Huang, Shengbing

Abstract BACKGROUND. The mammalian target of rapamycin (mTOR) kinase forms two multiprotein complexes, mTORC1 and mTORC2, which regulate cell growth, cell survival, and autophagy. We determined whether mTOR inhibition can induce cytoprotective autophagy resulting in anti-cancer drug resistance, and whether inhibition of autophagy using the lysomotrophic agent, chloroquine, can restore chemosensitivity. EXPERIMENTAL METHODS. Inhibition of mTOR was accomplished in human colon cancer cells (HT-29, DLD1) using lentiviral shRNA or by AZD8055, a TORC1 and TORC2 kinase inhibitor. These cells were then incubated with 5-FU (0-100 µM) or CPT-11 (0-20 µM) alone and in combination with chloroquine. LC3I/II expression, phospho-histone H2A.X (a DNA damage marker), and caspase cleavage were analyzed by Western blotting using highly specific antibodies, and clonogenic survival assays were also performed. RESULTS. AZD8055 was shown to attenuate caspase-3 and -9 cleavage and to suppress the expression of phospho-histone H2A.X induced by 5-FU or CPT-11 in colon cancer cells. AZD8055 was also found to antagonize the ability of 5-FU to inhibit clonogenic survival. Similarly, shRNA knockdown of mTOR attenuated 5-FU-induced caspase cleavage and phospho-histone H2A.X expression. Inhibition of mTOR by AZD8055 or shRNA enhanced the conversion of the autophagosome-associated protein light chain 3 (LC3) from a cytosolic (LC3I) to a membrane-bound (LC3II) form, consistent with induction of autophagy. Inhibition of autophagy by chloroquine was shown to attenuate the ability of AZD8055 to suppress 5-FU-induced caspase cleavage. Consistent with this result, chloroquine sensitized DLD1 cells to AZD8055 and 5-FU in a clonogenic survival assay. CONCLUSION. Inhibition of mTOR can protect colon cancer cells from DNA damage induced by cytotoxic chemotherapy. This cytoprotection was due, in part, to induction of pro-survival autophagy since chloroquine was able to partially restore chemosensitivity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5485. doi:10.1158/1538-7445.AM2011-5485

Abstract Purpose: Celecoxib has been shown to induce cell death in conjunction with its chemopreventive effects. However, the mechanisms by which celecoxib induces cell death are incompletely understood. We determined whether celecoxib can induce autophagy, a process of cell destruction whereby cytoplasmic proteins and organelles are sequestered in vacuoles and delivered to lysosomes for degradation and recycling. Both apoptosis and autophagy have been shown to be negatively regulated by pro-survival Bcl-2 proteins, and the small molecule Bcl-2/Bcl-xL antagonist (ABT-737), can enhance both processes. Since autophagy can be pro-survival or pro-death depending upon the cellular context, we determined whether autophagy inhibition can drive colon cancer cells into apoptosis. Methods: HCT116 (wild-type and Bax−/−), HT29, SW480 colorectal cancer cell lines were treated with celecoxib (0–120 µM) and/or ABT-737 (Abbott; 0–5 µM) in the presence or absence of a pan caspase inhibitor, z-VAD-fmk. Cell viability was determined using the MTS assay and apoptosis was analyzed by Annexin V FITC labeling. Immunoblotting was used to analyze caspase cleavage and LC3 conversion. GFP-LC3B cellular localization was determined by confocol microscopy. Autophagic flux was determined by LC3 immunoblotting in the presence of the lysosome inhibitor, bafilomycin A1. Knockdown of Bcl-xL, Vps34 or Atg8/LC3B were achieved by lentiviral shRNA or siRNA. Results: Celecoxib was shown to induce apoptosis that was attenuated by ectopic Bcl-2 or Bax knockout. ABT-737 synergistically enhanced celecoxib-induced apoptosis as shown by caspase cleavage and Annexin V labeling. Celecoxib also induced autophagy, as shown by conversion of the autophagosome-associated protein light chain 3 (LC3) from a cytosolic (LC3I) to a membrane-bound (LC3II) form as shown by immunoblotting, and a punctate fluorescence pattern of ectopic GFP-LC3 protein. Celecoxib-induced conversion of LC3 that was due to autophagy induction, as shown using the lysosome inhibitor, Bafilomycin A1 that produced an accumulation of LC3II. Furthermore, ABT-737 enhanced LC3 conversion by celecoxib. Autophagy inhibition was achieved using 3-methyladenine (3-MA), wortmannin or knockdown of Vps34 or Atg8/LC3B using siRNA. Inhibition of autophagy enhanced apoptosis as shown by caspase cleavage and Bid truncation in cells treated with celecoxib plus ABT-737. Furthermore, the addition of 3-MA to celecoxib plus ABT-737 produced a 5-fold increase in apoptosis that was associated with caspase cleavage and cytochrome c release. Conclusions: Celecoxib induces both apoptosis and autophagy that can both be potentiated by ABT-737. Inhibition of autophagy was shown to potently drive cells into apoptosis, suggesting a novel therapeutic strategy against colon cancer cells. Citation Information: Cancer Prev Res 2010;3(1 Suppl):B46.

