Yu, Chunrong

Abstract Background: Autophagy and apoptosis regulate cancer cell viability in response to cytotoxic stress; however, the functional relationship between these processes is unclear. During autophagy, the multifunctional p62/sequestosome 1 protein shuttles ubiquitinated proteins to the lysosome. Previously, we found that ectopic p62 can promote chemotherapy-induced apoptosis. Since Bcl-2 can regulate both autophagy and apoptosis, we determined whether ABT-263-induced apoptosis is regulated by p62. Methods: Human colorectal cancer cell lines (HCT116, DLD1) were incubated with ABT-263 (0-2 μM) and caspase cleavage (immunoblotting), caspase-8 activation (luminescence), apoptosis (annexin V labeling), and autophagy (localization of GFP-LC3 by confocal microscopy) were analyzed. Caspase-8 was suppressed by siRNA or using caspase-8-deficient Jurkat cells. P62 mutants were generated by site-directed mutagenesis. Caspase-8 self aggregation and colocalization with p62 were studied in cells with lentiviral mCherry-p62, transfected with a non-cleavable caspase-8 fused with fluorescent Venus proteins (BiFC). Results: We demonstrate that ABT-263 induces autophagy and a caspase-8-mediated apoptosis, shown using caspase-8 siRNA or caspase-8-deficient cells. Regulation of this process by p62 was demonstrated by the ability of ectopic p62 expression to enhance apoptosis and caspase-8 activation that were disrupted by mutations at p62 functional domains. In contrast, p62 shRNA was shown to attenuate ABT-263-induced apoptosis. ABT-263 treatment enhanced binding between caspase-8 and p62, and triggered caspase-8 self aggregation that colocalized with p62, shown by Biomolecular Fluorescence Complementation (BiFC) assay using confocal microscopy. Consistent with ectopic p62, accumulation of p62 by the lysosomal autophagy inhibitors, chloroquine or bafilomycin A1, enhanced ABT-263-mediated caspase cleavage. Conclusion: p62 can regulate caspase-8 self aggregation/activation by ABT-263 to enable crosstalk between autophagy and apoptosis. Citation Format: Shengbing Huang, Jae Myung Park, Chunrong Yu, Frank A. Sinicrope. p62/sequestosome 1 regulates caspase-8 activation by the Bcl-2 antagonist ABT-263 to enable crosstalk between autophagy and apoptosis. [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 1733. doi:10.1158/1538-7445.AM2013-1733

Abstract Despite the high incidence and mortality of prostate cancer, the etiology of this disease is not fully understood. In this study, we develop functional evidence for CBP and PTEN interaction in prostate cancer based on findings of their correlate expression in the human disease. Cbppc−/−;Ptenpc+/− mice exhibited higher cell proliferation in the prostate and an early onset of high-grade prostatic intraepithelial neoplasia. Levels of EZH2 methyltransferase were increased along with its Thr350 phosphorylation in both mouse Cbp−/−; Pten+/− and human prostate cancer cells. CBP loss and PTEN deficiency cooperated to trigger a switch from K27-acetylated histone H3 to K27-trimethylated bulk histones in a manner associated with decreased expression of the growth inhibitory EZH2 target genes DAB2IP, p27KIP1, and p21CIP1. Conversely, treatment with the histone deacetylase inhibitor panobinostat reversed this switch, in a manner associated with tumor suppression in Cbppc−/−;Ptenpc+/− mice. Our findings show how CBP and PTEN interact to mediate tumor suppression in the prostate, establishing a central role for histone modification in the etiology of prostate cancer and providing a rationale for clinical evaluation of epigenetic-targeted therapy in patients with prostate cancer. Cancer Res; 74(7); 2050–61. ©2014 AACR.

