bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1653: Regulation of autophagy and apoptosis by a natural triterpenoid in diffuse large B-cell lymphoma.
Authors: Doonan Bently , Hossain Azim , Radwan Faisal , Haque Azizul
Published: August 2014

Diffuse large B-cell lymphoma (DLBCL), a subtype of non-Hodgkin's lymphoma, is the most common lymphoid malignancy in the western world. Treatment of DLBCL has been greatly improved in recent years with the addition of the monoclonal antibody Rituximab to the gold standard

CHOP chemotherapy regimen, but these treatments are often ineffective in patients with highly aggressive disease or in patients of advanced age. A real problem given the population of DLBCL patients is largely 60 years of age or older, these chemotherapeutics also present the issue of severe toxicity to the patient(s). Thus, studies have focused lately on natural products that can selectively kill malignant lymphomas while displaying a reduced host toxicity profile. Here, we report that a natural extract from the Ganoderma lucidum mushroom, GA-DM, induces apoptosis in human DLBCL cell lines and primary tumors, and activates an orchestrated autophagy/apoptosis pathway in the murine B-cell lymphoma cell line A20. Antitumor efficacy of

GA-DM was also investigated in vivo in a murine B-cell lymphoma model using the

A20 cell line, where GA-DM (50mg/kg) treatment reduced both the number of tumor metastases and the overall tumor burden in diseased mice. These findings support the potential use of GA-DM as a novel chemotherapeutic in the treatment of DLBCL and could improve the treatment of higher risk patients with advanced disease who can't tolerate current chemotherapy treatment.

Citation Format: Bently Doonan, Azim Hossain, Faisal Radwan, Azizul Haque. Regulation of autophagy and apoptosis by a natural triterpenoid in diffuse large B-cell lymphoma. [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 1653. doi:10.1158/1538-7445.AM2013-1653
Abstract 1654: Cisplatin resistance is associated with autophagy in SPARC expressing medulloblastoma cells.
Authors: Pannuru Padmavathi , Dontula Ranadheer , Engelhard Herbert , Ozer Howard , Lakka Sajani S.
Published: August 2014

Medulloblastoma is the most common malignant brain tumor in children and is associated with a poor outcome. Our previous studies indicate that SPARC expression suppressed medulloblastoma tumor growth in vitro and in vivo. We were interested in gaining further insight into the potential of SPARC expression as a novel approach to sensitize medulloblastoma to chemotherapeutic agents. In this study, we demonstrate that SPARC: expression resulted in enhanced resistance of cancer cells to cisplatin, a commonly used chemotherapeutic drug in the treatment of medulloblastoma. We also demonstrate that autophagy was involved in SPARC expression mediated resistance to cisplatin. We further demonstrated that suppression of autophagy by autophagy inhibitors, 3-methyladenosine (3MA) and small interfering RNAs (siRNAs) targeting Atg5 in SPARC expressed cells enhanced the sensitivity of medulloblastoma cells to cisplatin compared to cells treated with cisplatin alone. Moreover, sensitization to cisplatin by inhibition of autophagy is SPARC expressed cells was accompanied by induction of apoptosis indicating that suppression of autophagy indeed renders tumor cells more sensitive to cisplatin. Further we show overexpression of High mobility group box 1 (HMGB1 in SPARC expressed cells rendered medulloblastoma cells resistant to cell death; whereas depletion of HMGB1 using RNA interference suppressed autophagy and increased the sensitivity of medulloblastoma cells to cisplatin as assessed by microtubule-associated protein 1 light chain 3 (LC3) lipidation, redistribution of LC3 into cytoplasmic puncta, and accumulation of autophagosomes and autolysosomes. Further our data suggest a role for HMGB1 in the regulation of autophagy through the MEK-ERK pathway in SPARC over expressed cells. The results of our study revealed a new mechanism for enhancing cisplatin sensitivity in SPARC expressed cells. These findings have important clinical implications for the design of experimental treatment protocols for medulloblastoma.

Citation Format: Padmavathi Pannuru, Ranadheer Dontula, Herbert Engelhard, Howard Ozer, Sajani S. Lakka. Cisplatin resistance is associated with autophagy in SPARC expressing medulloblastoma 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 1654. doi:10.1158/1538-7445.AM2013-1654

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 1657: Targeting Cullin-RING ligases by MLN4924 induces autophagy via modulating the HIF1-REDD1-TSC1-mTORC1-DEPTOR axis in breast cancer cells.
Authors: Zhao Yongchao , Xiong Xiufang , Jia Lijun , Sun Yi
Published: August 2014

MLN4924, a newly discovered small molecule inhibitor of NEDD8-Activating Enzyme (NAE), inactivates Cullin-RING E3 ubiquitin ligases (CRLs) by blocking cullin neddylation. As a result, MLN4924 causes accumulation of several key substrates of CRLs and effectively suppresses tumor cell growth by inducing apoptosis and senescence. However, the role of MLN4924 in induction of autophagy and its biological significance are totally unknown. Here we showed that MLN4924 effectively induces autophagy in both time- and dose-dependent manners in breast cancer cells. Mechanistically, by inactivating CRLs, MLN4924 causes accumulation of DEPTOR and HIF1α. The siRNA knockdown and gene KO studies showed that DEPTOR and the HIF1α-REDD1-TSC1 axis are responsible for MLN4924-induced autophagy via inhibiting mTORC1. Biologically, autophagy is a survival signal to tumor cells, and blockage of autophagy via siRNA knockdown, gene KO and small molecule inhibitor remarkably enhanced MLN4924-induced apoptosis. Our study reveals an uncharacterized mechanism of MLN4924 action and provides the proof-of-concept evidence for strategic drug combination of MLN4924 with an autophagy inhibitor for maximal killing of tumor cells via enhancing apoptosis.