Abstract Autophagy is a homeostatic, catabolic degradation process whereby cellular proteins and organelles are engulfed by autophagosomes, digested in lysosomes, and recycled to sustain cellular metabolism. Autophagy has dual roles in cancer, acting as both a tumor suppressor by preventing the accumulation of damaged proteins and organelles and as a mechanism of cell survival that can promote the growth of established tumors. Tumor cells activate autophagy in response to cellular stress and/or increased metabolic demands related to rapid cell proliferation. Autophagy-related stress tolerance can enable cell survival by maintaining energy production that can lead to tumor growth and therapeutic resistance. As shown in preclinical models, inhibition of autophagy restored chemosensitivity and enhanced tumor cell death. These results established autophagy as a therapeutic target and led to multiple early phase clinical trials in humans to evaluate autophagy inhibition using hydroxychloroquine in combination with chemotherapy or targeted agents. Targeting autophagy in cancer will provide new opportunities for drug development, because more potent and specific inhibitors of autophagy are needed. The role of autophagy and its regulation in cancer cells continues to emerge, and studies aim to define optimal strategies to modulate autophagy for therapeutic advantage. Mol Cancer Ther; 10(9); 1533–41. ©2011 AACR.

Abstract BACKGROUND. Autophagy is a mechanism of cellular destruction whereby cytoplasmic proteins and organelles are sequestered in vacuoles and delivered to lysosomes for degradation and recycling. Autophagy is not always pro-death but can also be pro-survival under conditions of cellular stress. Autophagy and apoptosis can be regulated by Bcl-2 family proteins. We determined whether the anti-neoplastic agent celecoxib can induce autophagy and/or apoptosis, and if a small molecule Bcl-2/Bcl-xL antagonist, e.g., ABT-737, can enhance either/both processes. Furthermore, we determined whether autophagy inhibition can drive colon cancer cells into apoptosis. EXPERIMENTAL METHODS. Human colon cancer cell lines (HCT116, SW480 ± Bcl-2, HT-29 ± Bcl-xL lentiviral shRNA) were incubated with celecoxib (0-120 µM) alone and in combination with ABT-737 (0- 5 µM). Autophagy was studied by LC3I/II expression (Western blotting) and using a GFP-LC3B chimeric plasmid. Additionally, autophagy was imaged by monodansylcadaverine (MDC) staining of autophagic vacuoles and acridine orange staining of acidic vesicles by fluorescent confocal microscopy. Apoptosis was analyzed by caspase cleavage (Western blotting) by annexin V labeling and FACS. RESULTS. Celecoxib triggered conversion of the autophagosome-associated protein light chain 3 (LC3) from a cytosolic (LC3I) to a membrane-bound (LC3II) form as shown by Western blotting, and a change in the localization of the fluorescence pattern of an ectopic GFP-LC3B protein. Celecoxib-induced conversion of LC3 was due to autophagy induction, as shown using the lysosome inhibitor, Bafilomycin A1 that produced an accumulation of LC3II. Celecoxib-induced autophagy and apoptosis were attenuated by ectopic Bcl-2, but were augmented by Bcl-xL knockdown. ABT-737 was shown to enhance celecoxib-induced autophagy as shown by LC3 conversion, acridine orange staining of acidic vesicles, and MDC staining of autophagic vacuoles. Celecoxib also induced apoptosis, as shown by caspase cleavage and annexin V labeling, that was synergistically enhanced by ABT-737. Given that autophagy can be pro-survival under conditions of cellular stress, we determined whether inhibition of autophagy can drive cells into apoptosis. Use of the autophagy inhibitor 3-methyladenine (3-MA) was shown to block LC3 conversion and to enhance celecoxib-induced apoptosis that was further augmented by ABT-737. Similarly, Vps34 or Atg8/LC3 siRNA were shown to enhance caspase cleavage in cells treated with celecoxib plus ABT-737. CONCLUSION. Celecoxib induces autophagy and apoptosis that can both be potentiated by a Bcl-2/ Bcl-xL antagonist. Furthermore, inhibition of autophagy was shown to drive cells into apoptosis, suggesting a novel therapeutic strategy against colon cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4858.

