Huang, Shengbing

Abstract Oncogenic BRAFV600E mutations activate MAPK signaling and are associated with treatment resistance and poor prognosis in patients with colorectal cancer. In BRAFV600E-mutant colorectal cancers, treatment failure may be related to BRAFV600E-mediated apoptosis resistance that occurs by an as yet undefined mechanism. We found that BRAFV600E can upregulate anti-apoptotic MCL-1 in a gene dose-dependent manner using colorectal cancer cell lines isogenic for BRAF. BRAFV600E-induced MCL-1 upregulation was confirmed by ectopic BRAFV600E expression that activated MEK/ERK signaling to phosphorylate (MCL-1Thr163) and stabilize MCL-1. Upregulation of MCL-1 was mediated by MEK/ERK shown by the ability of ERK siRNA to suppress MCL-1. Stabilization of MCL-1 by phosphorylation was shown by a phosphorylation-mimicking mutant and an unphosphorylated MCL-1 mutant that decreased or increased MCL-1 protein turnover, respectively. MEK/ERK inhibition by cobimetinib suppressed MCL-1 expression/phosphorylation and induced proapoptotic BIM to a greater extent than did vemurafenib in BRAFV600E cell lines. MCL-1 knockdown versus control shRNA significantly enhanced cobimetinib-induced apoptosis in vitro and in HT29 colon cancer xenografts. The small-molecule MCL-1 inhibitor, A-1210477, also enhanced cobimetinib-induced apoptosis in vitro that was due to disruption of the interaction of MCL-1 with proapoptotic BAK and BIM. Knockdown of BIM attenuated BAX, but not BAK, activation by cobimetinib plus A-1210477. In summary, BRAFV600E-mediated MEK/ERK activation can upregulate MCL-1 by phosphorylation/stabilization to confer apoptosis resistance that can be reversed by MCL-1 antagonism combined with cobimetinib, suggesting a novel therapeutic strategy against BRAFV600E-mutant CRCs. Mol Cancer Ther; 15(12); 3015–27. ©2016 AACR.

Abstract Introduction: Liver metastasis of pancreatic or colorectal tumors is a major cause of cancer-related death worldwide. When cancer cells reach the liver parenchyma and make a direct contact with the surrounding extracellular matrix (ECM), β1-integrin-FAK signaling is activated allowing the cells attach, migration, and proliferate. Thus, the β1-integrin-FAK signaling axis is key for cancer cells to colonize the liver. However, mechanisms governing β1-integrin activation and how this pathway promotes liver metastasis remain incompletely understood. Aim: We sought to test the hypothesis that vasodilator-stimulated phosphoprotein (VASP), an actin binding protein, regulates the β1-integrin-FAK signaling axis, thereby, promoting liver metastasis of GI cancer. Methods: We employed lentiviruses encoding different VASP shRNAs to knockdown (KD) VASP in human GI cancer cells. We used a Matrigel-based 3-dimensional (3D) cell culture model and intrasplenic tumor injection mouse model to assess the role of VASP for cancer cells in vitro and in vivo. Immunofluorescence (IF) was used to study localization of VASP and active β1-integrin (using Huts-4 antibody), and Western blot analysis (WB) was used to quantitate protein levels of active β1-integrin and YAP1/TAZ. Additionally, we performed VASP immunohistochemistry (IHC) to analyze VASP immunoreactivity in 116 colorectal or pancreatic adenocarcinomas of patients. Results: In the Matrigel-based 3D-culture model, a single cancer cell gave arise to a 3D-cancer spheroid after being cultured for 6-8 days. Using KM12L4, HCT116, or HT29 colorectal cancer cells, and L3.6 or MIA PaCa-1 pancreatic cancer cells, we found that VASP KD reduced the sizes of the 3D-cancer spheroids grown on Matrigel (P<0.05 by t-test for each cell line). IF and WB show that VASP KD reduced levels of Huts-4 (active β1-integrin) in KM12L4, HCT116 colorectal cancer cells and in L3.6, MIA PaCa-1 pancreatic cancer cells. VASP KD also reduced the protein levels of P-FAK and YAP1/TAZ in the 4 cell lines tested. Additionally, YAP1/TAZ protein levels were reduced by PF-228, a FAK inhibitor that blocks the β1-integrin-FAK signaling. In the intrasplenic tumor implanation mouse model, VASP KD suppressed liver metastasis of GI cancer as well as reduced Huts-4 and YAP1 signals of their liver metastases in mice (P<0.05 by t-test). IHC of patient's samples reveals that VASP protein levels were upregulated in cancer cells in 81% of colorectal tumors (51/63) and pancreatic tumors (43/53), with higher VASP levels being associated with liver metastasis of colorectal cancer (P<0.01 by Fisher exact test). Conclusions: VASP promotes ECM-mediated β1-integrin-FAK-YAP1/TAZ signaling axis of GI cancer. We propose that VASP and the β1-integrin-FAK-YAP1/TAZ signaling axis represent potential new therapeutic targets for liver metastasis of pancreatic or colorectal cancer. Citation Format: Xiaoyu Xiang, Jinhua Piao, Selvaraj Muthusamy, Lei Wang, Yibin Deng, Shengbing Huang, Raul Urrutia, Jinping Lai, Ningling Kang. Vasodilator-stimulated phosphoprotein promotes β1-integrin-FAK-YAP1/TAZ signaling axis that is required for liver metastasis of GI cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1633.