Citation Format: Yongchao Zhao, Xiufang Xiong, Lijun Jia, Yi Sun. Targeting Cullin-RING ligases by MLN4924 induces autophagy via modulating the HIF1-REDD1-TSC1-mTORC1-DEPTOR axis in 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 1657. doi:10.1158/1538-7445.AM2013-1657
Abstract 1661: Autophagy induced by cathepsin S inhibition causes early ROS production for oxidative DNA damage and subsequent cell death.
Authors: Huang Chien-Chang , Chang Jang-Yang
Published: August 2014

Cathepsin S plays multiple roles in MHC class II antigen presentation, extracellular matrix degradation, angiogenesis, and tumorogenesis. Pharmacological inhibition of cathepsin S activity not only inhibits tumor invasion and angiogenesis but also reduces cell viability. Herein, we investigate the molecular executioner which responsible for targeting cathepsin S-induced cell death and its association with autophagy. Surprisingly, distinct from the classical regulation of autophagy by ROS, autophagy, rather than mitochondrial process, is the genuine regulator of early ROS production which responsible for consequent oxidative DNA damage and cell death. Molecular silencing of autophagy-dependent ATG genes and pharmacologic inhibition of autophagy with 3-MA and wortmannin significantly reduced ROS production, oxidative DNA damage, and cell death. Collectively, this study reveals a non-canonical molecular pathway where after the inhibition of cathepsin S, autophagy up-regulates ROS production for oxidative DNA damage and subsequently contributes to autophagy-dependent cell death. (This work was supported by the following grants: Department of Health, Taiwan DOH99-TD-C-111-004, National Health Research Institutes CA-101-PP-22, and National Research Program for Biopharmaceuticals 100CAP015-5)

Citation Format: Chien-Chang Huang, Jang-Yang Chang. Autophagy induced by cathepsin S inhibition causes early ROS production for oxidative DNA damage and subsequent 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 1661. doi:10.1158/1538-7445.AM2013-1661
Abstract 1668: Induction of Autophagy by a novel agent promotes the growth arrest in breast cancer cells.
Authors: Subramani Reddy Ramadevi , Pahari Pallab , Damodaran Chendil
Published: August 2014

Breast cancer is a highly complex disease due to its heterogeneity in nature; that often impedes the current therapeutic options. Autophagy is a catabolic process which plays a vital role in both progressions as well as in the treatment of cancer. In fact, the role of autophagy in cancer is still unclear. Our lab is focusing on small molecules, which may induce autophagy that may result in growth arrest in breast cancer cells. In our preliminary screening studies, we have identified a small molecule, Nimocinol Acetate (NA) which induces autophagy that resulted in inhibition of growth of estrogen-positive (MCF-7) and estrogen-negative (MDA-MB-231) breast cancer cells. Molecular analysis showed the significant induction of LC-3B and ATG-3 in NA treated cells as compared to other autophagy-regulators like Atg5, Atg7, Atg12, and beclin-1, which convey the importance of ATG3 and LC3B markers in induction of autophagy. The immunofluorescence analysis confirmed the formation of acidic autophagic vesicles upon NA treatment. To further ascertain the involvement of lysosomes and autophagosomes, we specifically blocked ATG-3 and LC-3B expression by siRNA and then subjected to immunofluorescence assay. The observed results confirmed the contribution and activation of these two molecules is required for the induction of autophagy. Interestingly, no significant changes were obseverved either in the cell cycle regulation or in induction of apoptosis, which revealed that the activation of autophagy may be responsible for NA mediated growth inhibition in breast cancer cells. Currently, we are planning to conduct xenograft studies, which may suggest the in vivo efficacy and pharmacokinetic potential of NA. The present study concludes that NA could be a potential agent either alone or in combination with other chemotherapeutic agents with a novel strategy to combat breast cancer.

Citation Format: Ramadevi Subramani Reddy, Pallab Pahari, Chendil Damodaran. Induction of Autophagy by a novel agent promotes the growth arrest in 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 1668. doi:10.1158/1538-7445.AM2013-1668
Abstract 1669: DIRAS family members inhibit cell growth and proliferation while inducing autophagy in human ovarian cancer cells.
Authors: Sutton Margie N. , Lu Zhen , Bast Robert C.
Published: August 2014

