Repurposing ‘old’ drugs can facilitate rapid clinical translation but necessitates novel mechanistic insight. Warfarin, a vitamin K “antagonist” used clinically for the prevention of thrombosis for over 50 years, has been shown to have anti-cancer effects. We hypothesized that the molecular mechanism underlying its anti-tumor activity is unrelated to its effect on coagulation, but is due to inhibition of the Axl receptor tyrosine kinase on tumor cells. Activation of Axl by its ligand Gas6, a vitamin K-dependent protein, is inhibited at doses of warfarin that do not affect coagulation. Here we show that inhibiting Gas6-dependent Axl activation with low dose warfarin or with other tumor-specific Axl targeting agents, blocks the progression and spread of pancreatic cancer. Warfarin also inhibited Axl-dependent tumor cell migration, invasiveness and proliferation while increasing apoptosis and sensitivity to chemotherapy. We conclude that Gas6-induced Axl signaling is a critical driver of pancreatic cancer progression and its inhibition with low dose warfarin or other Axl targeting agents may improve outcome in patients with Axl-expressing tumors.

Myeloid derived suppressor cells (MDSC) contribute significantly to the malignant characters conferred by hypoxic tumor microenvironments. However, selective biomarkers of MDSC function in this critical setting have not been defined. Here we report that miR-210 expression is elevated by HIF-1α in MDSC localized to tumors, compared to splenic MDSC from tumor-bearing mice. In tumor MDSC, we determined that HIF-1α was bound directly to a transcriptionally active hypoxia-response element in the miR-210 proximal promoter. miR-210 overexpression was sufficient to enhance MDSC-mediated T cell suppression under normoxic conditions, while targeting hypoxia-induced miR-210 was sufficient to decrease MDSC function against T cells. Mechanistic investigations revealed that miR-210 modulated MDSC function by increasing arginase activity and NO production, without affecting ROS, IL-6 or IL-10 production or expression of PD-L1. In splenic MDSC, miR-210 regulated Arg1, Cxcl12 and IL-16 at the levels of both mRNA and protein, the reversal of which under normoxic conditions decreased T cell suppressive effects and IFN-gamma production. Interestingly, miR-210 overexpression or targeting IL-16 or CXCL12 enhanced the immune suppressive activity of MDSC in vivo resulting in increased tumor growth. Taken together, these results provide a preclinical rationale to explore miR-210 inhibitory oligonucleotides as adjuvants to boost immunotherapeutic responses in cancer patients.

The treatment options remain limited for patients with melanoma who are wild-type for both BRAF and NRAS (WT/WT). We demonstrate that a subgroup of WT/WT melanomas display high basal phosphorylation of ErbB3 that is associated with autocrine production of the ErbB3 ligand neuregulin-1 (NRG1). In WT/WT melanoma cells displaying high levels of phospho-ErbB3, knockdown of NRG1 reduced cell viability and was associated with decreased phosphorylation of ErbB3, its coreceptor ErbB2, and its downstream target, AKT. Similar effects were observed by targeting ErbB3 with either siRNAs or the neutralizing ErbB3 monoclonal antibodies huHER3-8 and NG33. In addition, pertuzumab-mediated inhibition of ErbB2 heterodimerization decreased AKT phosphorylation, cell growth in vitro, and xenograft growth in vivo. Pertuzumab also potentiated the effects of MEK inhibitor on WT/WT melanoma growth in vitro and in vivo. These findings demonstrate that targeting ErbB3–ErbB2 signaling in a cohort of WT/WT melanomas leads to tumor growth reduction. Together, these studies support the rationale to target the NRG1–ErbB3–ErbB2 axis as a novel treatment strategy in a subset of cutaneous melanomas. Cancer Res; 75(17); 1–14. ©2015 AACR.

The treatment options remain limited for melanoma patients who are wild-type for both BRAF and NRAS (WT/WT). We demonstrate that a subgroup of WT/WT melanomas display high basal phosphorylation of ErbB3 that is associated with autocrine production of the ErbB3 ligand, neuregulin-1 (NRG1). In WT/WT melanoma cells displaying high levels of phospho-ErbB3, knockdown of NRG1 reduced cell viability and was associated with decreased phosphorylation of ErbB3, its co-receptor ErbB2 and its downstream target, AKT. Similar effects were observed by targeting ErbB3 with either small interfering RNAs or the neutralizing ErbB3 monoclonal antibodies, huHER3-8 and NG33. Additionally, pertuzumab-mediated inhibition of ErbB2 heterodimerization decreased AKT phosphorylation, cell growth in vitro and xenograft growth in vivo. Pertuzumab also potentiated the effects of MEK inhibitor on WT/WT melanoma growth in vitro and in vivo. These findings demonstrate that targeting ErbB3-ErbB2 signaling in a cohort of WT/WT melanomas leads to tumor growth reduction. Together these studies support the rationale to target the NRG1-ErbB3-ErbB2 axis as a novel treatment strategy in a subset of cutaneous melanomas.

Mast cells (MCs) are immune cells located next to the intestinal epithelium with regulatory function in maintaining the homeostasis of the mucosal barrier. We have investigated MC activities in colon inflammation and cancer in mice either wild-type or MC-deficient (KitW-sh) reconstituted or not with bone marrow-derived MCs. Colitis was chemically induced with dextran sodium sulfate (DSS). Tumors were induced by administering azoxymethane (AOM) intraperitoneally before DSS. Following DSS withdrawal KitW-sh mice showed reduced weight gain and impaired tissue repair compared to their WT littermates or KitW-sh mice reconstituted with bone marrow-derived MCs. MCs were localized in areas of mucosal healing rather than damaged areas where they degraded interleukin-33, an alarmin released by epithelial cells during tissue damage. KitW-sh mice reconstituted with MC deficient for mouse mast cell protease 4 did not restore normal mucosal healing or reduce efficiently inflammation after DSS withdrawal. In contrast to MCs recruited during inflammation-associated wound healing, MCs adjacent to transformed epithelial cells acquired a pro-tumorigenic profile. In AOM- and DSS-treated WT mice, high MC density correlated with high-grade carcinomas. In similarly treated KitW-sh mice, tumors were less extended and displayed lower histological grade. Our results indicate that the action of MCs and their interaction with epithelial cells is dependent on the inflammatory stage, initiation, chronic or resolution, and the activation of the tissue repair program. Selective targeting of MCs for prevention or treatment of inflammation-associated colon cancer should be timely pondered to allow tissue repair at pre-malignant stages or to reduce aggressiveness at the tumor stage.