Abstract Background. Oncogenic BRAFV600E mutations in colorectal cancer (CRC) are associated with poor prognosis and treatment resistance. BRAF mutations activate MAP kinase signaling and may confer apoptosis resistance by upregulating anti-apoptotic BCL-2/BCL-XL/MCL-1 proteins. Results. Ectopic expression of mutant BRAFV600E was shown to potently activate MEK/ ERK that mediated the phosphorylation (T163) and upregulation of MCL-1, but not BCL-2 or BCL-XL. MEK/ERK-mediated phosphorylation of MCL-1 (T163) increased its protein stability, shown by an MCL-1 phospho-mimic mutant. In human colon cancers, mutant vs wild-type BRAF was associated with increased MCL-1 expression by IHC. MEK/ERK inhibition by cobimetinib induced pro-apoptotic BIM in BRAF mutant CRC cell lines (HT-29, RKO), and suppressed phospho-MCL-1/MCL-1 proteins as did ERK siRNA. Suppression of MCL-1 by shRNA or using a small molecule MCL-1 inhibitor (A-1210477) synergistically enhanced cobimetinib-induced apoptosis (Table) that was associated with BAK/BAX activation. A-1210477 antagonized MCL-1 by disrupting its interaction with BAK and BIM proteins, shown by immunoprecipitation. Conclusion. BRAF/MEK/ERK induces upregulation of MCL-1 via phosphorylation/stabilization to confer apoptosis resistance that can be overcome by combined ERK and MCL-1 inhibition, suggesting a novel therapeutic strategy against BRAF mutant CRCs. Drug-induced apoptosis (annexin V) in CRC cell linesDrugsHT-29 % apoptosisRKO % apoptosisDMSO16.842.11A-1210477 (2.5 μM)29.5222.88Cobimetinib(5 μM)28.0421.05Combination53.2250.93 Citation Format: Hisato Kawakami, Shengbing Huang, Frank A. Sinicrope. MEK/ERK and MCL-1 inhibition synergistically reverse apoptosis resistance in colon cancer cells with BRAF(V600E)-mediated MCL-1-upregulation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2115.