Among the three members of the DIRAS family, DIRAS3 (also known as AplasiaRasHomolog Member I; ARHI) has been best characterized. ARHI is a maternally expressed imprinted tumor suppressor gene that encodes a 26 kDa GTPase with 60% homology to Ras and Rap. ARHI is downregulated in many tumor types including ovarian cancer where its re-expression at physiologic levels inhibits cancer cell growth, reduces motility, induces autophagy and promotes tumor dormancy. ARHI induces autophagy by multiple mechanism including:1) blocking PI3K-Akt signaling and inhibiting mTOR activity, 2) displacing Bcl-2 from Beclin and participating directly in the autophagy initiation complex; and 3) inducing expression of Atg4 and participating directly in the formation of autophagic vesicles, co-localizing with MAP-LC3-II in the vesicle membrane. In human cells, knockdown experiments have demonstrated that ARHI is essential for the induction of autophagy. During evolution, murine cells have lost the ARHI (DIRAS3) gene, yet can still undergo autophagy. Mice and humans express two closely related Ras-related GTPases,DIRAS1 and DIRAS2. These 22 kDa GTPases, have 30-40% overall homology with H-Ras, and 50-60% homology with ARHI (DIRAS3) with the only major difference being the truncation of the N-terminal extension. In this study, we show that transfection of DIRAS1, 2 or 3 with a selectable marker inhibits clonogenic growth of SKOv3 human ovarian cancer cells. Whereas DIRAS3 produced complete inhibition, DIRAS 1 produced 50% inhibition and D IRAS2 35% inhibition of clonogenic growth. One possible mechanism for the growth inhibition observed could be attributed to the induction of autophagy which has been shown by the formation of LC3 punctae in SKOV3 cells and the increased conversion of LC3-I to LC3-II by western blot analysis when DIRAS1 and DIRAS2 were overexpressed by transfection. These findings suggest that these DIRAS family homologs may have similar functions in limiting human tumor progression and in inducing autophagy. Future studies will examine whether DIRAS1 and DIRAS2 serve as surrogates forARHI (DIRAS3) in murine cells.

Citation Format: Margie N. Sutton, Zhen Lu, Robert C. Bast. DIRAS family members inhibit cell growth and proliferation while inducing autophagy in human ovarian 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 1669. doi:10.1158/1538-7445.AM2013-1669
Abstract 1676: Chemotherapy-induced immunogenic modulation of tumor cells enhances killing by cytotoxic T lymphocytes and is distinct from immunogenic cell death.
Authors: Hodge James W. , Garnett Charlie T. , Farsaci Benedetto , Palena Claudia , Tsang Kwong-Yok , Ferrone Soldano , Gameiro Sofia R.
Published: August 2014

Certain chemotherapeutic regimens trigger cancer cell death while inducing dendritic cell maturation and subsequent immune responses. However, chemotherapy-induced immunogenic cell death (ICD) has thus far been restricted to select agents. In contrast, several chemotherapeutic drugs modulate antitumor immune responses, despite not inducing classic ICD. In addition, in many cases tumor cells do not die after treatment. Here, using docetaxel, one of the most widely used cancer chemotherapeutic agents, as a model, we examined phenotypic and functional consequences of tumor cells that do not die from immunogenic cell death. Docetaxel treatment of tumor cells did not induce ATP or HMGB1 secretion, or cell death. However, calreticulin exposure was observed in all cell lines examined after chemotherapy treatment. Killing by CEA, MUC-1, or PSA-specific CD8+ CTLs was significantly enhanced after docetaxel treatment. This killing was associated with increases in components of antigen-processing machinery, and mediated largely by calreticulin membrane translocation, as determined by functional knockdown of calreticulin, PERK, or calreticulin-blocking peptide. A docetaxel-resistant cell line was selected (MDR-1+, CD133+) by continuous exposure to docetaxel. These cells, while resistant to direct cytostatic effects of docetaxel, were not resistant to the chemomodulatory effects that resulted in enhancement of CTL killing. Here, we provide an operational definition of “immunogenic modulation,” where exposure of tumor cells to nonlethal/sublethal doses of chemotherapy alters tumor phenotype to render the tumor more sensitive to CTL killing. These observations are distinct and complementary to immunogenic cell death and highlight a mechanism whereby chemotherapy can be used in combination with immunotherapy.

Citation Format: James W. Hodge, Charlie T. Garnett, Benedetto Farsaci, Claudia Palena, Kwong-Yok Tsang, Soldano Ferrone, Sofia R. Gameiro. Chemotherapy-induced immunogenic modulation of tumor cells enhances killing by cytotoxic T lymphocytes and is distinct from immunogenic 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 1676. doi:10.1158/1538-7445.AM2013-1676
Abstract 1682: Radiation-induced autophagy is dependent on Sirtuin 3.
Authors: Shukla Sadhanshu , Smart DeeDee
Published: August 2014

Sirtuin family members (Sirtuin 1 to 7) are highly conserved proteins, both structurally and functionally, and have either NAD+ dependent enzymatic activity (Sirtuin 1,2, 3, 5 and 7) for deacetylation of proteins or ADP ribosyl transferase activity (Sirtuin 4 and 6). Their functions in metabolic processes and chromatin remodeling have been conserved throughout evolution. Sirtuin 1 and Sirtuin 2 have been shown to be linked with several neurological disorders. The role of other Sirtuins in neurodegenerative disorders is not known. Radiation therapy (RT), one of the preferred and widely accepted treatments for intracranial primary and secondary metastatic tumors, may lead to long-term irreversible neurotoxicity in susceptible individuals with cognitive dysfunction similar to that observed in other neurodegenerative disorders. There are ample data which demonstrate that aberrant autophagic process and dysfunctional mitochondria are important factors leading to several neurological diseases, such as Huntington's disease and Parkinson's dementia. Among the Sirtuins, Sirtuin 3, 4 and 5 are localized within mitochondria, however, their role in radiation-induced neurotoxicity has not been evaluated.

