Small molecules that restore the expression of growth inhibitory microRNAs downregulated in tumors may have potential as anticancer agents. miR-34a functions as a tumor suppressor and is downregulated or silenced commonly in a variety of human cancers including hepatocellular carcinoma (HCC). In this study, we used an HCC cell-based miR-34a luciferase reporter system to screen for miR-34a modulators that could exert anticancer activity. One compound identified as a lead candidate, termed Rubone, was identified through its ability to specifically upregulate miR-34a in HCC cells. Rubone activated miR-34a expression in HCC cells with wild type or mutated p53 but not in cells with p53 deletions. Notably, Rubone lacked growth inhibitory effects on non-tumorigenic human hepatocytes. In a mouse xenograft model of HCC, Rubone dramatically inhibited tumor growth, exhibiting stronger anti-HCC activity than sorafenib both in vitro and in vivo. Mechanistic investigations showed that Rubone decreased expression of Cyclin D1, Bcl-2 and other miR-34a target genes and that it enhanced the occupancy of p53 on the miR-34a promoter. Taken together, our results offer a preclinical proof of concept for Rubone as a lead candidate for further investigation as a new class of HCC therapeutic based on restoration of miR-34a tumor suppressor function.

Cell-based drug screenings indicate that tumors displaying c-MET gene amplification are “addicted” to MET signaling and therefore are very sensitive to MET-targeted agents. However, these screenings were conducted in the absence of the MET ligand, hepatocyte growth factor (HGF), which is abundant in the tumor microenvironment. Sensitivity of six MET-addicted human tumor cells to three MET kinase inhibitors (JNJ-38877605, PHA-665752, crizotinib) and one antagonistic anti-MET antibody (DN30 Fab) was analyzed in the absence or presence of HGF, in a stroma–tumor coculture system, and by combining anti-MET drugs with an HGF neutralizing antibody (ficlatuzumab) in human HGF knock-in mice bearing c-MET–amplified tumors. In all models examined, HGF promoted resistance to MET-targeted agents, affecting both their potency and efficacy. HGF-induced resistance was due to restoration of physiologic GAB1–mediated PI3K activation that compensated for loss of aberrant HER3-dependent PI3K signaling. Ficlatuzumab restored sensitivity to MET-targeted agents in coculture systems and overcame resistance to JNJ-38877605, crizotinib, and DN30 Fab in human HGF knock-in mice. These data suggest that c-MET–amplified tumor cells—which normally exhibit ligand-independent, constitutive MET activation—become dependent on HGF for survival upon pharmacologic MET inhibition. Because HGF is frequently overexpressed in human cancer, this mechanism may represent a major cause of resistance to anti-MET therapies. The ability of ficlatuzumab to overcome HGF-mediated resistance generates proof of principle that vertical inhibition of both a tyrosine kinase receptor and its ligand can be therapeutically beneficial and opens new perspectives for the treatment of MET-dependent tumors. Cancer Res; 74(22); 1–12. ©2014 AACR.

Cell-based drug screenings indicate that tumors displaying c-MET gene amplification are ‘addicted’ to MET signaling and therefore very sensitive to MET-targeted agents. However, these screenings were conducted in the absence of the MET ligand, Hepatocyte Growth Factor (HGF), which is abundant in the tumor microenvironment. Sensitivity of 6 MET-addicted human tumor cells to 3 MET kinase inhibitors (JNJ-38877605, PHA-665752, crizotinib) and 1 antagonistic anti-MET antibody (DN30 Fab) was analyzed in the absence or presence of HGF, in a stroma-tumor co-culture system, and by combining anti-MET drugs with a HGF-neutralizing antibody (ficlatuzumab) in human HGF knock-in mice bearing c-MET-amplified tumors. In all models examined, HGF promoted resistance to MET-targeted agents, affecting both their potency and efficacy. HGF-induced resistance was due to restoration of physiologic, GAB-1-mediated PI3K activation that compensated for loss of aberrant, HER3-dependent PI3K signaling. Ficlatuzumab restored sensitivity to MET-targeted agents in co-culture systems and overcame resistance to JNJ-38877605, crizotinib and DN30 Fab in human HGF knock-in mice. These data suggest that c-MET-amplified tumor cells -which normally exhibit ligand-independent, constitutive MET activation- become dependent on HGF for survival upon pharmacological MET inhibition. Since HGF is frequently overexpressed in human cancer, this mechanism may represent a major cause of resistance to anti-MET therapies. The ability of ficlatuzumab to overcome HGF-mediated resistance generates proof-of-principle that vertical inhibition of both a tyrosine kinase receptor and its ligand can be therapeutically beneficial, and opens new perspectives for the treatment of MET-dependent tumors.

Acquired resistance to TGFβ is a key step in the early stages of tumorigenesis. Mutations in TGFβ signaling components are rare, and little is known about the development of resistance in breast cancer. On the other hand, an activated Notch pathway is known to play a substantial role in promoting breast cancer development. Here, we present evidence of crosstalk between these two pathways through HEYL. HEYL, a basic helix–loop–helix transcription factor and a direct target of Notch signaling, is specifically overexpressed in breast cancer. HEYL represses TGFβ activity by binding to TGFβ-activated Smads. HeyL−/− mice have defective mammary gland development with fewer terminal end buds. On the other hand, HeyL transgenic mice show accelerated mammary gland epithelial proliferation and 24% of multiparous mice develop mammary gland cancer. Therefore, repression of TGFβ signaling by Notch acting through HEYL may promote initiation of breast cancer. Cancer Res; 74(22); 1–10. ©2014 AACR.

Acquired resistance to transforming growth factor-β (TGF-β) is a key step in the early stages of tumorigenesis. Mutations in TGF-β signaling components are rare, and little is known about development of resistance in breast cancer. On the other hand, an activated Notch pathway is known to play a substantial role in promoting breast cancer development. Here, we present evidence of crosstalk between these two pathways through HEYL. HEYL, a basic helix-loop-helix (bHLH) transcription factor and a direct target of Notch signaling, is specifically overexpressed in breast cancer. HEYL represses TGF-β activity by binding to TGF-β-activated Smads. HeyL-/- mice have defective mammary gland development with fewer terminal end buds. On the other hand, HeyL transgenic mice show accelerated mammary gland epithelial proliferation and 24% of multiparous mice develop mammary gland cancer. Therefore, repression of TGF-β signaling by Notch acting through HEYL may promote initiation of breast cancer.