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 The mammalian target of rapamycin (mTOR) consists of two distinct complexes, mTORC1 and mTORC2. mTOR plays a pivotal role in Hepatocellular Carcinoma (HCC). Previous studies indicated that inhibition of mTORC1 only moderately enhanced histone deacetylase inhibitors (HDACis)-mediated anti-tumor activity. This limited effect may be attributable to the increase of AKT activation through S6K1-IRS1-PI3K negative feedback loop triggered by selective mTORC1 inhibition. While as AKT is the downstream effector of mTORC2, we hypothesize that dual targeting of mTORC1/C2 would be more efficient in suppressing AKT and in enhancing anti-tumor activity of HDACi. In this study, the interactions between mTOR kinase inhibitors (mTORKis) and HDACis were examined in vitro using established HCC cell lines and in vivo using patient-derived HCC tumor xenografts on SCID mice. Our results demonstrate that inhibition of both mTORC1/2 not only effectively blocked mTORC1 signaling, but also abrogated negative-feedback activation of AKT caused by mTORC1 inhibition. Furthermore, inactivation of mTORC1/C2 by mTORKis substantially enhanced HDACi-induced anti-tumor activity. Either dysfunction of mTORC2 by knockdown of Rictor (a component of mTORC2), or diminution of AKT1 significantly lowered the threshold of HDACi-induced apoptosis. Lastly, in vivo study indicated that the combination of AZD8055 and Vorinostat yielded much greater tumor-growth inhibition compared to either agent alone in primary HCC xenografts without obvious adverse effects. Conclusion: Dual mTORC1/2 inhibition is more efficient than selective mTORC1 inhibition in enhancing anti-tumor activity of HDACi due to abrogation of negative-feedback activation of AKT induced by selective mTORC1 inhibition. Our findings suggest that combining regimen of mTORKi and HDACi may be an effective therapeutic strategy for HCC. 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 5363. doi:10.1158/1538-7445.AM2011-5363

Abstract We have reported previously the activity of the insulin-like growth factor-I (IGF-IR)/insulin receptor (InsR) inhibitor, BMS-554417, in breast and ovarian cancer cell lines. Further studies indicated treatment of OV202 ovarian cancer cells with BMS-554417 increased phosphorylation of HER-2. In addition, treatment with the pan-HER inhibitor, BMS-599626, resulted in increased phosphorylation of IGF-IR, suggesting a reciprocal cross-talk mechanism. In a panel of five ovarian cancer cell lines, simultaneous treatment with the IGF-IR/InsR inhibitor, BMS-536924 and BMS-599626, resulted in a synergistic antiproliferative effect. Furthermore, combination therapy decreased AKT and extracellular signal-regulated kinase activation and increased biochemical and nuclear morphologic changes consistent with apoptosis compared with either agent alone. In response to treatment with BMS-536924, increased expression and activation of various members of the HER family of receptors were seen in all five ovarian cancer cell lines, suggesting that inhibition of IGF-IR/InsR results in adaptive up-regulation of the HER pathway. Using MCF-7 breast cancer cell variants that overexpressed HER-1 or HER-2, we then tested the hypothesis that HER receptor expression is sufficient to confer resistance to IGF-IR-targeted therapy. In the presence of activating ligands epidermal growth factor or heregulin, respectively, MCF-7 cells expressing HER-1 or HER-2 were resistant to BMS-536924 as determined in a proliferation and clonogenic assay. These data suggested that simultaneous treatment with inhibitors of the IGF-I and HER family of receptors may be an effective strategy for clinical investigations of IGF-IR inhibitors in breast and ovarian cancer and that targeting HER-1 and HER-2 may overcome clinical resistance to IGF-IR inhibitors. [Mol Cancer Ther 2008;7(9):2589–98]

Abstract AZD6244 (ARRY 142886) is a potent and selective mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor currently in early clinical trials. We examined the activity of AZD6244 in a panel of non–small cell lung cancer and a panel of cell lines representing many cancer types using in vitro growth assays. AZD6244 induced G0-G1 cell cycle arrest in sensitive cell lines that primarily included cells containing the BRAF V600E mutation. In these cells, G0-G1 arrest is accompanied by the up-regulation of the cell cycle inhibitors p21WAF1 and p27Kip1 and down-regulation of cyclin D1. In the majority of cell lines tested, including those with K-ras or non-V600E BRAF mutations, AZD6244 induced the accumulation of phospho-MEK, an effect not observed in the most sensitive BRAF V600E-containing cells. Accumulation of phospho-MEK in non–V600E-containing cell lines is due to abrogation of negative feedback pathways. BRAF V600E disrupts negative feedback signaling, which results in enhanced baseline phospho-MEK expression. Exogenous expression of BRAF V600E disrupts feedback inhibition but does not sensitize cells to AZD6244. Specific suppression of endogenous BRAF V600E does not confer resistance to AZD6244 but enhances sensitivity to AZD6244. Thus, our findings show that BRAF V600E marks cells with an in vitro requirement for MAPK signaling to support proliferation. These cells are exquisitely sensitive to AZD6244 (IC50, <100 nmol/L), have high baseline levels of phospho-MEK, and lack feedback inhibition between ERK and Raf. These data suggest an approach to identifying cells that may be sensitive to AZD6244 and other MEK inhibitors. [Cancer Res 2008;68(15):6145–53]