Using Sirtuin 3 knockout (KO) mice generated through genomic deletion allowed us the opportunity to study its role in autophagy and mitochondrial function during exposure to radiation. Preliminary reports show Sirtuin 3 in the adult mouse brain to be localized primarily within hippocampus and subcortical plate. Through flow cytometry and immunofluorescence stainin of mouse embryonic fibroblasts (MEFs) derived from Sirtuin 3 KO mice, we demonstrate that loss of Sirtuin 3 alters rates of basal autophagy. Furthermore, immunoblot analysis of autophagic marker LC3B I &II in the presence and absence of hydrolase inhibitors (E64d & Pepstatin A) exhibits that loss of Sirtuin 3 stimulates autophagy. Radiation treatment of Sirtuin 3 wild-type and knockout MEFs exhibited dose dependent induction of autophagy. Staining with mitotracker red and JC1 illustrates that MEFs lacking Sirtuin 3 retain mitochondria with abnormal morphology and disturbed membrane potential which can further enhanced by radiation treatment and exposure to the ionophore Carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Enrichment of lower form of LC3B in Sirtuin 3 KO cells upon treatment with CCCP and radiation suggests that loss of Sirtuin 3 sensitizes cells towards enhanced autophagy via mitophagy. Taken together, this study indicates that Sirtuin 3 is an important player in deciding cell fate via mitophagy during radiation treatment and has important implications in further studies of radiation-induced neurotoxicity.

Citation Format: Sadhanshu Shukla, DeeDee Smart. Radiation-induced autophagy is dependent on Sirtuin 3. [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 1682. doi:10.1158/1538-7445.AM2013-1682
Abstract 1685: Inactivation of a transcriptional variant of MAP1LC3A gene in human cancers.
Authors: Inoue Jun , Bai Hua , Kawano Tatsuyuki , Inazawa Johji
Published: August 2014

Autophagy functions to clear damaged or unfolded cellular components in the lysosomes as a survival pathway used by established cancers to sustain metabolism and tolerate stress. Autophagy, however, also suppress cancer initiation in liver and the accumulation of p62/SQSTM1 protein, a substrate for autophagic degradation, contributes to its tumorigenesis, suggesting that autophagy-related gene(s) may be actually inactivated in a part of human cancers at least. Recently, we have identified that a transcriptional variant of microtubule-associated protein 1 light chain 3A (LC3Av1), a member of human LC3 gene family, was frequently inactivated by aberrant DNA methylation in human cancers. Induction of wild-type LC3Av1 led to the decrease of p62 protein level, resulting in the inhibition of tumor growth, but not its mutant with the C-terminal glycine (lipidation site) changed to alanine. Thus, these results suggest that the impairment of autophagy through LC3Av1-silencing due to DNA methylation may be associated with tumorigenesis in various types of human cancer.

Citation Format: Jun Inoue, Hua Bai, Tatsuyuki Kawano, Johji Inazawa. Inactivation of a transcriptional variant of MAP1LC3A gene in human 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 1685. doi:10.1158/1538-7445.AM2013-1685
Abstract 1687: Frequent inactivation of the Mieap-regulated mitochondrial quality control in colorectal cancer.
Authors: Kamino Hiroki , Nakamura Yasuyuki , Kitamura Noriaki , Futamura Manabu , Yoshida Masaki , Murai Ryuya , Saito Yuri , Sano Hitoya , Kanai Yae , Moriya Yoshihiro , Arakawa Hirofumi
Published: August 2014

Recently, we discovered a novel mechanism for mitochondrial quality control (MQC), in which Mieap, a p53-inducible protein, controls MQC by repairing or eliminating unhealthy mitochondria in a process known as MALM (Mieap-induced Accumulation of Lysosome-like organelles within Mitochondria) or MIV (Mieap-Induced Vacuole), respectively (1, 2). We also reported that two mitochondrial outer membrane proteins, BNIP3 and NIX, are essential mediators of the MALM (2, 3). To examine the role of Mieap-regulated MQC pathway in tumorigenesis, we analyzed the status of p53, Mieap, BNIP3, and NIX in 57 cases of primary colorectal cancer. Of the 57 patients, Mieap and BNIP3 promoter methylation was observed in 5 (8.8%) and 26 (45.6%) patients, respectively. A p53 mutation was detected in 4 (18%) of the 22 patients with either Mieap or BNIP3 promoter methylation whereas it was found in 9 (52%) of the 17 patients with neither Mieap nor BNIP3 promoter methylation, implying that the p53/Mieap/BNIP3 pathway in the Mieap-regulated MQC is inactivated in at least 80% of colorectal cancer cases. Hypoxia induced MALM in the colorectal cancer cell line LS174T (wild-type p53, Mieap-positive, BNIP3-positive). In vitro knockdown of p53, Mieap, or BNIP3 in the LS174T cells severely inhibited hypoxia-induced MALM, leading to the accumulation of unhealthy mitochondria and increased mitochondrial reactive oxygen species (mtROS) generation. Furthermore, the mtROS dramatically enhanced cancer migration and invasion under hypoxic conditions. These results suggest that the Mieap-regulated MQC function plays a critical role in tumor suppression in colorectal cancer.

