Reversal of Mutant KRAS-Mediated Apoptosis Resistance by Concurrent Noxa/Bik Induction and Bcl-2/Bcl-xL Antagonism in Colon Cancer Cells

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.

[1]  K. Kinzler,et al.  Glucose Deprivation Contributes to the Development of KRAS Pathway Mutations in Tumor Cells , 2009, Science.

[2]  Sara Schmitt,et al.  From bortezomib to other inhibitors of the proteasome and beyond. , 2013, Current pharmaceutical design.

[3]  A. Letai,et al.  Failure to induce apoptosis via BCL-2 family proteins underlies lack of efficacy of combined MEK and PI3K inhibitors for KRAS-mutant lung cancers. , 2014, Cancer research.

[4]  Wenhua Gao,et al.  Elimination of Mcl-1 is required for the initiation of apoptosis following ultraviolet irradiation. , 2003, Genes & development.

[5]  Next-generation proteasome inhibitor approved in multiple myeloma , 2012, Nature Biotechnology.

[6]  C. Tse,et al.  ABT-263: a potent and orally bioavailable Bcl-2 family inhibitor. , 2008, Cancer research.

[7]  C. Tse,et al.  Activity of the Bcl-2 Family Inhibitor ABT-263 in a Panel of Small Cell Lung Cancer Xenograft Models , 2008, Clinical Cancer Research.

[8]  C. Billard.  BH3 Mimetics: Status of the Field and New Developments , 2013, Molecular Cancer Therapeutics.

[9]  W. McBride,et al.  The Proteasome in Cancer Biology and Treatment , 2001, Radiation research.

[10]  P. Auberger,et al.  Cleavage of Mcl-1 by caspases impaired its ability to counteract Bim-induced apoptosis , 2004, Oncogene.

[11]  F. Sinicrope,et al.  BH3 mimetic ABT-737 potentiates TRAIL-mediated apoptotic signaling by unsequestering Bim and Bak in human pancreatic cancer cells. , 2008, Cancer research.

[12]  S. Cory,et al.  The Bcl2 family: regulators of the cellular life-or-death switch , 2002, Nature Reviews Cancer.

[13]  B. Dörken,et al.  Induction of cell death by the BH3‐only Bcl‐2 homolog Nbk/Bik is mediated by an entirely Bax‐dependent mitochondrial pathway , 2003, The EMBO journal.

[14]  D. Lane,et al.  Drugging the p53 pathway: understanding the route to clinical efficacy , 2014, Nature Reviews Drug Discovery.

[15]  Julian Downward,et al.  Determination of synthetic lethal interactions in KRAS oncogene-dependent cancer cells reveals novel therapeutic targeting strategies , 2012, Cell Research.

[16]  T. Chou.  Drug combination studies and their synergy quantification using the Chou-Talalay method. , 2010, Cancer research.

[17]  John Calvin Reed,et al.  Tumor suppressor p53 is a direct transcriptional activator of the human bax gene , 1995, Cell.

[18]  Michael J. Emanuele,et al.  A Genome-wide RNAi Screen Identifies Multiple Synthetic Lethal Interactions with the Ras Oncogene , 2009, Cell.

[19]  Lorenzo Galluzzi,et al.  Mitochondrial membrane permeabilization in cell death. , 2007, Physiological reviews.

[20]  A. Kater,et al.  Tipping the Noxa/Mcl-1 Balance Overcomes ABT-737 Resistance in Chronic Lymphocytic Leukemia , 2011, Clinical Cancer Research.

[21]  H. Lenz,et al.  Predictive and prognostic markers in the treatment of metastatic colorectal cancer (mCRC): personalized medicine at work. , 2015, Hematology/oncology clinics of North America.

[22]  H. Xin,et al.  Enhanced killing of melanoma cells by simultaneously targeting Mcl-1 and NOXA. , 2006, Cancer research.

