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-RafS338) and impede therapy. This study identified the interaction of C-Raf with S308 phosphorylated DAPK (pDAPKS308), which together became co-localized in the mitochondria to facilitate mitochondrial remodelling. Combined use of the Raf inhibitors sorafenib and GW5074 had synergistic anti-cancer effects in vitro and in vivo, but targeted mitochondrial function, rather than the canonical Raf signaling pathway. C-Raf depletion in knock-out MEFC-Raf-/- or siRNA knock-down 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 pDAPKS308, 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-RafS338 and pDAPKS308 translocation from mitochondria to cytoplasm, leading to mitochondrial dysfunction and ROS generation. Second, ROS facilitated PP2A-mediated de-phosphorylation of pDAPKS308 to DAPK. PP2A then dissociated from the C-Raf-DAPK complex and induced profound cancer cell death. Increased pDAPKS308 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 anti-cancer effect, pDAPKS308 may serve as a predictive biomarker for Raf inhibitors combination therapy, suggesting an ideal pre-clinical model that is worthy of clinical translation.

The repertoire of antigens associated with the development of an autoimmune response in breast cancer has relevance to detection and treatment strategies. We have investigated the occurrence of autoantibodies associated with the development of triple-negative breast cancer (TNBC) in the before diagnosis setting and in samples collected at the time of diagnosis of TNBC. Lysate arrays containing protein fractions from the TNBC MDA-MB-231 cell line were hybridized with TNBC plasmas from the Women’s Health Initiative cohort, collected before clinical diagnosis and with plasmas from matched controls. An immune response directed against spliceosome and glycolysis proteins was observed with case plasmas as previously reported in estrogen receptor+ breast cancer. Importantly, autoantibodies directed against networks involving BRCA1, TP53, and cytokeratin proteins associated with a mesenchymal/basal phenotype were distinct to TNBC before diagnosis samples. Concordant autoantibody findings were observed with mouse plasma samples collected before occurrence of palpable tumors from a C3(1)-T triple negative mouse model. Plasma samples collected at the time of diagnosis of stage II TNBC and from matched healthy controls were subjected to proteomic analysis by mass spectrometry to identify Ig-bound proteins yielding a predominance of cytokeratins, including several associated with a mesenchymal/basal phenotype among cases compared with controls. Our data provide evidence indicative of a dynamic repertoire of antigens associated with a humoral immune response reflecting disease pathogenesis in TNBC. Cancer Res; 75(16); 1–9. ©2015 AACR.

The repertoire of antigens associated with the development of an autoimmune response in breast cancer has relevance to detection and treatment strategies. We have investigated the occurrence of autoantibodies associated with the development of triple negative breast cancer (TNBC) in the prior to diagnosis setting and in samples collected at the time of diagnosis of TNBC. Lysate arrays containing protein fractions from the TNBC MDA-MB-231 cell line were hybridized with TNBC plasmas from the Women’s Health Initiative cohort, collected prior to clinical diagnosis and with plasmas from matched controls. An immune response directed against spliceosome and glycolysis proteins was observed with case plasmas as previously reported in ER+ breast cancer. Importantly autoantibodies directed against networks involving BRCA1, TP53 and cytokeratin proteins associated with a mesenchymal/basal phenotype were distinct to TNBC prior to diagnosis samples. Concordant autoantibody findings were observed with mouse plasma samples collected prior to occurrence of palpable tumors from a C3(1)-T triple negative mouse model. Plasma samples collected at the time of diagnosis of stage II TNBC and from matched healthy controls were subjected to proteomic analysis by mass spectrometry to identify Ig bound proteins yielding a predominance of cytokeratins including several associated with a mesenchymal/basal phenotype among cases compared to controls. Our data provide evidence indicative of a dynamic repertoire of antigens associated with a humoral immune response reflecting disease pathogenesis in TNBC.

STAT3 offers an attractive target for cancer therapy but small molecule inhibitors with appealing pharmacologic properties have been elusive. Here we report hydroxamic acid-based and benzoic acid-based inhibitors (SH5-07 and SH4-54, respectively) with robust bioactivity. Both inhibitors blocked STAT3 DNA binding activity in vitro and in human glioma, breast, and prostate cancer cells and in v-Src-transformed murine fibroblasts. STAT3-dependent gene transcription was blocked along with Bcl-2, Bcl-xL, Mcl-1, Cyclin D1, c-Myc and Survivin expression. Nuclear magnetic resonance analysis of STAT3-inhibitor complexes defined interactions with the SH2 and DNA binding domains of STAT3. Ectopic expression of the SH2 domain in cells was sufficient to counter the STAT3 inhibitory effects of SH4-54. Neither compound appreciably affected STAT1 or STAT5 DNA binding activities, STAT3-independent gene transcription or activation of a panel of oncogenic kinases in malignant cells.
Each compound decreased the proliferation and viability of glioma, breast and prostate cancer cells and v-Src-transformed murine fibroblasts harboring constitutively active STAT3. Further, in mouse xenograft models of glioma and breast cancer, administration of SH5-07 or SH4-54 effectively inhibited tumor growth. Our results offer preclinical proof of concept for SH5-07 and SH4-54 as candidates for further development as cancer therapeutics.