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

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

In normal melanocytes, TGF-β signaling has a cytostatic effect. However, in primary melanoma cells, TGF-β-induced cytostasis is diminished, thus allowing melanoma growth. Later, a second phase of TGF-β signaling supports melanoma EMT-like changes, invasion and metastasis. In parallel with these "present-absent-present" TGF-β signaling phases, cell surface protein EWI motif-containing protein 2 (EWI-2 or IgSF8) is "absent-present-absent" in melanocytes, primary melanoma, and metastatic melanoma, respectively, suggesting that EWI-2 may serve as a negative regulator of TGF-β signaling. Using melanoma cell lines and melanoma short-term cultures, we performed RNAi and overexpression experiments and found that EWI-2 negatively regulates TGF-β signaling and its downstream events including cytostasis (in vitro and in vivo), EMT-like changes, cell migration, CD271-dependent invasion, and lung metastasis (in vivo). When EWI-2 is present, it associates with cell surface tetraspanin proteins CD9 and CD81 - molecules not previously linked to TGF-β signaling. Indeed, when associated with EWI-2, CD9 and CD81 are sequestered and have no impact on TβR2-TβR1 association or TGF-β signaling. However, when EWI-2 is knocked down, CD9 and CD81 become available to provide critical support for TβR2-TβR1 association, thus markedly elevating TGF-β signaling. Consequently, all of those TGF-β-dependent functions specifically arising due to EWI-2 depletion are reversed by blocking or depleting cell surface tetraspanin proteins CD9 or CD81. These results provide new insights into regulation of TGF-β signaling in melanoma, uncover new roles for tetraspanins CD9 and CD81, and strongly suggest that EWI-2 could serve as a favorable prognosis indicator for melanoma patients.

Topics: Animals; Antigens, CD; Benzamides; Cell Line, Tumor; Cell Movement; Cell Proliferation; Dioxoles; HEK293 Cells; Humans; Melanoma; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, SCID; Neoplasm Invasiveness; Neoplasm Transplantation; Nerve Tissue Proteins; Prognosis; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Nerve Growth Factor; Receptors, Transforming Growth Factor beta; RNA Interference; RNA, Small Interfering; Signal Transduction; Tetraspanin 24; Tetraspanin 28; Tetraspanin 29; Transforming Growth Factor beta; Transplantation, Heterologous

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