Abstract Background. Oncogenic KRAS mutations occur in nearly one-half of colorectal cancers (CRC) where they contribute to de novo apoptosis resistance and treatment failure. Inhibition of MEK/ERK is a promising strategy to sensitize KRAS mutant tumors to apoptosis that may involve upregulation of pro-apoptotic BH3-only proteins, including BIM. However, BH3-only proteins can be neutralized by anti-apoptotic Bcl-xL that we found was upregulated in isogenic KRAS mutant compared to wild-type HCT116 and DLD1 CRC cells. Aims. We determined the ability and mechanism by which a novel MEK/ERK inhibitor, GDC-0623, can induce BIM to promote apoptotic signaling. A strategy of concurrent induction of BH3-only proteins and antagonism of Bcl-xL was studied to overcome mutant KRAS-mediated apoptosis resistance. Results. Using isogenic KRAS HCT116 cells and KRAS mutant SW620 cells, we found that GDC-0623 can potently induce the BH3-only protein BIM, and to a lesser extent BIK, in both KRAS wild-type and mutant cells. Only KRAS mutant cells remained resistant to GDC-0623-induced apoptosis. Knockdown of Bcl-xL or use of a BH3 mimetic drug, ABT-263, sensitized KRAS mutant cells to GDC-0623-mediated apoptosis. GDC-0623 and ABT-263 were shown to interact synergistically to promote apoptosis in KRAS mutant cells. Dependence on BIM or BIK for synergy between GDC-0623 and ABT-263 was shown by shRNA knockdown that each attenuated drug-induced apoptosis. Mechanistically, ABT-263 treatment was shown to release BIM from its binding to Bcl-xL using immunoprecipitation. The ability of GDC-0623 to induce apoptosis occurred via BIM that was phosphorylated by MEK/ERK activation leading to BIM degradation. GDC-0623 was shown to dephosphorylate BIM at serine 69 in cells with wild-type BIM or a phospho-mimic mutation (S69E). However, GDC-0623's ability to dephosphorylate BIM was lost in cells containing a phosphorylation-defective BIM mutation (S69G) that showed delayed BIM degradation. Conclusion. GDC-0623 was shown to dephosphorylate BIM to enhance its accumulation and to promote apoptosis. Concurrent induction of BIM/BIK and antagonism of Bcl-xL can overcome mutant KRAS-mediated apoptosis resistance, suggesting a promising therapeutic strategy against KRAS mutant CRCs. Note: This abstract was not presented at the meeting. Citation Format: Aziz Zaanan, Koichi Okamoto, Hisato Kawakami, Shengbing Huang, Frank Sinicrope. MEK/ERK inhibitor GDC-0623 dephosphorylates and accumulates BIM that enables a synergistic apoptosis with the Bcl-xL antagonist, ABT-263, in mutant KRAS colorectal cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2937. doi:10.1158/1538-7445.AM2015-2937

Abstract BACKGROUND. Proteosome inhibition is associated with induction of the proapoptotic BH3 protein NOXA. Therefore, we hypothesize that the combination of the irreversible proteosome inhibitor, carfilzomib, will cooperatively enhance apoptosis induction by the BH3 mimetic drug, ABT-263. Given that nononcogenic addiction to proteosome activity has been reported in KRAS mutant cells, we utilized isogenic colorectal cancer (CRC) cell lines in which the endogenous wild-type (wt) or mutant alleles had been inactivated through targeted homologous recombination. Since proteasome blockade results in proteotoxic stress, we determined whether an autophagy inhibitor can enhance chemosensitivity. EXPERIMENTAL METHODS. Isogenic KRAS mutant (MUT) CRC cells (HCT116, DLD1; obtained from B. Vogelstein, JHU) were incubated with carfilzomib (25,50nM) alone or combined with ABT-263 (1 µM) and apoptosis was measured by caspase cleavage and annexin V+ labeling. Synergy was measured by calculation of the combination index. Effect of carfilzomib on Bcl-2 family proteins was determined by immunoblotting. Interaction of NOXA with MCL-1 was examined by immunoprecipitation. Knockdown of NOXA was achieved by lentiviral shRNA. Cells were incubated with drugs alone or combined with chloroquine, and autophagy (LC3I/II conversion) and apoptosis were analyzed. RESULTS. Carfilzomib was shown to potently and synergistically enhance ABT-263-induced apoptosis (2-fold) in both KRAS MUT and wild type (WT) cell lines, with the effect being modestly higher in wild type cells, as shown by annexin V+ staining and caspase cleavage. Carfilzomib induced NOXA expression in both KRAS MUT and WT cells, and reduced MCL-1 expression. Induced NOXA was shown to bind and sequester MCL-1 proteins. Knockdown of NOXA attenuated drug-induced apoptosis. Autophagy inhibition by chloroquine accumulated LC3II, but only modestly increased carfilzomib-induced cell death. CONCLUSION. Irreversible proteasome inhibition by carfilzomib synergistically enhanced ABT-263-induced cell death that is mediated by NOXA upregulation and its sequestration/neutralization of MCL-1. The finding of similar efficacy in KRAS MUT vs WT cells suggests that this combination may represent a novel therapeutic strategy in this tumor subset. Citation Format: Shengbing Huang, Koichi Okamoto, Frank A. Sinicrope. Carfilzomib synergistically enhances ABT-263-induced apoptosis due to NOXA induction in KRAS wild type and mutant colorectal cancer cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 337. doi:10.1158/1538-7445.AM2014-337

