ABSTRACT
Melanoma is the most lethal form of skin cancer and successful treatment of metastatic melanoma remains challenging. BRAF/MEK inhibitors only show a temporary benefit due to rapid occurrence of resistance, whereas immunotherapy is mainly effective in selected subsets of patients. Thus, there is a need to identify new targets to improve treatment of metastatic melanoma. To this extent, we searched for markers that are elevated in melanoma and are under regulation of potentially druggable enzymes. Here, we show that the pro-proliferative transcription factor FOXM1 is elevated and activated in malignant melanoma. FOXM1 activity correlated with expression of the enzyme Pin1, which we found to be indicative of a poor prognosis. In functional experiments, Pin1 proved to be a main regulator of FOXM1 activity through MEK-dependent physical regulation during the cell cycle. The Pin1-FOXM1 interaction was enhanced by BRAF(V600E), the driver oncogene in the majority of melanomas, and in extrapolation of the correlation data, interference with\ Pin1 in BRAF(V600E)-driven metastatic melanoma cells impaired both FOXM1 activity and cell survival. Importantly, cell-permeable Pin1-FOXM1-blocking peptides repressed the proliferation of melanoma cells in freshly isolated human metastatic melanoma ex vivo and in three-dimensional-cultured patient-derived melanoids. When combined with the BRAF(V600E)-inhibitor PLX4032 a robust repression in melanoid viability was obtained, establishing preclinical value of patient-derived melanoids for prognostic use of drug sensitivity and further underscoring the beneficial effect of Pin1-FOXM1 inhibitory peptides as anti-melanoma drugs. These proof-of-concept results provide a starting point for development of therapeutic Pin1-FOXM1 inhibitors to target metastatic melanoma.
Subject(s)
Forkhead Box Protein M1/genetics , Melanoma/drug therapy , NIMA-Interacting Peptidylprolyl Isomerase/genetics , Proto-Oncogene Proteins B-raf/genetics , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Humans , Indoles/administration & dosage , Melanoma/genetics , Melanoma/pathology , Molecular Targeted Therapy , Mutation , Neoplasm Metastasis , Protein Kinase Inhibitors/administration & dosage , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Signal Transduction , Sulfonamides/administration & dosage , VemurafenibABSTRACT
Extension of an inverted repeat transgene cassette, containing partial nucleoprotein (N) gene sequences from four different tomato-infecting Tospovirus spp. with a partial N gene sequence from the tomato strain of Tomato yellow ring virus (TYRV-t), renders transgenic Nicotiana benthamiana plants additionally resistant to this strain but not to the soybean strain of this Tospovirus sp. (TYRV-s), both strains exhibiting 14.4% nucleotide sequence divergence in their N genes. Surprisingly, coinoculation of the TYRV-t-resistant transgenic lines with both TYRV-t and TYRV-s resulted in rescue of the former. Mass-spectrometric analysis of the viral ribonucleocapsids accumulating in the transgenic plants showed the presence of the N proteins of both strains excluding hetero-encapsidation as rescue mechanism and indicating suppression of TYRV-t N gene transcript breakdown by RNA interference. Prior (Potato virus X [PVX]-vector-mediated) expression of the TYRV-s silencing suppressor (NS(s)) gene also allowed TYRV-t to break the resistance. This phenomenon was also observed when the homologous (TYRV-t) NS(s) gene was provided from a PVX replicon, demonstrating that TYRV can break RNA-mediated host resistance upon a priori expression of its NS(s) protein. Remarkably, mixed inoculation of TYRV-t with other Tospovirus spp. or nonrelated viruses did not result in resistance breaking, indicating that the rescuing activity of NS(s)-though based on suppressing RNA silencing-is species-dependent.
