RESUMEN
BRAFV600E is the most frequent oncogenic mutation identified in papillary thyroid cancer (PTC). In PTC patients who do not respond to standard treatment, BRAF inhibitors are currently tested as alternative strategies. However, as observed for other targeted therapies, patients eventually develop drug resistance. The mechanisms of BRAF inhibitors response are still poorly understood in a thyroid cancer (TC) context. In this study, we investigated in BRAFV600E mutated TC cell lines the effects of Vemurafenib and Dabrafenib, two BRAF inhibitors currently used in a clinical setting. We assessed cell proliferation, and the expression and activity of the thyroid function related transporter NIS following the treatment with BRAF inhibitors. In addition, we investigated the global gene expression by microarray, the relevant modulated biological processes by gene set enrichment analysis (GSEA), and TC specific gene signatures related to MAPK pathway activation, thyroid differentiation, and transcriptional profile associated with BRAFV600E or RAS mutation. We found that both inhibitors induce antiproliferative and redifferentiative effects on TC cells, as well as a rewiring of the MAPK pathway related to RAS signaling. Our results suggest a possible mechanism of drug response to the BRAF inhibitors Vemurafenib or Dabrafenib, supporting very recent findings in TC patients treated with targeted therapies.
Asunto(s)
Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Neoplasias de la Tiroides/metabolismo , Proteínas ras/metabolismo , Biomarcadores de Tumor , Línea Celular Tumoral , Biología Computacional/métodos , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Mutación , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Neoplasias de la Tiroides/tratamiento farmacológico , Neoplasias de la Tiroides/etiología , Neoplasias de la Tiroides/patología , TranscriptomaRESUMEN
INTRODUCTION: Serine/threonine kinase 11 (LKB1/STK11) is one of the most mutated genes in NSCLC accounting for approximately one-third of cases and its activity is impaired in approximately half of KRAS-mutated NSCLC. At present, these patients cannot benefit from any specific therapy. METHODS: Through CRISPR/Cas9 technology, we systematically deleted LKB1 in both wild-type (WT) and KRAS-mutated human NSCLC cells. By using these isogenic systems together with genetically engineered mouse models we investigated the cell response to ERK inhibitors both in vitro and in vivo. RESULTS: In all the systems used here, the loss of LKB1 creates vulnerability and renders these cells particularly sensitive to ERK inhibitors both in vitro and in vivo. The same cells expressing a WT LKB1 poorly respond to these drugs. At the molecular level, in the absence of LKB1, ERK inhibitors induced a marked inhibition of p90 ribosomal S6 kinase activation, which in turn abolished S6 protein activation, promoting the cytotoxic effect. CONCLUSIONS: This work shows that ERK inhibitors are effective in LKB1 and LKB1/KRAS-mutated tumors, thus offering a therapeutic strategy for this prognostically unfavorable subgroup of patients. Because ERK inhibitors are already in clinical development, our findings could be easily translatable to the clinic. Importantly, the lack of effect in cells expressing WT LKB1, predicts that treatment of LKB1-mutated tumors with ERK inhibitors should have a favorable toxicity profile.
Asunto(s)
Antineoplásicos , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Antineoplásicos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Mutación , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Thyroid carcinoma (TC) comprises several histotypes with different aggressiveness, from well (papillary carcinoma, PTC) to less differentiated forms (poorly differentiated and anaplastic thyroid carcinoma, PDTC and ATC, respectively). Previous reports have suggested a functional role for cancer-associated fibroblasts (CAFs) or senescent TC cells in the progression of PTC. In this study, we investigated the presence of CAFs and senescent cells in proprietary human TCs including PTC, PDTC, and ATC. Screening for the driving lesions BRAFV600E and N/H/KRAS mutations, and gene fusions was also performed to correlate results with tumor genotype. In samples with unidentified drivers, transcriptomic profiles were used to establish a BRAF- or RAS-like molecular subtype based on a gene signature derived from The Cancer Genome Atlas. By using immunohistochemistry, we found co-occurrence of stromal CAFs and senescent TC cells at the tumor invasive front, where deposition of collagen (COL1A1) and expression of lysyl oxidase (LOX) enzyme were also detected, in association with features of local invasion. Concurrent high expression of CAFs and of the senescent TC cells markers, COL1A1 and LOX was confirmed in different TC histotypes in proprietary and public gene sets derived from Gene Expression Omnibus (GEO) repository, and especially in BRAF mutated or BRAF-like tumors. In this study, we show that CAFs and senescent TC cells co-occur in various histotypes of BRAF-driven thyroid tumors and localize at the tumor invasive front.
RESUMEN
Copper, the highly toxic micronutrient, plays two essential roles: it is a catalytic and structural cofactor for Cu-dependent enzymes, and it acts as a secondary messenger. In the cells, copper is imported by CTR1 (high-affinity copper transporter 1), a transmembrane high-affinity copper importer, and DMT1 (divalent metal transporter). In cytosol, enzyme-specific chaperones receive copper from CTR1 C-terminus and deliver it to their apoenzymes. DMT1 cannot be a donor of catalytic copper because it does not have a cytosol domain which is required for copper transfer to the Cu-chaperons that assist the formation of cuproenzymes. Here, we assume that DMT1 can mediate copper way required for a regulatory copper pool. To verify this hypothesis, we used CRISPR/Cas9 to generate H1299 cell line with CTR1 or DMT1 single knockout (KO) and CTR1/DMT1 double knockout (DKO). To confirm KOs of the genes qRT-PCR were used. Two independent clones for each gene were selected for further studies. In CTR1 KO cells, expression of the DMT1 gene was significantly increased and vice versa. In subcellular compartments of the derived cells, copper concentration dropped, however, in nuclei basal level of copper did not change dramatically. CTR1 KO cells, but not DMT1 KO, demonstrated reduced sensitivity to cisplatin and silver ions, the agents that enter the cell through CTR1. Using single CTR1 and DMT1 KO, we were able to show that both, CTR1 and DMT1, provided the formation of vital intracellular cuproenzymes (SOD1, COX), but not secretory ceruloplasmin. The loss of CTR1 resulted in a decrease in the level of COMMD1, XIAP, and NF-κB. Differently, the DMT1 deficiency induced increase of the COMMD1, HIF1α, and XIAP levels. The possibility of using CTR1 KO and DMT1 KO cells to study homeodynamics of catalytic and signaling copper selectively is discussed.