title: Novel Cancer Therapeutics with Allosteric Modulation of the Mitochondrial C-Raf-DAPK Complex by Raf Inhibitor Combination Therapy.
abstract: Mitochondria are the powerhouses of cells. Mitochondrial C-Raf is a potential cancer therapeutic target, as it regulates mitochondrial function and is localized to the mitochondria by its N-terminal domain. However, Raf inhibitor monotherapy can induce S338 phosphorylation of C-Raf (pC-Raf(S338)) and impede therapy. This study identified the interaction of C-Raf with S308 phosphorylated DAPK (pDAPK(S308)), which together became colocalized in the mitochondria to facilitate mitochondrial remodeling. Combined use of the Raf inhibitors sorafenib and GW5074 had synergistic anticancer effects in vitro and in vivo, but targeted mitochondrial function, rather than the canonical Raf signaling pathway. C-Raf depletion in knockout MEF(C-Raf-/-) or siRNA knockdown ACHN renal cancer cells abrogated the cytotoxicity of combination therapy. Crystal structure simulation showed that GW5074 bound to C-Raf and induced a C-Raf conformational change that enhanced sorafenib-binding affinity. In the presence of pDAPK(S308), this drug-target interaction compromised the mitochondrial targeting effect of the N-terminal domain of C-Raf, which induced two-hit damages to cancer cells. First, combination therapy facilitated pC-Raf(S338) and pDAPK(S308) translocation from mitochondria to cytoplasm, leading to mitochondrial dysfunction and reactive oxygen species (ROS) generation. Second, ROS facilitated PP2A-mediated dephosphorylation of pDAPK(S308) to DAPK. PP2A then dissociated from the C-Raf-DAPK complex and induced profound cancer cell death. Increased pDAPK(S308) modification was also observed in renal cancer tissues, which correlated with poor disease-free survival and poor overall survival in renal cancer patients. Besides mediating the anticancer effect, pDAPK(S308) may serve as a predictive biomarker for Raf inhibitors combination therapy, suggesting an ideal preclinical model that is worthy of clinical translation.
authors.affiliation: Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan, Republic of China. Division of Urology, Department of Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China.
authors.forename: Yi-Ta
authors.initials: YT
authors.lastname: Tsai
authors.affiliation: Division of Urology, Department of Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China.
authors.forename: Mei-Jen
authors.initials: MJ
authors.lastname: Chuang
authors.affiliation: Division of Urology, Department of Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China.
authors.forename: Shou-Hung
authors.initials: SH
authors.lastname: Tang
authors.affiliation: Division of Urology, Department of Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China.
authors.forename: Sheng-Tang
authors.initials: ST
authors.lastname: Wu
authors.affiliation: Division of Urology, Department of Surgery, E-Da Hospital, Kaohsiung, Taiwan, Republic of China.
authors.forename: Yu-Chi
authors.initials: YC
authors.lastname: Chen
authors.affiliation: Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan, Republic of China. Division of Urology, Department of Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China. Graduate Institutes of Life Sciences, National Defense Medical Center, Taipei, Taiwan, Republic of China.
authors.forename: Guang-Huan
authors.initials: GH
authors.lastname: Sun
authors.affiliation: Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan, Republic of China.
authors.forename: Pei-Wen
authors.initials: PW
authors.lastname: Hsiao
authors.affiliation: Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan, Republic of China.
authors.forename: Shih-Ming
authors.initials: SM
authors.lastname: Huang
authors.affiliation: Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan, Republic of China.
authors.forename: Hwei-Jen
authors.initials: HJ
authors.lastname: Lee
authors.affiliation: Graduate Institutes of Life Sciences, National Defense Medical Center, Taipei, Taiwan, Republic of China. Graduate Institute of Pathology and Parasitology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China.
authors.forename: Cheng-Ping
authors.initials: CP
authors.lastname: Yu
authors.affiliation: Graduate Institutes of Life Sciences, National Defense Medical Center, Taipei, Taiwan, Republic of China. Graduate Institute of Pathology and Parasitology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China.
authors.forename: Jar-Yi
authors.initials: JY
authors.lastname: Ho
authors.affiliation: Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas. The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, Texas.
authors.forename: Hui-Kuan
authors.initials: HK
authors.lastname: Lin
authors.affiliation: Division of Urology, Department of Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China. Graduate Institutes of Life Sciences, National Defense Medical Center, Taipei, Taiwan, Republic of China.
authors.forename: Ming-Rong
authors.initials: MR
authors.lastname: Chen
authors.affiliation: Division of Urology, Department of Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China.
authors.forename: Chung-Chih
authors.initials: CC
authors.lastname: Lin
authors.affiliation: Division of Urology, Department of Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China. Taipei City Hospital, Taipei, Taiwan, Republic of China.
authors.forename: Sun-Yran
authors.initials: SY
authors.lastname: Chang
authors.affiliation: Division of Urology, Department of Surgery, E-Da Hospital, Kaohsiung, Taiwan, Republic of China.
authors.forename: Victor C
authors.initials: VC
authors.lastname: Lin
authors.affiliation: Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan, Republic of China. Division of Urology, Department of Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China. Graduate Institutes of Life Sciences, National Defense Medical Center, Taipei, Taiwan, Republic of China.
authors.forename: Dah-Shyong
authors.initials: DS
authors.lastname: Yu
authors.affiliation: Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan, Republic of China. Division of Urology, Department of Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China. Graduate Institutes of Life Sciences, National Defense Medical Center, Taipei, Taiwan, Republic of China. Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan, Republic of China. tlcha@ndmctsgh.edu.tw.
authors.forename: Tai-Lung
authors.initials: TL
authors.lastname: Cha
mesh_terms: D000368:Aged; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D064550:Death-Associated Protein Kinases / Q000235:genetics*; D018572:Disease-Free Survival; D004357:Drug Synergism; D005260:Female; D055786:Gene Knockout Techniques; D006801:Humans; D007211:Indoles / Q000008:administration & dosage; D007680:Kidney Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D008297:Male; D051379:Mice; D008875:Middle Aged; D008928:Mitochondria / Q000187:drug effects / Q000473:pathology; D009536:Niacinamide / Q000008:administration & dosage / Q000031:analogs & derivatives; D010636:Phenols / Q000008:administration & dosage; D010671:Phenylurea Compounds / Q000008:administration & dosage; D010766:Phosphorylation / Q000187:drug effects; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics*; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D000077157:Sorafenib; D023041:Xenograft Model Antitumor Assays
chemical_list: D007211:Indoles; D010636:Phenols; D010671:Phenylurea Compounds; D017382:Reactive Oxygen Species; D009536:Niacinamide; D000077157:Sorafenib; C579096:DAPK1 protein, human; D064550:Death-Associated Protein Kinases; D019908:Proto-Oncogene Proteins c-raf; C489251:5-iodo-3-((3,5-dibromo-4-hydroxyphenyl)methylene)-2-indolinone