4-(5-benzo(1-3)dioxol-5-yl-4-pyridin-2-yl-1h-imidazol-2-yl)benzamide and Skin-Neoplasms

Topics: Adult; Aged; Animals; Apoptosis; Benzamides; Cadherins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Dioxoles; Epithelial-Mesenchymal Transition; Female; Humans; Intracellular Signaling Peptides and Proteins; Male; Melanoma; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Neoplasm Invasiveness; Prognosis; Signal Transduction; Skin Neoplasms; Smad4 Protein; Transforming Growth Factor beta1; Vimentin

Recent data implicate elevated transforming growth factor-β (TGFβ) signalling in BRAF inhibitor drug-resistance mechanisms, but the potential for targeting TGFβ signalling in cases of advanced melanoma has not been investigated. We show that mutant BRAFV600E confers an intrinsic dependence on TGFβ/TGFβ receptor 1 (TGFBR1) signalling for clonogenicity of murine melanocytes. Pharmacological inhibition of the TGFBR1 blocked the clonogenicity of human mutant BRAF melanoma cells through SMAD4-independent inhibition of mitosis, and also inhibited metastasis in xenografted zebrafish. When investigating the therapeutic potential of combining inhibitors of mutant BRAF and TGFBR1, we noted that unexpectedly, low-dose PLX-4720 (a vemurafenib analogue) promoted proliferation of drug-naïve melanoma cells. Pharmacological or pharmacogenetic inhibition of TGFBR1 blocked growth promotion and phosphorylation of SRC, which is frequently associated with vemurafenib-resistance mechanisms. Importantly, vemurafenib-resistant patient derived cells retained sensitivity to TGFBR1 inhibition, suggesting that TGFBR1 could be targeted therapeutically to combat the development of vemurafenib drug-resistance.

Topics: Animals; Animals, Genetically Modified; Antineoplastic Agents; Benzamides; Cell Line, Tumor; Cell Proliferation; Dioxoles; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Humans; Indoles; Melanocytes; Melanoma; Mice, Nude; Mitosis; Mutation; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins B-raf; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; RNA Interference; Signal Transduction; Skin Neoplasms; Smad4 Protein; Sulfonamides; Time Factors; Transfection; Transforming Growth Factor beta1; Vemurafenib; Xenograft Model Antitumor Assays; Zebrafish

CRIPTO-1 (CR-1) is involved in the pathogenesis and progression of human carcinoma of different histological origin. In this study we addressed the expression and the functional role of CR-1 in cutaneous melanoma.. Expression of CR-1 protein in melanomas and melanoma cell lines was assessed by immunohistochemistry, western blotting and/or flow cytometry. Levels of mRNA were evaluated by real-time PCR. Invasion assays were performed in Matrigel-coated modified Boyden chambers.. Expression of CR-1 protein and/or mRNA was found in 16 out of 37 primary human cutaneous melanomas and in 12 out of 21 melanoma cell lines. Recombinant CR-1 protein activated in melanoma cells c-Src and, at lesser extent, Smad signalling. In addition, CR-1 significantly increased the invasive ability of melanoma cells that was prevented by treatment with either the ALK4 inhibitor SB-431542 or the c-Src inhibitor saracatinib (AZD0530). Anti-CR-1 siRNAs produced a significant inhibition of the growth and the invasive ability of melanoma cells. Finally, a close correlation was found in melanoma cells between the levels of expression of CR-1 and the effects of saracatinib on cell growth.. These data indicate that a significant fraction of cutaneous melanoma expresses CR-1 and that this growth factor is involved in the invasion and proliferation of melanoma cells.

Topics: Activin Receptors, Type I; Benzamides; Benzodioxoles; Blotting, Western; Cell Adhesion; Cell Movement; Cell Proliferation; CSK Tyrosine-Protein Kinase; Dioxoles; Flow Cytometry; GPI-Linked Proteins; Humans; Immunoenzyme Techniques; Intercellular Signaling Peptides and Proteins; Melanoma; Neoplasm Invasiveness; Neoplasm Proteins; Protein-Tyrosine Kinases; Quinazolines; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Skin Neoplasms; Smad Proteins; src-Family Kinases; Tumor Cells, Cultured

Pharmacological inhibitors of the transforming growth factor β (TGFβ) type I receptor (ALK5) have shown promise in blocking growth of xenotransplanted cancer cell lines but the effect on a multistage cancer model is not known. To test this, we treated mouse skin with SB431542 (SB), a well-characterized ALK5 inhibitor, during a two-stage skin carcinogenesis assay. Topical SB significantly reduced the total number, incidence and size of papillomas compared with 12-O-tetradecanoylphorbol 13-acetate (TPA) promotion alone, and this was linked to increased epidermal apoptosis, decreased proliferation and decreased cutaneous inflammation during promotion. In contrast, the frequency of conversion to squamous cell carcinoma (SCC) was 2-fold higher in papillomas treated with SB. Although there was no difference in tumor cell proliferation in early premalignant lesions, those that formed after SB treatment exhibited reduced squamous differentiation and an altered inflammatory microenvironment similar to SCC. In an inducible epidermal RAS transgenic model, treatment with SB enhanced proliferation and cutaneous inflammation in skin but decreased expression of keratin 1 and increased expression of simple epithelial keratin 18, markers of premalignant progression. In agreement with increased frequency of progression in the multistage model, SB treatment resulted in increased tumor formation with a more malignant phenotype following long-term RAS induction. In contrast to the current paradigm for TGFβ in carcinogenesis, these results demonstrate that cutaneous TGFβ signaling enables promotion of benign tumors but suppresses premalignant progression through context-dependent regulation of epidermal homeostasis and inflammation.

Topics: Animals; Benzamides; Dioxoles; Disease Progression; Genes, ras; Mice; Papilloma; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Skin Neoplasms; Smad2 Protein; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta1