RESUMEN
BACKGROUND: Breast cancer comprises clinically and molecularly distinct tumor subgroups that differ in cell histology and biology and show divergent clinical phenotypes that impede phase III trials, such as those utilizing cathepsin K inhibitors. Here we correlate the epithelial-mesenchymal-like transition breast cancer cells and cathepsin K secretion with activation and aggregation of platelets. Cathepsin K is up-regulated in cancer cells that proteolyze extracellular matrix and contributes to invasiveness. Although proteolytically activated receptors (PARs) are activated by proteases, the direct interaction of cysteine cathepsins with PARs is poorly understood. In human platelets, PAR-1 and -4 are highly expressed, but PAR-3 shows low expression and unclear functions. METHODS: Platelet aggregation was monitored by measuring changes in turbidity. Platelets were immunoblotted with anti-phospho and total p38, Src-Tyr-416, FAK-Tyr-397, and TGFß monoclonal antibody. Activation was measured in a flow cytometer and calcium mobilization in a confocal microscope. Mammary epithelial cells were prepared from the primary breast cancer samples of 15 women with Luminal-B subtype to produce primary cells. RESULTS: We demonstrate that platelets are aggregated by cathepsin K in a dose-dependent manner, but not by other cysteine cathepsins. PARs-3 and -4 were confirmed as the cathepsin K target by immunodetection and specific antagonists using a fibroblast cell line derived from PARs deficient mice. Moreover, through co-culture experiments, we show that platelets activated by cathepsin K mediated the up-regulation of SHH, PTHrP, OPN, and TGFß in epithelial-mesenchymal-like cells from patients with Luminal B breast cancer. CONCLUSIONS: Cathepsin K induces platelet dysfunction and affects signaling in breast cancer cells.
Asunto(s)
Plaquetas/metabolismo , Neoplasias de la Mama/metabolismo , Catepsina K/metabolismo , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales , Animales , Plaquetas/efectos de los fármacos , Neoplasias de la Mama/sangre , Neoplasias de la Mama/patología , Calcio/metabolismo , Catepsina K/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Femenino , Proteínas Hedgehog/metabolismo , Humanos , Hidrólisis , Ligandos , Proteínas de la Membrana/antagonistas & inhibidores , Ratones , Fosforilación , Activación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Proteolisis , Receptores de Trombina/antagonistas & inhibidores , Trombina/metabolismo , Trombina/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
Cancer progression is associated with an evolving tissue interface of direct epithelial-tumor microenvironment interactions. In biopsies of human breast tumors, extensive alterations in molecular pathways are correlated with cancer staging on both sides of the tumor-stroma interface. These interactions provide a pivotal paracrine signaling to induce malignant phenotype transition, the epithelial-mesenchymal transition (EMT). We explored how the direct contact between platelets-fibrin bundles primes metastasis using platelet-rich plasma (PRP) as a source of growth factors and mimics the provisional fibrin matrix between actively growing breast cancer cells and the tumor stroma. We have demonstrated PRP functions, modulating cell proliferation that is tumor-subtype and cancer cell-type-specific. Epithelial and stromal primary cells were prepared from breast cancer biopsies from 21 women with different cancer subtypes. Cells supplemented with PRP were immunoblotted with anti-phospho and total Src-Tyr-416, FAK-Try-925, E-cadherin, N-cadherin, TGF-ß, Smad2, and Snail monoclonal antibodies. Breast tumor cells from luminal B and HER2 subtypes showed the most malignant profiles and the expression of thrombin and other classes of proteases at levels that were detectable through FRET peptide libraries. The angiogenesis process was investigated in the interface obtained between platelet-fibrin-breast tumor cells co-cultured with HUVEC cells. Luminal B and HER2 cells showed robust endothelial cell capillary-like tubes ex vivo. The studied interface contributes to the attachment of endothelial cells, provides a source of growth factors, and is a solid substrate. Thus, replacement of FBS supplementation with PRP supplementation represents an efficient and simple approach for mimicking the real multifactorial tumor microenvironment.