1. Miyamoto Y et al. PLoS ONE 6: e16054, 2011

2. Kitamura N et al. PLoS ONE 6: e16060, 2011

3. Nakamura Y et al. PLoS ONE 7: e30767, 2012

Citation Format: Hiroki Kamino, Yasuyuki Nakamura, Noriaki Kitamura, Manabu Futamura, Masaki Yoshida, Ryuya Murai, Yuri Saito, Hitoya Sano, Yae Kanai, Yoshihiro Moriya, Hirofumi Arakawa. Frequent inactivation of the Mieap-regulated mitochondrial quality control in colorectal 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 1687. doi:10.1158/1538-7445.AM2013-1687
Abstract 1692: SR16388 impairs protein homeostasis and induces autophagy in diverse human cancer cell lines.
Authors: Sambucetti Lidia C. , Zhou Wei , Calaoagan Joy , Kara Laurie , Liu Xiaohe , Sato Barbara , Samuelson Steve , Collins Nathan , Laderoute Keith
Published: August 2014

SR16388 is a novel amino steroid that targets estrogen-binding proteins including genomic estrogen receptors (ERs). Preclinical studies have demonstrated that SR16388 is a well-tolerated, orally active compound with compelling properties as an experimental antitumor agent: SR16388 has been demonstrated to 1) inhibit the proliferation and viability of diverse human cancer cell lines in vitro; 2) have antiangiogenic activity in vivo; and 3) reduce the growth of human tumor xenografts in mice. SR16388, which binds to and potently inhibits ERα and ERβ in breast cancer cells, also inhibits the widely expressed orphan nuclear receptor ERRα that has been implicated in the development of various human malignancies. ERRα is thought to regulate tumor cell energy metabolism associated with energy stress within solid tumor microenvironments.

We have been exploring potential mechanisms that could explain SR16388’s broad anti-proliferative effects toward human tumor cell lines with different genotypes and tissues of origin. In these studies, we observed that SR16388 can initiate a cell-cycle arrest at G1 or G2 and ultimately cause cytotoxicity (e.g., by apoptosis) even in cells that do not express ERs or ERRα. We recently observed that SR16388 can induce persistent macroautophagy (autophagy) in diverse tumor cells, including cells from hematopoietic malignancies (e.g., multiple myeloma/MM cells) in addition to carcinomas such as prostate and breast cancers. We demonstrated induction of autophagy by using established assays (e.g., LC3I/II processing; fluorescent reporters for acidic vacuole formation; long-lived protein degradation). Here we present results demonstrating that SR16388 can rapidly and potently induce autophagy in cultures of MM cells (U266, RPMI-8226) and PC3 prostate cancer cells. Induction of autophagy in PC3 cells, in particular, was detected in tumor xenografts as early as 3 hours after dosing of mice with a therapeutic dose level of SR16388. We will present data supporting the hypothesis that SR16388 exerts its anti-proliferative and anti-tumor effects in part through the induction of stress pathways modulated by perturbations of protein homeostasis. These effects could yield therapeutic benefits that derive from targets within solid tumor microenvironments.

Citation Format: Lidia C. Sambucetti, Wei Zhou, Joy Calaoagan, Laurie Kara, Xiaohe Liu, Barbara Sato, Steve Samuelson, Nathan Collins, Keith Laderoute. SR16388 impairs protein homeostasis and induces autophagy in diverse human cancer cell lines. [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 1692. doi:10.1158/1538-7445.AM2013-1692
Abstract 1699: Suppressor of cytokine signaling 6 (SOCS6) promotes mitochondrial fission through E3 ubiquitin ligase complex activity.
Authors: Lin Huan-Yu , Lin Shiu-Ting , Wang Mei-June , Chen Jeou-Yuan
Published: August 2014

Mitochondria are highly motile organelles that constantly undergo fission and fusion. Impairment of mitochondrial dynamics is associated with mitochondrial dysfunction and is frequently linked to the pathogenesis of neurodegenerative diseases and cancer. We have previously shown that biallelic inactivation of the suppressor of cytokine signaling 6 (SOCS6) is a frequent event in human gastric cancer, and that overexpression of SOCS6 inhibits cell growth as well as colony formation in soft agar, suggesting a potential role of SOCS6 as a tumor suppressor. We also showed that SOCS6 is targeted to mitochondria, and induces mitochondrial fragmentation accompanied with intrinsic apoptosis. SOCS6 induces mitochondrial fragmentation is in part mediated through its interaction with DRP1 and regulation of DRP1 fission activity. In this study, we further showed that SOCS6 interacts with the Elongin B/C and Cullin 5 forming an ECS E3 ubiquitin ligase complex, and that formation of an intact ECS E3 ligase complex is important for SOCS6-mediated mitochondrial fragmentation. First, the interaction of SOCS6 with Elongin B/C and/or Cullin 5 prolonged SOCS6 stability. Second, mutations of the conserved Leu500 and Cys504 residues in SOCS6 BC-box which are critical for Elongin B/C binding yielded a SOCS6 mutant failed to induce mitochondrial fragmentation and apoptosis. Most importantly, ablation of Elongin C activity protected cells from SOCS6-mediated mitochondrial fission. Taken together, our data suggest an important role of SOCS6 in modulating mitochondrial dynamics that is dependent on ECS E3 ubiquitin ligase complex activity.

