RESUMEN
Activating BRAF mutations occurs in 50-60% of malignant melanomas. Although initially treatable, the development of resistance to BRAF-targeted therapies (BRAFi) is a major challenge and limits their efficacy. We have previously shown that the BRAFV600E signaling pathway mediates the expression of EZH2, an epigenetic regulator related to melanoma progression and worse overall survival. Therefore, we wondered whether inhibition of EZH2 would be a way to overcome resistance to vemurafenib. We found that the addition of an EZH2 inhibitor to vemurafenib improved the response of melanoma cells resistant to BRAFi with regard to decreased viability, cell-cycle arrest and increased apoptosis. By next-generation sequencing, we revealed that the combined inhibition of BRAF and EZH2 dramatically suppresses pathways of mitosis and cell cycle. This effect was linked to the downregulation of Polo-kinase 1 (PLK1), a key regulator of cell cycle and proliferation. Subsequently, when we inhibited PLK1, we found decreased cell viability of melanoma cells resistant to BRAFi. When we inhibited both BRAF and PLK1, we achieved an improved response of BRAFi-resistant melanoma cells, which was comparable to the combined inhibition of BRAF and EZH2. These results thus reveal that targeting EZH2 or its downstream targets, such as PLK1, in combination with BRAF inhibitors are potential novel therapeutic options in melanomas with BRAF mutations.
Asunto(s)
Resistencia a Antineoplásicos , Melanoma , Neoplasias Cutáneas , Humanos , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Indoles/farmacología , Indoles/uso terapéutico , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/patología , Mutación , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , Vemurafenib/farmacología , Vemurafenib/uso terapéutico , Quinasa Tipo Polo 1RESUMEN
In IgA vasculitis (IgAV) perivascular deposition of IgA1 immune complexes (IgA-ICs) is traditionally considered the fundamental trigger for polymorphonuclear neutrophil (PMN)-mediated damage. We propose that IgA-IC deposition, although mandatory, is not sufficient alone for IgAV. Serum IgA-IC levels and IgA-IC binding to PMNs were quantified in IgAV patients and controls. Activation of PMNs was evaluated by neutrophil extracellular trap (NET) release, adherence, and cytotoxicity assays and in a flow system to mirror conditions at postcapillary venules. In vitro results were related to findings in biopsies and a mouse vasculitis model. During acute IgAV flares we observed elevated serum levels of IgA-ICs and increased IgA-IC binding to circulating PMNs. This IgA-IC binding primed PMNs with consequent lowering of the threshold for NETosis, demonstrated by significantly higher release of NETs from PMNs activated in vitro and PMNs from IgAV patients with flares compared with surface IgA-negative PMNs after flares. Blocking of FcαRI abolished these effects, and complement was not essential. In the flow system, marked NETosis only occurred after PMNs had adhered to activated endothelial cells. IgA-IC binding enhanced this PMN tethering and consequent NET-mediated endothelial cell injury. Reflecting these in vitro findings, we visualized NETs in close proximity to endothelial cells and IgA-coated PMNs in tissue sections of IgAV patients. Inhibition of NET formation and knockout of myeloperoxidase in a murine model of IC vasculitis significantly reduced vessel damage in vivo. Binding of IgA-ICs during active IgAV primes PMNs and promotes vessel injury through increased adhesion of PMNs to the endothelium and enhanced NETosis.
Asunto(s)
Vasculitis por IgA , Vasculitis , Animales , Complejo Antígeno-Anticuerpo/metabolismo , Células Endoteliales , Inmunoglobulina A , Ratones , Neutrófilos , Peroxidasa/metabolismoRESUMEN
The key players in different chronic inflammatory skin diseases are cytokines belonging to the IL-17 group, IL-17 receptors and a T helper cell population, Th17 cells. Successful therapeutic strategies that target either IL-17 or the major IL-17 receptor IL-17RA have confirmed the immune-pathogenic pathway. To study the IL-17-ligand - receptor axis at the molecular level, a number of cutaneous cell types from healthy human subjects has been cultured and analyzed for the expression of IL-17 receptors. IL-17RA was the most abundantly expressed receptor type in keratinocytes, epidermal stem cells, fibroblasts, mesenchymal stem cells, hemo- and lymphovascular endothelial cells. IL-17RC and IL-17RD showed moderate expression, while the genes for IL-17RB and IL-17RE were poorly expressed. In none of the investigated cell types, IL-17 ligands caused an increased expression level of the five receptor types in time- and dose-dependent experiments. No evidence for IL-17A, -C, -E or -F induced signal transduction cascades could be obtained by a qRT-PCR and western blot analyses. Further studies are necessary to identify relevant co-stimulating factors from IL-17 subtypes under physiological and pathophysiological conditions.
Asunto(s)
Queratinocitos/efectos de los fármacos , Receptores de Interleucina-17/metabolismo , Células Th17/metabolismo , Células Cultivadas , Humanos , Interleucina-17/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal , Piel/metabolismoRESUMEN
Many melanomas are associated with activating BRAF mutation. Targeted therapies by inhibitors of BRAF and MEK (BRAFi, MEKi) show marked antitumor response, but become limited by drug resistance. The mechanisms for this are not fully revealed, but include miRNA. Wishing to improve efficacy of BRAFi and knowing that certain miRNAs are linked to resistance to BRAFi, we wanted to focus on miRNAs exclusively associated with response to BRAFi. We found increased expression of miR-129-5p during BRAFi treatment of BRAF- mutant melanoma cells. Parallel to emergence of resistance we observed mir-129-5p expression to become suppressed by BRAF/EZH2 signaling. In functional analyses we revealed that miR-129-5p acts as a tumor suppressor as its overexpression decreased cell proliferation, improved treatment response and reduced viability of BRAFi resistant melanoma cells. By protein expression analyses and luciferase reporter assays we confirmed SOX4 as a direct target of mir-129-5p. Thus, modulation of the miR-129-5p-SOX4 axis could serve as a promising novel strategy to improve response to BRAFi in melanoma.