Abstract Purpose: To evaluate the effects of combining the multiple receptor tyrosine kinase inhibitor AEE788 and histone deacetylase (HDAC) inhibitors on cytotoxicity in a broad spectrum of cancer cell lines, including cisplatin-resistant ovarian adenocarcinoma cells. Experimental Design: Multiple cancer cell lines were treated in vitro using AEE788 and HDAC inhibitors (LBH589, LAQ824, and trichostatin A), either alone or in combination. Effects on cytotoxicity were determined by growth and morphologic assays. Effects of the combination on cell signaling pathways were determined by Western blotting, and the results were confirmed using pathway-specific inhibitors and transfection of constitutively active proteins. Results: Cell treatment with AEE788 and HDAC inhibitors (LBH589, LAQ824, and trichostatin A) in combination resulted in synergistic induction of apoptosis in non–small cell lung cancer (MV522, A549), ovarian cancer (SKOV-3), and leukemia (K562, Jurkat, and ML-1) cells and in OV202hp cisplatin-resistant human ovarian cancer cells. AEE788 alone or in combination with LBH589 inactivated mitogen-activated protein kinase (MAPK) and Akt cascades. Inhibition of either MAPK and/or Akt enhanced LBH589-induced apoptosis. In contrast, constitutively active MAPK or Akt attenuated LBH589 or LBH589 + AEE788–induced apoptosis. Increased apoptosis was correlated with enhanced reactive oxygen species (ROS) generation. The free radical scavenger N-acetyl-l-cysteine not only substantially suppressed the ROS accumulation but also blocked the induction of apoptosis mediated by cotreatment with AEE788 and LBH589. Conclusion: Collectively, these results show that MAPK and Akt inactivation along with ROS generation contribute to the synergistic cytotoxicity of the combination of AEE788 and HDAC inhibitors in a variety of human cancer cell types. This combination regimen warrants further preclinical and possible clinical study for a broad spectrum of cancers.

Abstract This study was undertaken to characterize preclinical cytotoxic interactions for human malignancies between the multikinase inhibitor sorafenib (BAY 43-9006) and proteasome inhibitors bortezomib or MG132. Multiple tumor cell lines of varying histiotypes, including A549 (lung adenocarcinoma), 786-O (renal cell carcinoma), HeLa (cervical carcinoma), MDA-MB-231 (breast), K562 (chronic myelogenous leukemia), Jurkat (acute T-cell leukemia), MEC-2 (B-chronic lymphocytic leukemia), and U251 and D37 (glioma), as well as cells derived from primary human glioma tumors that are likely a more clinically relevant model were treated with sorafenib or bortezomib alone or in combination. Sorafenib and bortezomib synergistically induced a marked increase in mitochondrial injury and apoptosis, reflected by cytochrome c release, caspase-3 cleavage, and poly(ADP-ribose) polymerase degradation in a broad range of solid tumor and leukemia cell lines. These findings were accompanied by several biochemical changes, including decreased phosphorylation of vascular endothelial growth factor receptor-2, platelet-derived growth factor receptor-β, and Akt and increased phosphorylation of stress-related c-Jun NH2-terminal kinase (JNK). Inhibition of Akt was required for synergism, as a constitutively active Akt protected cells against apoptosis induced by the combination. Alternatively, the JNK inhibitor SP600125 could also protect cells from apoptosis induced by the combination, indicating that both inhibition of Akt and activation of JNK were required for the synergism. These findings show that sorafenib interacts synergistically with bortezomib to induce apoptosis in a broad spectrum of neoplastic cell lines and show an important role for the Akt and JNK pathways in mediating synergism. Further clinical development of this combination seems warranted. [Mol Cancer Ther 2006;5(9):2378–87]