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Background: The mechanisms by which stress hormones impact triple-negative breast cancer (TNBC) etiology and treatment are unclear. We have previously shown that stress hormones, cortisol, and catecholamines induce rapid DNA damage and impact DNA repair in NIH 3T3 fibroblasts. This study investigates whether stress hormones increase DNA damage in breast cancer cells and if this impacts drug efficacy. Methods: We first screened a panel of 39 breast cancer cell lines for expression of adrenergic and glucocorticoid receptors and examined if stress hormones induce DNA damage and alter cell cycle regulation in vitro. A TNBC xenograft model was used to assess the impact of restraint stress on tumour growth and chemosensitivity to paclitaxel. Results: We found that stress hormones induced DNA damage, phosphorylation of ATR, which was accompanied by an up-regulation of the G1 cell kinase inhibitor p21 and a cell cycle halt of TNBCs in the G1 phase. p21 knockdown abrogated G1 arrest by stress hormones. We also demonstrated that stress significantly decreased efficacy of paclitaxel. Conclusion: We describe a novel mechanism through which stress hormones can induce drug resistance to paclitaxel, which may have profound implications for treating drug resistance in patients with TNBC. Fil: Reeder, A.. University of Pittsburgh; Estados Unidos Fil: Attar, M.. University of Pittsburgh; Estados Unidos Fil: Nazario, L.. University of Pittsburgh; Estados Unidos Fil: Bathula, C.. University of Pittsburgh; Estados Unidos Fil: Zhang, A.. University of Pittsburgh; Estados Unidos Fil: Hochbaum, Daniel. University of Pittsburgh; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Roy, E.. University of Pittsburgh; Estados Unidos Fil: Cooper, K. L.. University of Pittsburgh; Estados Unidos Fil: Oesterreich, S.. University of Pittsburgh; Estados Unidos Fil: Davidson, N. E.. University of Pittsburgh; Estados Unidos Fil: Neumann, C. A.. University of Pittsburgh; Estados Unidos Fil: Flint, M. S.. University of Pittsburgh; Estados Unidos. University of Brighton; Reino Unido

TWIST1, an epithelial–mesenchymal transition (EMT) transcription factor, is critical for oncogene-driven non–small cell lung cancer (NSCLC) tumorigenesis. Given the potential of TWIST1 as a therapeutic target, a chemical–bioinformatic approach using connectivity mapping (CMAP) analysis was used to identify TWIST1 inhibitors. Characterization of the top ranked candidates from the unbiased screen revealed that harmine, a harmala alkaloid, inhibited multiple TWIST1 functions, including single-cell dissemination, suppression of normal branching in 3D epithelial culture, and proliferation of oncogene driver-defined NSCLC cells. Harmine treatment phenocopied genetic loss of TWIST1 by inducing oncogene-induced senescence or apoptosis. Mechanistic investigation revealed that harmine targeted the TWIST1 pathway through its promotion of TWIST1 protein degradation. As dimerization is critical for TWIST1 function and stability, the effect of harmine on specific TWIST1 dimers was examined. TWIST1 and its dimer partners, the E2A proteins, which were found to be required for TWIST1-mediated functions, regulated the stability of the other heterodimeric partner posttranslationally. Harmine preferentially promoted degradation of the TWIST1-E2A heterodimer compared with the TWIST-TWIST1 homodimer, and targeting the TWIST1-E2A heterodimer was required for harmine cytotoxicity. Finally, harmine had activity in both transgenic and patient-derived xenograft mouse models of KRAS-mutant NSCLC. These studies identified harmine as a first-in-class TWIST1 inhibitor with marked anti-tumor activity in oncogene-driven NSCLC including EGFR mutant, KRAS mutant and MET altered NSCLC. Implications: TWIST1 is required for oncogene-driven NSCLC tumorigenesis and EMT; thus, harmine and its analogues/derivatives represent a novel therapeutic strategy to treat oncogene-driven NSCLC as well as other solid tumor malignancies. Mol Cancer Res; 15(12); 1764–76. ©2017 AACR.