Abstract Signal transducers and activators of transcription 3 (STAT3) is constitutively active in human pancreatic cancer cells and can promote cell growth and apoptosis resistance that contribute to tumorigenesis. We determined if sorafenib, a multikinase inhibitor, can induce apoptosis by targeting STAT3 signaling to enhance apoptosis induction by tumor necrosis factor–related apoptosis-inducing ligand (TRAIL). Human pancreatic cancer cell lines (PANC-1 and BxPC-3) were preincubated with sorafenib (Nexavar) alone or followed by TRAIL. Apoptosis was determined by Annexin V labeling, caspase cleavage, and Bax/Bak activation. Protein expression was analyzed by immunoblotting. Knockdown of STAT3, Mcl-1, and Bim were achieved by lentiviral small hairpin RNA. Adenoviral dominant-negative or retroviral constitutively active (CA) STAT3 were also used. Sorafenib inhibited constitutive STAT3 phosphorylation (Tyr705) and suppressed Mcl-1 and Bcl-xL proteins in a dose- and time-dependent manner. CA-STAT3 overexpression was shown to attenuate caspase-3 cleavage and suppression of Mcl-1 by sorafenib. STAT3 knockdown or a DN STAT3 was shown to downregulate Mcl-1 and Bcl-xL and to sensitize cells to TRAIL-mediated apoptosis. Treatment with sorafenib enhanced TRAIL-induced Annexin V staining and release of mitochondrial cytochrome c and AIF. Because the BH3-only Bim protein is a potent inducer of mitochondrial apoptosis, Bim knockdown was shown to attenuate caspase-3, caspase-9 cleavage, and Bax/Bak activation by sorafenib plus TRAIL. The suppression of STAT3 by genetic means or using sorafenib was shown to downregulate Mcl-1 and Bcl-xL and to sensitize cells to TRAIL-mediated apoptosis. These data indicate that targeting STAT3 may enhance treatment efficacy against pancreatic cancer. Mol Cancer Ther; 9(3); 742–50

Abstract Purpose: Prosurvival Bcl-2 proteins inhibit the mitochondrial and death receptor-mediated apoptotic pathways. Obatoclax is a small-molecule antagonist of the BH3-binding groove of Bcl-2 proteins that may enhance tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) sensitivity and efficacy. Experimental Design: Human pancreatic cancer cell lines were incubated with obatoclax and/or TRAIL and cell viability, Annexin V labeling, caspase cleavage, and cytochrome c release were measured. In drug-treated cell lines, protein-protein interactions were studied by immunoprecipitation. Bax/Bak activation was analyzed using conformation-specific antibodies. Lentiviral short hairpin RNA was used to knockdown Bim, Bid, and apoptosis-inducing factor (AIF) expression. Results: Obatoclax reduced the viability of PANC-1 and BxPC-3 cell lines and synergistically enhanced TRAIL-mediated cytotoxicity. Obatoclax enhanced TRAIL-mediated apoptosis, as shown by Annexin V labeling, which was accompanied by caspase activation (caspase-8, -9, and -3) and cleavage of Bid. Obatoclax potentiated TRAIL-mediated Bax/Bak activation and the release of mitochondrial cytochrome c, Smac, and AIF. Mechanisms underlying the apoptotic effect of obatoclax include displacement of Bak from its sequestration by Bcl-xL or Mcl-1 and release of Bim from Bcl-2 or Mcl-1. Bid knockdown by short hairpin RNA attenuated caspase cleavage and cytotoxicity of obatoclax plus TRAIL. Bim knockdown failed to inhibit the cytotoxic effect of obatoclax alone or combined with TRAIL yet attenuated TRAIL-mediated cytotoxicity. AIF knockdown attenuated cytotoxicity of the drug combination. Conclusions: Obatoclax potentiates TRAIL-mediated apoptosis by unsequestering Bak and Bim from Bcl-2/Bcl-xL or Mcl-1 proteins. This drug combination enhances Bid-mediated cross-talk between the mitochondrial and death receptor-mediated apoptotic pathways and may represent a novel therapeutic strategy against pancreatic cancer.

