell survival, including Src and the caspase-8 inhibitory protein FLIP. Collectively, these results support an important role for CaM in DR5-induced DISC formation, thus regulating DR5-mediated survival and apoptotic signaling in pancreatic cancer cells.

In summary, we have demonstrated that CaM antagonists sensitize pancreatic cancer cell to TRA 8-induced apoptosis, and that CaM binding to DR5 regulates DR5-induced DISC formation. Defining the function of CaM in regulating DR5-induced signaling pathways may lead to novel therapeutic strategies including the use of CaM antagonists with TRA-8 as combination therapy for pancreatic cancer.

Citation Format: Kaiyu Yuan, Tong Zhou, Jay McDonald, Yabing Chen. Inhibition of calmodulin enhances TRA-8-induced apoptosis in pancreatic 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 2953. doi:10.1158/1538-7445.AM2013-2953
Abstract 2959: Celarstrol enhances TNF-induced neuroblastoma cells death by promoting ROS-mediated programmed necrosis.
Authors: Zhao Ming , Lin Song , Cheng Xiang , Chen Guozhu , Yu Xiaodan
Published: August 2014

Celastrol is an active ingredient of the traditional Chinese medicinal plant Tripterygium Wilfordii, which exhibits significant antitumor activity in different cancer models in vitro and in vivo. Previous studies have showed that celastrol kills cancer cells by inducing apoptosis, in this study, we reported that celastrol has potential to enhance TNF-induced necroptosis in SH-SY5Y neuroblastoma cells. Low dose of TNFα plus celastrol induced remarkable cell death which could be prevented by RIP1 kinase inhibitor Necrostatin-1, but exacerbated by caspase inhibitor Z-VAD-FMK. The mechanism for celastrol to enhance TNF-induced necroptosis involves promoting prolonged JNK activation and inducing ROS accumulation. TNFα plus celastrol induced cell death and mitochondrial outer membrane permeabilization (MOMP) could be reversed by ROS scavenger BHA. In addition, Mdivi-1, an inhibitor of mitochondrial fission protein Drp-1, could also block TNFα plus celastrol induced cell death and MOMP, which is consistent with previous elucidation that RIP1/RIP3 mediated cell death signal through Drp-1 to induced necroptosis. The effect of celastrol on enhancing cancer cells programmed necrosis may provide a new way to overcome the resistance chemotherapy induced by evading apoptosis in some cancer cells.

Citation Format: Ming Zhao, Song Lin, Xiang Cheng, Guozhu Chen, Xiaodan Yu. Celarstrol enhances TNF-induced neuroblastoma cells death by promoting ROS-mediated programmed necrosis. [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 2959. doi:10.1158/1538-7445.AM2013-2959

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 296: 12-O-tetradecanoylphorbol-13-acetate (TPA) induces cell scattering by upregulating krüppel-like factor 4 (KLF4) through the PKCα-ERK signaling axis.
Authors: Lai Jun-Kai , Wu Han-Chung , Chang Chia-Che
Published: August 2014

Krüppel-like factor 4 (KLF4) is a C2H2 zinc-finger transcription factor known to regulate multiple cellular processes including cell proliferation, differentiation and stemness. Cell scattering is characterized by disruption of E-cadherin-mediated intercellular adhesion and acquisition of fibroblast-like morphology with increased motility and invasiveness, a cellular process reminiscent of epithelial-to-mesenchymal transition (EMT). We have previously demonstrated the involvement of KLF4 in hepatocyte growth factor (HGF)-induced cell scattering (Lai et al., J. Cell Sci., in press). Still, the question as to whether KLF4 is generally involved in cell-scattered response induced by other stimuli such as 12-O-Tetradecanoylphorbol-13-Acetate (TPA) has never been formerly addressed and thus the main focus of this study. Using human hepatocellular carcinoma cell line HepG2 as the cellular model, we herein presented evidence that KLF4 was clearly up-regulated following TPA stimulation. RT-PCR analysis and luciferase reporter assay revealed that both the mRNA and promoter activity of KLF4 was markedly increased by TPA, indicating that TPA up-regulated KLF4 at the level of transcription. Of note, knockdown of KLF4 severely impaired the level of TPA-induced HepG2 cell scattering, highlighting the requirement of KLF4 in TPA-induced scattered response. Additionally, we found that the promoting effect of TPA on KLF4 expression was clearly blocked when cells were pre-treated with the pan-PKC inhibitor Gö6983 as well as the PKCα-specific inhibitor Gö6976. Likewise, TPA-induced cell scattering and KLF4 up-regulation were ablated when ERK activity was inhibited by U0126. Taken together, our results defined the involvement of KLF4 in TPA-induced cell scattered response. Particularly, TPA engages the PKCα-ERK signaling axis to induce transcriptional up-regulation of KLF4, leading to the initiation of cell scattering. Our findings further implicate the possible involvement of KLF4 in other TPA-induced cellular responses such as cell migration and invasion.