Epithelial cells are largely immotile under normal circumstances, but become motile during development, repair of tissue damage and during cancer metastasis. Numerous growth factors act to initiate epithelial cell movements. Hepatocyte growth factor (HGF) induces many epithelial cell lines to begin crawling. A number of small GTPases act downstream of HGF to alter cell shape and promote movement. Arf6 is one of these GTPases that can alter the cortical actin cytoskeleton and promote epithelial movement. Activation of Arf6 in MDCK cells by its guanine nucleotide exchange factor cytohesin 2/ARNO produces a scattering response strikingly reminiscent of the action of HGF. We have previously shown that IPCEF1, a scaffold that binds to cytohesin 2, is required for cytohesin-induced scattering. We report here that IPCEF1 is actually the C-terminal half of CNK3. CNKs are scaffolds involved in signal transduction downstream of Ras. We have found that both MDCK and CaCo-2 cells express a fused CNK3/IPCEF1 protein. Knockdown of this protein impairs HGF-induced Arf6 activation and migration in response to HGF treatment.

Cellular DNA is organized into chromosomes and capped by a unique nucleoprotein structure, the telomere. Both oxidative stress and telomere shortening/dysfunction cause aging-related degenerative pathologies and increase cancer risk. However, a direct connection between oxidative damage to telomeric DNA, comprising

The ARF-GEF ARNO promotes motility by activating ARF6 and a subsequent downstream activation of Rac. ARNO is shown to associate with the Rac GEF Dock180 via its coiled-coil domain. Knockdown of scaffold proteins that bind ARNO disrupts the formation of this complex and disrupts ARF-to-Rac signaling., ARFs are small GTPases that regulate vesicular trafficking, cell shape, and movement. ARFs are subject to extensive regulation by a large number of accessory proteins. The many different accessory proteins are likely specialized to regulate ARF signaling during particular processes. ARNO/cytohesin 2 is an ARF-activating protein that promotes cell migration and cell shape changes. We report here that protein–protein interactions mediated by the coiled-coil domain of ARNO are required for ARNO induced motility. ARNO lacking the coiled-coil domain does not promote migration and does not induce ARF-dependent Rac activation. We find that the coiled-coil domain promotes the assembly of a multiprotein complex containing both ARNO and the Rac-activating protein Dock180. Knockdown of either GRASP/Tamalin or IPCEF, two proteins known to bind to the coiled-coil of ARNO, prevents the association of ARNO and Dock180 and prevents ARNO-induced Rac activation. These data suggest that scaffold proteins can regulate ARF dependent processes by biasing ARF signaling toward particular outputs.

Background The transition of epithelial cells from their normal non-motile state to a motile one requires the coordinated action of a number of small GTPases. We have previously shown that epithelial cell migration is stimulated by the coordinated activation of Arf and Rac GTPases. This crosstalk depends upon the assembly of a multi-protein complex that contains the Arf-activating protein cytohesin 2/ARNO and the Rac activating protein Dock180. Two scaffolding proteins that bind directly to cytohesin 2 organize this complex. Results We now have found that Rac activation in response to hepatocyte growth factor (HGF) requires cytohesin 2 and Dock180. GRASP/Tamalin is one of the scaffolds that builds the complex containing cytohesin 2 and Dock180. We determine here that the Ala/Pro rich region of GRASP directly interacts with the SH3 domain of Dock180. By binding to both cytohesin 2/ARNO and Dock180, GRASP bridges the guanine nucleotide exchange factors (GEFs) that activate Arf and Rac, thereby promoting Arf-to-Rac signaling. Furthermore, we find that knockdown of GRASP impairs hepatocyte growth factor (HGF)-stimulated Rac activation and HGF-stimulated epithelial migration. Conclusions GRASP binds directly both cytohesin 2 and Dock180 to coordinate their activities, and by doing so promotes crosstalk between Arf and Rac.