Citation Format: Huan-Yu Lin, Shiu-Ting Lin, Mei-June Wang, Jeou-Yuan Chen. Suppressor of cytokine signaling 6 (SOCS6) promotes mitochondrial fission through E3 ubiquitin ligase complex activity. [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 1699. doi:10.1158/1538-7445.AM2013-1699
Abstract 1701: PENAO, a novel mitochondria-targeted agent, has shown potent antitumor effect on glioblastoma in vitro and in vivo.
Authors: Shen Han , Luk Peter P. , Chung Sylvia A. , Decollogne Stephanie , Dilda Pierre J. , Hogg Philip J. , McDonald Kerrie L.
Published: August 2014

Despite advances in multimodal therapy, the prognosis in the great majority of patients with glioblastoma (GBM) is still dismal as almost all tumors relapse and result in disease-related death. New therapeutic strategies are urgently needed. PENAO, (4-(N-(S-penicillaminylacetyl)amino) phenylarsonous acid) is a novel mitochondria-targeted agent that inactivates adenine nucleotide translocase (ANT) located on the inner-membrane of mitochondria. PENAO blocks ANT delivery of ATP to mitochondrial-bound hexokinase II. This blockage inhibits glucose metabolism and triggers the mitochondrial permeability transition pore to leak which results in proliferation arrest and apoptotic cell death.

In vitro, we tested the efficacy of PENAO on a panel of glioblastoma cell lines including immortalized and primary glioblastoma neural stem (GNS) cell lines. PENAO demonstrated potent anti-proliferative activity in all cells. The half-maximal inhibitory concentration (IC50) values for PENAO were in the range of 0.3-6 μM. Blocking ANT with PENAO resulted in inhibition of glioblastoma cell migration at concentration of 1.4 μM. PENAO (0.3-5 μM) increased caspase 3 and 9 activity levels indicative of the intrinsic apoptotic pathway. Proper functioning of ANT is also of importance for maintaining mitochondrial integrity. Low micromolar concentrations of PENAO inhibited oxygen utilization, increased the cytosolic levels of superoxide and depolarized the mitochondrial trans-membrane potential, which are responsible for the activation of mitochondria-mediated apoptosis in glioblastoma cells.

In vivo, PENAO was found to cross the intact blood-brain-barrier in mice and continuous administration of 1 mg/kg/day PENAO to 10 mice bearing subcutaneous glioblastoma tumors for 28 days resulted in 8 partial and 2 complete tumor remissions. No signs or symptoms of treatment toxicity were noticed.

To further inhibit glucose metabolism of glioblastoma, we tested the effectiveness of PENAO in combination with 2-deoxy-D-glucose, a glucose analogue that inhibits glycolysis via competitive inhibition of hexokinase. This dual-targeting of glucose metabolism by inhibiting both mitochondrial respiration and glycolysis led to synergistic effects and improved efficacy when measuring proliferation arrest in vitro. By targeting the glioblastoma aberrant metabolism of glucose better treatments for this cancer may arise.

Citation Format: Han Shen, Peter P. Luk, Sylvia A. Chung, Stephanie Decollogne, Pierre J. Dilda, Philip J. Hogg, Kerrie L. McDonald. PENAO, a novel mitochondria-targeted agent, has shown potent antitumor effect on glioblastoma in vitro and in vivo. [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 1701. doi:10.1158/1538-7445.AM2013-1701
Abstract 1704: Linking mitochondrial epigenetics to tumorigenesis and the cancer theory of Warburg.
Authors: Seyfried Thomas N.
Published: August 2014

Otto Warburg first proposed that respiratory insufficiency was the common metabolic hallmark of all tumor cells. Respiratory insufficiency is the precursor to aerobic fermentation (Warburg effect), and can also be linked to dysregulated proliferation and to the genomic abnormalities seen in the majority of cancers. It is well documented that tumorigenicity can be suppressed in a broad range of tumor types when cytoplasm from enucleated normal cells, containing normal mitochondria, is fused with nucleated tumor cells to form cybrids. Tumorigenicity is also suppressed in a broad range of tumors following transplantation of the nucleus from tumor cells into enucleated eggs or somatic cells, indicating that tumor nuclei are capable of directing development despite the persistence of tumor-associated mutations. Tumor cell mutations seem to abort development, rather than induce tumorigenesis. In contrast to the tumor suppressive effects of cytoplasm containing normal mitochondria, either cell death or tumor cells arise when nuclei from non-tumorigenic normal cells are fused with cytoplasm from tumor cells. The results from these studies indicate that nuclear genomic defects alone cannot produce tumors, and that normal mitochondria can suppress tumorigenesis. The findings, when viewed collectively, support Warburg's central theory and suggest that mitochondrial epigenetics drives tumorigenesis. Supported in part from the Boston College Res. Expense Fund.

Citation Format: Thomas N. Seyfried. Linking mitochondrial epigenetics to tumorigenesis and the cancer theory of Warburg. [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 1704. doi:10.1158/1538-7445.AM2013-1704
Abstract 1708: Metabolic regulation of the ubiquitin+proteasome system.
Authors: Driscoll James J. , Jagannathan Sajjeev
Published: August 2014