Abstract Purpose: The intrinsic drug resistance of colorectal cancers is related in part to overexpression of prosurvival Bcl-2 family proteins. We determined the effects of ABT-737, a small-molecule inhibitor of Bcl-2/Bcl-xL but not Mcl-1, on apoptosis induction alone and in combination with CPT-11 and explored mechanisms underlying their cooperativity. Experimental Design: Human colorectal carcinoma cell lines (HCT116 wild-type and Bax-/-, HT-29, and RKO) were incubated with ABT-737 alone and combined with CPT-11 or bortezomib, and cell viability, caspase cleavage, and Annexin V labeling were measured. In drug-treated cell lines, protein-protein interactions were analyzed by immunoprecipitation. Lentiviral short hairpin RNA was used to knockdown Noxa expression. Results: ABT-737 induced apoptosis in a dose-dependent manner and its coadministration with the topoisomerase I inhibitor, CPT-11, resulted in a synergistic cytotoxic effect. Apoptosis induction by the drug combination was associated with enhanced caspase-8, caspase-9, and caspase-3 activation and poly(ADP-ribose) polymerase cleavage that were completely abrogated in Bax knockout cells. ABT-737 unsequestered the BH3-only protein Bim from its complex with Bcl-xL or Bcl-2 and disrupted the interaction of Bcl-xL with Bak. CPT-11 treatment up-regulated Noxa expression, as did bortezomib, and enhanced Noxa/Mcl-1 complexes. CPT-11 also disrupted the Mcl-1/Bak interaction. Knockdown of Noxa using short hairpin RNA lentiviral constructs was shown to significantly attenuate the cytotoxic effect of CPT-11 or bortezomib combined with ABT-737 and inhibited caspase-3 cleavage. Conclusions: Induction of Noxa by CPT-11 or bortezomib can sensitize colorectal cancer cells expressing Mcl-1 to ABT-737. Up-regulation of Noxa may therefore represent an important strategy to enhance the therapeutic efficacy of ABT-737 against colorectal cancer and other solid tumors.

Abstract Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) has been shown to induce mitochondrial apoptotic signaling that can be negatively regulated by prosurvival Bcl-2 proteins. ABT-737 is a small-molecule BH3 mimetic that binds to and antagonizes Bcl-2/Bcl-xL but not Mcl-1. We show that ABT-737 can synergistically enhance TRAIL-mediated cytotoxicity in human pancreatic cancer cell lines. ABT-737 was shown to enhance TRAIL-induced apoptosis as shown by DNA fragmentation, activation of caspase-8 and Bid, and cleavage of caspase-3 and poly(ADP-ribose) polymerase. A Bax conformational change induced by TRAIL was enhanced by ABT-737. ABT-737 disrupted the interaction of Bak with Bcl-xL in both cell lines. Furthermore, ABT-737 untethered the proapoptotic BH3-only protein Bim from its sequestration by Bcl-xL or Bcl-2. Bim small hairpin RNA (shRNA) was shown to attenuate caspase-3 cleavage and to reduce the cytotoxic effects of TRAIL plus ABT-737 compared with shRNA control cells. Finally, Mcl-1 shRNA potentiated caspase-3 cleavage by ABT-737 and enhanced its cytotoxic effects. Taken together, ABT-737 augments TRAIL-induced cell killing by unsequestering Bim and Bak and enhancing a Bax conformational change induced by TRAIL. These findings suggest a novel strategy to enhance cross-talk between the extrinsic and intrinsic apoptotic pathways to improve therapeutic efficacy against pancreatic cancer. [Cancer Res 2008;68(8):2944–51]

Epigenetic inheritance of heterochromatin structure is an important cellular process whose mechanism remains elusive. In this article, we describe the identification of nine enhancers of the silencing defect of a Saccharomyces cerevisiae -PCNA mutant by screening a library of ≈4,700 viable yeast deletion mutants. Of the nine mutants identified, six ( hir1 , hir3 , sas2 , sas4 , sas5 , and sir1 ) were previously known to reduce silencing synergistically with a mutation in Cac1p, the large subunit of chromatin assembly factor 1 (CAF-1). The predicted gene products that are affected in three other mutants ( nam7 , msh2 , and rtt106 ) have not been implicated previously in silencing. Characterization of the rtt106 Δ allele revealed that it synergistically reduced heterochromatin silencing when combined with a mutation in Cac1p but not with a mutation in Asf1p (a histone H3 and H4 chaperone). Moreover, Rtt106p interacted with histones H3 and H4 both in vitro and in vivo , and it displayed a nucleosome assembly activity in vitro . Furthermore, Rtt106p interacts with CAF-1 physically through Cac1p. These biochemical and genetic data indicate that Rtt106p is a previously uncharacterized histone chaperone connecting S phase to epigenetic inheritance.