Exploring association of melanoma-specific Bcl-xL with tumor immune microenvironment

Resumo

Melanoma represents the deadliest form of skin cancer. Features of melanoma are the poorly responsiveness and to standard chemotherapeutics and the high aggressiveness. Molecular mechanisms leading to melanoma development and progression are the focus of intense investigation, aimed at understanding its pathobiology and at developing new treatment strategies. A number of driver mutations have been identified and the most common mutations affect signaling of the Ras/Raf/mitogen- activated protein kinase pathways. In addition, the Bcl-2 family network is found deregulated in melanoma. Among Bcl-2 family proteins, B-cell lymphoma-extra Large (Bcl-xL) has emerged as multifaceted factor acting not only as a canonical anti-apoptotic factor, but also as a promoter of tumor progression. In melanoma Bcl-Xl has been found deregulated. In the last years, the importance of the microenvironment for the tumor progression and response to therapy was established, underlying the importance of a continuous crosstalk between tumor cells and microenvironment, through secretion of tumorigenic mediators. In this scenario, tumor-associated macrophages (TAM) play a pivotal role affecting the nature of the tumor microenvironment. TAM infiltration is directly correlated to melanoma thickness and to increased angiogenesis and micro- vessel density, through modulation of tumor pro-inflammatory factors. Moreover, elevated number of TAMs in the tumor microenvironment is often correlated with poor prognosis in melanoma. Thus, macrophages represent promising therapeutic targets, and their depletion can represent an effective therapeutic intervention in the management of primary and metastatic melanoma. Macrophages can be classified in “classically activated macrophages” or M1, and “alternatively activated macrophages” or M2. TAM display an M2-like phenotype and are closely associated partners of malignant cells for migration/invasion, angiogenesis, immune suppression, metastasis formation and drug resistance. To prove the effect induced by the overexpression of Bcl-xL in the regulation of the tumor microenvironment human melanoma cells silencing or overexpressing Bcl-xL protein, THP-1 monocytic cells and monocyte-derived macrophages were used in this study. Protein array and specific neutralizing antibodies were used to analyse cytokines and chemokines secreted by melanoma cells. qRT-PCR, ELISA and Western Blot analyses were used to evaluate macrophage polarization markers and protein expression levels. Transwell chambers were used to evaluate migration of THP-1 and monocyte-derived macrophages. Mouse and zebrafish models were used to evaluate the ability of melanoma cells to recruit and polarize macrophages in vivo. We demonstrated that melanoma cells overexpressing Bcl-xL recruit macrophages at the tumor site and induce a M2 phenotype. In addition, we identified that interleukin-8 and interleukin-1β cytokines are involved in macrophage polarization, and the chemokine CCL5/RANTES in the macrophage’s recruitment at the tumor site. We also found that all these Bcl-xL-induced factors are regulated in a NF-B dependent manner in human and zebrafish melanoma models. Our findings confirmed the pro-tumoral function of Bcl-xL in melanoma through its effects on macrophages demonstrating the ability of Bcl-xL to regulate macrophages polarization and recruitment via the secretion of specific factors, establishing tumor microenvironmental conditions that favour melanoma development.