The ubiquitin+proteasome system (UPS) is a highly complex network that maintains protein homeostasis and cell viability through the highly regulated and selective turnover of targeted proteins. The proteasome is the major intracellular protease in eukaryotes and serves as the catalytic core of the UPS. The cytotoxic effect of proteasome inhibition has been translated therapeutically to dramatically improve the overall survival of patients diagnosed with the plasma cell malignancy multiple myeloma (MM). Bortezomib has emerged as the standard-of-care therapy for MM to catapult the UPS into a position of prominence in cancer biology and drug development but significant obstacles remain since many patients do not respond to bortezomib, clinical responses are not sustained and drug resistance inevitably emerges through unknown mechanisms. Gene profiling was performed as an unbiased approach to address the precise molecular events that promote bortezomib-resistance. Results indicated that effectors of energy metabolism were differentially expressed in cells resistant to bortezomib. Subunits of the cellular energy sensor AMP-activated protein kinase (AMPK) were expressed at lower levels in resistant cells relative to the parental cells. Real-time, cell-based metabolic measurements demonstrated that bortezomib rapidly and dramatically reduced tumor cell oxygen-consumption while bortezomib-resistant cells were less sensitive to metabolic deregulation. Genetic ablation of the AMPKα1/α2 encoding-genes also reduced the effect of bortezomib to promote autophagosome formation as well as bortezomib-induced cytotoxicity. Similarly, knockout of the AMPK-substrate and autophagy-initiating kinase ULK1 also reduced bortezomib-induced autophagosome formation and cell death. Proteasome inhibition promoted ULK-1 association with the autophagosome initiators ATG13, FIP200 and beclin-1. The AMPK activator AICAR further enhanced bortezomib-induced cytotoxicity in myeloma, lymphoma, leukemic and solid tumor cells. Proteasome inhibitors induce AMPK+ULK-mediated autophagosome formation that is coupled to apoptosis and can be exploited as a novel anti-cancer therapeutic strategy.

Citation Format: James J. Driscoll, Sajjeev Jagannathan. Metabolic regulation of the ubiquitin+proteasome system. [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 1708. doi:10.1158/1538-7445.AM2013-1708
Abstract 1710: An anti-apoptotic Bcl-2 family protein index predicts the response of leukemic cells to the pan-Bcl-2 inhibitor S1.
Authors: Liu Yubo , Zhang Zhichao
Published: August 2014

Bcl-2-like proteins prevent cell death through expression level, post transcriptional modification, and protein-protein-interaction. Recent studies have shown that antiapoptotic Bcl-2-like members are overexpressed in many haematological malignancies. BH3 mimetics are promising anticancer agents by directly target these proteins and their interactions. S1 (8-oxo-3-thiomorpholin-4-yl-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile) is an authentic BH3 mimetic and a triple inhibitor of Bcl-2, Mcl-1, and Bcl-XL. In the present study, we found S1 exhibited a variant sensitivity with LD50 values ranging >2 logs (0.24-80.4 μM) in five established leukemia cell lines (Jurkat, K562, HL60, U937 and PTA-3920) and forty-one primary leukemic cells (including Acute myeloid leukemia (AML), Acute lymphoblastic leukemia (ALL), Chronic Myeloid Leukemia (CML), and Chronic lymphoblastic leukemia (CLL)). Through a semi-quantitative approach by analyzing western blotting bands via imaging software, it revealed that the index value phosphoralated Bcl-2 (pBcl-2) to Bcl-2 and Mcl-1 provided a highly significant linear correlation with S1 sensitivity (r = 0.69, P < 0.001 in primary cells, and r = 0.76, P = 0.05 in cell lines). In contrast, antiapoptotic Bcl-2-like protein levels, used individually or in combination could not predict S1 sensitivity. Phosphorylated Bcl-2 antagonized S1 by sequestering the Bak and Bim proteins that were released from Mcl-1 in leukemia cell lines. Furthermore, pBcl-2/Bak, pBcl-2/Bax, and pBcl-2/Bim complexes cannot be disrupted by S1. The shift of proapoptotic proteins from being complexed with Mcl-1 to being complexed with pBcl-2 is revealed for the first time, and it could be the mechanism underlying the index value described herein. This study defines the antiapoptotic role of pBcl-2 linked with the protein-protein interaction network within Bcl-2 family.

Citation Format: Yubo Liu, Zhichao Zhang. An anti-apoptotic Bcl-2 family protein index predicts the response of leukemic cells to the pan-Bcl-2 inhibitor S1. [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 1710. doi:10.1158/1538-7445.AM2013-1710

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 1714: A novel NPM-BAX pathway regulates death evasion and drug sensitivity in human hepatoma cells.
Authors: Hsieh Sen-Yung
Published: August 2014

Death evasion is crucial for both carcinogenesis and resistance to anticancer therapies. Recently we identified nucleophosmin (NPM) as a key factor counteracting death stimuli in human hepatocellular carcinoma (HCC) cells. Here we report the identification of a novel NPM-BCL2-associated X protein (BAX) pathway orchestrating death evasion in human HCC cells. Silencing of NPM expression significantly sensitized HCC cells, particularly those bearing inactivated p53 (Huh7, Hep3B, and Mahlavu), to UV irradiation, mitomycin C, doxorubicin, cisplatin, sorafenib, and lapatinib. This sensitizing effect was not further changed as p53 expression had been simultaneously silenced. Following cell stress, NPM and BAX were induced and exported out of nucleoli and nucleus, respectively. BAX was translocated to cytoplasm in cells with relatively high NPM level, or accumulated in the mitochondria in cells with relatively low NPM level and undergoing apoptosis. Subcellular fractionation revealed that silencing of NPM expression greatly enhanced mitochondrial translocation and oligomerization of BAX in Huh7 and Mahlavu cells. In situ proximity ligation assays and reciprocal co-immunoprecipitation evidenced direct interaction between NPM and BAX in the cytoplasm. Silencing of BAX expression abolished the sensitization effect exerted by silencing of NPM in HCC cells. Clinically, upregulation of NPM was significantly associated with advanced tumor stage and poor prognosis. Conclusion: Via directly blockading BAX mitochondrial translocation and activation, NPM helps human HCC cells evade death induction independently of p53-mediated cell death. Silencing of NPM significantly sensitized HCC cells to anticancer therapies. NPM is a potential co-target in combination with other therapies for HCC particularly harboring inactivated p53. Our findings are of clinical significance, since NPM upregualtion and p53 mutations are usually found in advanced human cancers, including HCC.

