RESUMO
Platinum (PT)-resistant Epithelial Ovarian Cancer (EOC) grows as a metastatic disease, disseminating in the abdomen and pelvis. Very few options are available for PT-resistant EOC patients, and little is known about how the acquisition of PT-resistance mediates the increased spreading capabilities of EOC. Here, using isogenic PT-resistant cells, genetic and pharmacological approaches, and patient-derived models, we report that Integrin α6 (ITGA6) is overexpressed by PT-resistant cells and is necessary to sustain EOC metastatic ability and adhesion-dependent PT-resistance. Using in vitro approaches, we showed that PT induces a positive loop that, by stimulating ITGA6 transcription and secretion, contributes to the formation of a pre-metastatic niche enabling EOC cells to disseminate. At molecular level, ITGA6 engagement regulates the production and availability of insulin-like growth factors (IGFs), over-stimulating the IGF1R pathway and upregulating Snail expression. In vitro data were recapitulated using in vivo models in which the targeting of ITGA6 prevents PT-resistant EOC dissemination and improves PT-activity, supporting ITGA6 as a promising druggable target for EOC patients.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Integrina alfa6 , Neoplasias Ovarianas , Regulação para Cima , Humanos , Integrina alfa6/metabolismo , Integrina alfa6/genética , Feminino , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/metabolismo , Regulação para Cima/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Platina/farmacologia , Platina/uso terapêutico , Carcinoma Epitelial do Ovário/tratamento farmacológico , Carcinoma Epitelial do Ovário/genética , Carcinoma Epitelial do Ovário/metabolismo , Carcinoma Epitelial do Ovário/patologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacosRESUMO
Characterization of serum glycoprotein N-glycans with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) in positive-ion mode needs a derivatization step to stabilize and neutralize the negative charge on sialic acids. The acidic sugars are attached to the end of glycoproteins, glycolipids or gangliosides. Here, we present a method for sialic acid stabilization via modification based on derivatization of carboxylic acid group activated with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) with methylamine. DMTMM substitutes in many processes N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide (EDC/NHS) as activation reagent due to its better performance and higher stability in water. Glycosylated proteins are used as solid phase support for glycan derivatization and purification from excess of derivatization reagents. We evaluated our glycan analysis method in murine sera and intestinal lavages. The stabilization of sialic acid enables a complete conservation of the glycan structures, in contrast to other methods where sialic acids are partially lost. In BALB/c mouse sera, we detected predominantly mono- and di-sialylated N-glycans with mostly N-Glycolylneuraminic acid (Neu5Gc) and only trace amounts of N-Acetyl neuraminic acid (Neu5Ac). BALB/c mouse intestinal lavages glycoproteins contained asialo N-glycans. DMTMM-mediated methylamidation of N-glycans for MALDI mass spectrometry analysis is a fast and cheap method for structurally conserved glycan derivatization.