[23]  John Calvin Reed,et al.  Mechanisms of apoptosis sensitivity and resistance to the BH3 mimetic ABT-737 in acute myeloid leukemia. , 2006, Cancer cell.

[24]  F. Sinicrope,et al.  p62/Sequestosome-1 Up-regulation Promotes ABT-263-induced Caspase-8 Aggregation/Activation on the Autophagosome* , 2013, The Journal of Biological Chemistry.

[25]  G. Perkins,et al.  Mcl-1 levels need not be lowered for cells to be sensitized for ABT-263/737-induced apoptosis , 2011, Cell Death and Disease.

[26]  T. Lister,et al.  The proteasome inhibitor bortezomib acts independently of p53 and induces cell death via apoptosis and mitotic catastrophe in B-cell lymphoma cell lines. , 2007, Cancer research.

[27]  C. Watson,et al.  p53-dependent apoptosis induced by proteasome inhibition in mammary epithelial cells , 2001, Cell Death and Differentiation.

[28]  S. Varambally,et al.  Tumor cell-selective regulation of NOXA by c-MYC in response to proteasome inhibition , 2007, Proceedings of the National Academy of Sciences.

[29]  F. Sinicrope,et al.  BH3 Mimetic Obatoclax Enhances TRAIL-Mediated Apoptosis in Human Pancreatic Cancer Cells , 2009, Clinical Cancer Research.

[30]  F. Sinicrope,et al.  Induction of Noxa Sensitizes Human Colorectal Cancer Cells Expressing Mcl-1 to the Small-Molecule Bcl-2/Bcl-xL Inhibitor, ABT-737 , 2008, Clinical Cancer Research.

[31]  M. Hendrix,et al.  Proteasome inhibitors trigger NOXA-mediated apoptosis in melanoma and myeloma cells. , 2005, Cancer research.

[32]  F. Sinicrope,et al.  Celecoxib-induced apoptosis is enhanced by ABT-737 and by inhibition of autophagy in human colorectal cancer cells , 2010, Autophagy.

[33]  Dongsheng Tu,et al.  K-ras mutations and benefit from cetuximab in advanced colorectal cancer. , 2008, The New England journal of medicine.

[34]  J. Hampe,et al.  Increased proteasome subunit protein expression and proteasome activity in colon cancer relate to an enhanced activation of nuclear factor E2-related factor 2 (Nrf2) , 2009, Oncogene.

[35]  R. Youle,et al.  Predominant requirement of Bax for apoptosis in HCT116 cells is determined by Mcl-1’s inhibitory effect on Bak , 2011, Oncogene.

[36]  A. Jemal,et al.  Annual Report to the Nation on the status of cancer, 1975‐2010, featuring prevalence of comorbidity and impact on survival among persons with lung, colorectal, breast, or prostate cancer , 2014, Cancer.

[37]  Derick R. Peterson,et al.  Obatoclax Interacts Synergistically with the Irreversible Proteasome Inhibitor Carfilzomib in GC- and ABC-DLBCL Cells In Vitro and In Vivo , 2012, Molecular Cancer Therapeutics.

[38]  P. Colman,et al.  BCL-2 family antagonists for cancer therapy , 2008, Nature Reviews Drug Discovery.

[39]  F. Sinicrope,et al.  Inhibition of mTOR Kinase by AZD8055 Can Antagonize Chemotherapy-induced Cell Death through Autophagy Induction and Down-regulation of p62/Sequestosome 1* , 2011, The Journal of Biological Chemistry.

[40]  E. Bilotti.  Carfilzomib: a next-generation proteasome inhibitor for multiple myeloma treatment. , 2013, Clinical journal of oncology nursing.

[41]  林田 健太郎,et al.  加齢に伴う難聴におけるnuclear factor E2-related factor 2 (Nrf2) の関与 , 2009 .

[42]  T. Tsuruo,et al.  Acceleration of apoptotic cell death after the cleavage of Bcl-XL protein by caspase-3-like proteases , 1998, Oncogene.