Citation Format: Sen-Yung Hsieh. A novel NPM-BAX pathway regulates death evasion and drug sensitivity in human hepatoma 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 1714. doi:10.1158/1538-7445.AM2013-1714
Abstract 1720: Maritoclax, a selective Mcl-1 inhibitor, sensitizes melanoma cells to the Bad BH3-mimetic ABT-737.
Authors: Pandey Manoj K. , Gowda Krishne , Doi Kenichiro , Sharma Arun , Wang Hong-Gang , Amin Shantu
Published: August 2014

Melanoma is the most aggressive form of skin cancer. Despite the development of the therapies it remains one of the most invasive and highly drug resistance cancers. The dysregulation of apoptosis are thought to be one barrier to effective systemic treatment of melanoma. The overexpression of anti-apoptotic Bcl-2 family proteins has been associated with inferior survival, poor prognosis and chemoresistance of several cancers including malignant melanoma. ABT-737, a small-molecule inhibitor of Bcl-2, Bcl-xL and Bcl-w, has demonstrated efficacy in several forms of hematological and non-hematological cancers. However, the efficacy of ABT-737 is diminished in cancer cells with high levels of Mcl-1, another anti-apoptotic member of the Bcl-2 family. The overexpression of Mcl-1 is frequently observed in melanoma and therefore, specifically targeting of Mcl-1 may overcome the resistance of ABT-737. In this study, we investigated the effects of Maritoclax, a novel Mcl-1-selective inhibitor, alone and in combination with ABT-737, on the survival of human melanoma cells. The treatments of Maritoclax reduce the cell viabilities of melanoma cells with an IC50 of between 2.2-5.0μM. Further, treatment of melanoma cells with 2.5 μM Maritoclax for 24 h showed significant decrease in Mcl-1 expression. We found that Maritoclax is able to induce apoptosis in melanoma cells in a caspase-dependent manner. Moreover, Maritoclax induces Mcl-1 degradation via the proteasome system, which is associated with its pro-apoptotic activity. Importantly, Maritoclax markedly enhances the efficacy of ABT-737 against melanoma cells. As a single agent, ABT-737 was not efficient in killing UACC 903 cells with IC50 values of >30 μm. However, in combination with a sub-optimal dose of Maritoclax (2.5μM), the efficacy of ABT-737 was enhanced (IC50 1.3 μm). Similarly, Maritoclax potentiated apoptotic effects of ABT-737 from 20%-71% and 18%-62% when measured by Live and Dead and annexin-V assay, respectively. Taken together, these results suggest that targeting of Mcl-1 by Maritoclax may represent a new therapeutic strategy to sensitize melanoma cells to ABT-737-induced apoptosis.

Citation Format: Manoj K. Pandey, Krishne Gowda, Kenichiro Doi, Arun Sharma, Hong-Gang Wang, Shantu Amin. Maritoclax, a selective Mcl-1 inhibitor, sensitizes melanoma cells to the Bad BH3-mimetic ABT-737. [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 1720. doi:10.1158/1538-7445.AM2013-1720
Abstract 1728: Bcl-XL overexpression prevents B Cell receptor driven autophagy in IgM+ lymphoma.
Authors: Carey Gregory B. , Roy Sanjit K. , Sesay Alphius
Published: August 2014

Our previous studies showed that B cell receptor (BCR) induced apoptosis in lymphoma was preceded by suppression of PI-3K [Carey GB, Scott DW (2001) J. Immunol, 166: 1618-1626]. PI-3K stimulates mTOR, a suppressor of autophagy and mitophagy. In addition, Bcl-XL is known to inhibit BCR-driven apoptosis in lymphoma, although the underlying mechanism is not clear. Therefore the present study examined autophagy in BCR induced apoptosis in lymphoma cells expressing high or low levels of Bcl-XL. WEHI-231 and WEHI-231 cells expressing Bcl-XL (WEHI-231/Bcl-XL) were treated with anti-IgM to cross-link the BCR and to determine the cellular processes leading to apoptosis. The results confirmed dramatic mitochondrial dysfunction followed by apoptosis in parental WEHI-231 compared to WEHI-231/BclXL. BCR-crosslinking resulted in a massive and rapid induction of the autophagic response in parental WEHI-231 compared to those expressing Bcl-XL. Furthermore, treatment with STF-62247, a small molecule inducer of autophagy, resulted in apoptosis in WEHI-231 but not WEHI-231/BclXL cells. These data suggest that BclXL prevents BCR-driven apoptosis in lymphoma via blocking autophagic and mitophagic responses. Hence, combinations of small molecule autophagy inducers and Bcl-XL antagonists may prove to be effective in addressing lymphoma. Supported by NCI Grant UH2CA158689 to G.B.C.

Citation Format: Gregory B. Carey, Sanjit K. Roy, Alphius Sesay. Bcl-XL overexpression prevents B Cell receptor driven autophagy in IgM+ lymphoma. [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 1728. doi:10.1158/1538-7445.AM2013-1728