Citation Format: Jun-Kai Lai, Han-Chung Wu, Chia-Che Chang. 12-O-tetradecanoylphorbol-13-acetate (TPA) induces cell scattering by upregulating krüppel-like factor 4 (KLF4) through the PKCα-ERK signaling axis. [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 296. doi:10.1158/1538-7445.AM2013-296
Abstract 2962: Histone H3.3 mutations drive paediatric glioblastoma through upregulation of MYCN.
Authors: Bjerke Lynn , Mackay Alan , Nandhabalan Meera , Burford Anna , Jury Alexa , Popov Sergey , Bax Dorine , Carvalho Diana , Taylor Katy , Vinci Mara , Bajrami Illirjana , McGonnell Imelda , Lord Chris , Reis Rui , Hargrave Darren , Ashworth Alan , Workman Paul , Jones Chris
Published: August 2014

Glioblastomas of children and young adults (pGBM) have a median survival of only 12-15 months and are clinically and biologically distinct from histologically similar cancers in older adults. They are defined by highly specific mutations in the gene encoding the histone H3.3 variant H3F3A, occurring either at or close to key residues marked by methylation for regulation of transcription - K27 and G34. The G34 mutation is specific to tumours of the cerebral hemispheres and is associated with a distinct age of incidence (16 yrs) and gene expression signature compared to K27 and wild-type tumours. ChIP-Seq for the activating K36 trimethylation mark (H3K36me3) mark of G34V mutant KNS42 pGBM cells identified 156 genes differentially bound and expressed compared to wild-type pGBM control. The transcriptional program induced recapitulates that of the developing forebrain, and involves numerous markers of stem cell maintenance, cell fate decisions and self-renewal. Critically, H3F3A G34 mutations cause profound upregulation of MYCN, a potent oncogene which is causative of glioblastomas when expressed in the correct developmental context. A synthetic lethality siRNA screen revealed this driving aberration to be selectively targetable in this patient population by inhibiting kinases responsible for stabilisation of the protein such as AURKA and CHK1. We thus provide the mechanistic explanation for how the first histone gene mutation in human disease biology acts to deliver MYCN, a potent tumorigenic initiator, into a stem cell compartment of the developing forebrain, selectively giving rise to incurable cerebral hemispheric glioblastoma. Employing synthetic lethal approaches to these mutant tumour cells provides a rational way to develop novel and highly selective treatment strategies.

Citation Format: Lynn Bjerke, Alan Mackay, Meera Nandhabalan, Anna Burford, Alexa Jury, Sergey Popov, Dorine Bax, Diana Carvalho, Katy Taylor, Mara Vinci, Illirjana Bajrami, Imelda McGonnell, Chris Lord, Rui Reis, Darren Hargrave, Alan Ashworth, Paul Workman, Chris Jones. Histone H3.3 mutations drive paediatric glioblastoma through upregulation of MYCN. [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 2962. doi:10.1158/1538-7445.AM2013-2962

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 2966: The role of histone demethylase GASC1 in promoting prostate cancer progression.
Authors: Zhang Lihong , Hou Jinling , Holowatyj Andreana , Yang Zeng-Quan
Published: August 2014

Prostate cancer affects 1 in 6 American men, and is the second leading cause of cancer-related deaths in males. The defiance of castration-resistant prostate cancer (CRPC) to currently available treatments represents the most challenging aspect of this disease. Although resistant to androgen deprivation treatment, most CRPCs continue to express the androgen receptor (AR) and remain dependent on AR signaling for growth and proliferation. Previously, we cloned a novel gene, GASC1 (gene amplified in squamous cell carcinoma 1), from 9p24 amplified cancer cells. Recently, the GASC1 protein has been identified as a key histone demethylase that controls chromatin-mediated regulation of gene expression. Importantly, the GASC1 protein interacts with the AR and serves as a co-activator of AR-induced transcription in prostate cells. In this study, we demonstrated that various aggressive tumors, including CRPCs, have upregulated GASC1. We used an shRNA approach to determine whether GASC1 knockdown would affect the proliferation and transformation of CRPC cell lines. We found that knocking down GASC1 inhibits growth and colony formation of CRPC cells (C4-2B and CWR22Rv1) in vitro. Previously, it was reported that the GASC1 protein interacts with the AR and functions as a co-activator of AR-induced transcription in LNCaP cells. The GASC1 protein contains N-terminal Jumonji catalytic domains and C-terminal Tudor and PHD domains; however, which domains are responsible for the association of GASC1 with the AR have not yet been identified. To address this question, we generated stable LNCaP cell lines expressing V5-tagged N-terminal Jumonji catalytic domains and C-Terminal PHD/Tudor domains, and performed Co-IP analyses. We found that the C-terminal region is essential for GASC1 association with the AR in prostate cancer cells. To explore the contribution of GASC1 to endogenous AR-regulated gene expression in AR-positive CRPC cells, we performed quantitative RT-PCR assays to measure the expression of both classical AR target genes (including PSA/KLK3, KLK2, FKBP5, and NDRG1) and CRPC-specific genes (including UBE2C, CDK1, CDC20, and CCNA2) in C4-2B cells with or without GASC1 knockdown. We found that knocking down GASC1 significantly reduced transcript levels of a set of classical and CRPC-specific AR target genes in either the presence or absence of the AR ligand. Our data demonstrates that GASC1 is a target of substantial relevance to aggressive prostate cancers, including CRPCs. As such, targeting GASC1 is a promising strategy to control CRPC or prevent its emergence.

Citation Format: Lihong Zhang, Jinling Hou, Andreana Holowatyj, Zeng-Quan Yang. The role of histone demethylase GASC1 in promoting prostate cancer progression. [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 2966. doi:10.1158/1538-7445.AM2013-2966