Abstract KRAS mutations are frequently detected in human colorectal cancer and contribute to de novo apoptosis resistance and ultimately therapeutic failure. To overcome KRAS-mediated apoptosis resistance, the irreversible proteasome inhibitor, carfilzomib, was evaluated and found to potently induce Noxa, which was dependent upon c-Myc, and Bik. Isogenic mutant versus wild-type KRAS carcinoma cells showed elevated Bcl-xL, confirmed by KRAS siRNA or ectopic expression. Upregulated Bcl-xL by mutant KRAS was mediated by ERK as indicated by ERK knockdown. Bcl-xL expression was regulated at the level of mRNA and protein as shown using actinomycin D and cyclohexamide, respectively. Suppression of Bcl-xL by shRNA sensitized mutant KRAS cells to carfilzomib. Concurrent Bcl-xL antagonism by the BH3 mimetic ABT-263 combined with carfilzomib synergistically enhanced apoptosis that was dependent on Bax or p53, and was attenuated by Noxa or Bik shRNA. In support of this strategy, ectopically expressed Noxa enhanced apoptosis by ABT-263. Carfilzomib-induced Noxa and Bik sequestered Mcl-1 and ABT-263 released Bik and Bak from Bcl-xL, suggesting a mechanism for drug synergy. These preclinical findings establish mutant KRAS-mediated Bcl-xL upregulation as a key mechanism of apoptosis resistance in KRAS-mutant colorectal cancer. Furthermore, antagonizing Bcl-xL enabled carfilzomib-induced Noxa and Bik to induce synergistic apoptosis that reversed KRAS-mediated resistance. Implications: This novel study reveals a promising treatment strategy to overcome apoptosis resistance in KRAS-mutant colorectal cancer by concurrent upregulation of Noxa/Bik and antagonism of Bcl-xL. Mol Cancer Res; 13(4); 659–69. ©2014 AACR.

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 Background. Autophagy maintains energy metabolism and enables tumor cell survival during cellular stress. Beclin 1 is involved in autophagosome formation during which membrane-bound LC3 binds to p62/sequestosome 1. We reported that ectopic p62 can promote 5-fluorouracil-induced cell death (JBC 286:40002-12, 2011). It is unknown whether these autophagy markers can predict clinical outcome. Materials & Methods. Beclin 1, LC3B, and p62 proteins were analyzed by immunohistochemistry in tissue microarrays containing stage II and III colon carcinomas (N=151) from patients receiving 5-FU or surgery alone. The mean weighted score (intensity x extent) was dichotomized for survival analysis using Cox models. Results. Increased levels of cytoplasmic LC3, Beclin 1, and p62 expression were detected in 21%, 14%, and 68% of cancers, respectively, and were not associated with tumor site, stage or grade. By multivariable analysis, increased LC3 was significantly associated with worse disease-free survival (DFS) (p=0.022), but not overall survival (OS) [p=0.069](Table). Beclin 1 was not prognostic, but increased p62 was significantly associated with improved DFS (p=0.038), but not OS (p= 0.176)[Table]. When restricted to 5-FU-treated patients, increased LC3 was associated with worse DFS [HR= 2.38 (1.19, 4.75, p=0.020), elevated Beclin 1 predicted worse OS [HR=2.83 (1.27, 6.34), p = 0.019], and p62 was not prognostic. Conclusion. LC3 and Beclin 1 overexpression were independently associated with adverse outcome. Increased p62 was associated with better outcome consistent with its ability to promote cell death. These data support strategies to inhibit autophagy for therapeutic advantage. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4112. doi:1538-7445.AM2012-4112