Purpose: To investigate the roles of melanoma-associated macrophages in melanoma resistance to BRAF inhibitors (BRAFi). Experimental Design: An in vitro macrophage and melanoma cell coculture system was used to investigate whether macrophages play a role in melanoma resistance to BRAFi. The effects of macrophages in tumor resistance were examined by proliferation assay, cell death assay, and Western blot analyses. Furthermore, two mouse preclinical models were used to validate whether targeting macrophages can increase the antitumor activity of BRAFi. Finally, the number of macrophages in melanoma tissues was examined by immunohistochemistry. Results: We demonstrate that in BRAF-mutant melanomas, BRAFi paradoxically activate the mitogen-activated protein kinase (MAPK) pathway in macrophages to produce VEGF, which reactivates the MAPK pathway and stimulates cell growth in melanoma cells. Blocking the MAPK pathway or VEGF signaling then reverses macrophage-mediated resistance. Targeting macrophages increases the antitumor activity of BRAFi in mouse and human tumor models. The presence of macrophages in melanomas predicts early relapse after therapy. Conclusions: Our findings demonstrate that macrophages play a critical role in melanoma resistance to BRAFi, suggesting that targeting macrophages will benefit patients with BRAF-mutant melanoma. Clin Cancer Res; 21(7); 1652–64. ©2015 AACR.

Summary: The presence of tumor‐associated macrophages (TAMs) in melanomas is correlated with a poor clinical prognosis. However, there is limited information on the characteristics and biological activities of human TAMs in melanomas. In this study, we developed an in vitro method to differentiate human monocytes to macrophages using modified melanoma‐conditioned medium (MCM). We demonstrate that factors from MCM‐induced macrophages (MCMI‐Mφ) express both M1‐Mφ and M2‐Mφ markers and inhibit melanoma‐specific T‐cell proliferation. Furthermore, microarray analyses reveal that the majority of genes up‐regulated in MCMI‐Mφ are associated with tumor invasion. The most strikingly up‐regulated genes are CCL2 and MMP‐9. Consistent with this, blockade of both CCL‐2 and MMPs diminish MCMI‐Mφ‐induced melanoma invasion. Finally, we demonstrated that both MCMI‐Mφ and in vivo TAMs express the pro‐invasive, melanoma‐associated gene, glycoprotein non‐metastatic melanoma protein B. Our study provides a framework for understanding the mechanisms of cross‐talk between TAMs and melanoma cells within the tumor microenvironment.

CD7 is a cell-surface molecule, expressed on T lymphocytes and NK cells, which functions as a costimulatory receptor for T cell proliferation. SECTM1 has been proposed as a ligand for CD7. However, the expression pattern of this molecule in human immune cells and role in human T cell function remain unclear. In the present study, using human rSECTM1, we demonstrate that SECTM1 strongly costimulates CD4 and CD8 T cell proliferation and induces IFN-γ production, likely via a CD7-dependent mechanism. In addition, SECTM1 synergizes with suboptimal anti-CD28 to strongly augment T cell functions. We found a robust induction of IL-2 production when SECTM1 and anti-CD28 signals were present with TCR ligation. Furthermore, addition of SECTM1 into a MLR significantly enhanced proliferation of alloantigen-activated T cells, whereas blockade of SECTM1 inhibited T cell proliferation in a two-way MLR assay. Simultaneously blocking the effect of SECTM1, along with CTLA-4/Fc, diminishes two-way MLR. Finally, we demonstrated that expression of SECTM1 is not detected in monocytes and imMoDCs at the protein level. However, it is strongly induced by IFN-γ in monocytes and imMoDCs, and this induction is STAT1-dependent. These results indicate that SECTM1 is a broadly expressed, IFN-γ-inducible molecule, which functions as a potent costimulatory ligand for T cell activation and is synergistic with anti-CD28.

Tumor-associated macrophages (TAMs) have essential roles in tumor progression and metastasis. Tumor cells recruit myeloid progenitors and monocytes to the tumor site, where they differentiate into TAMs; however, this process is not well studied in humans. Here we show that human CD7, a T-cell and NK cell receptor, is highly expressed by monocytes and macrophages. Expression of CD7 decreases in M-CSF-differentiated macrophages and in melanoma-conditioned medium–induced macrophages (MCMI/Mφ) in comparison to monocytes. A ligand for CD7, SECTM1 (secreted and transmembrane protein 1), is highly expressed in many tumors, including melanoma cells. We show that SECTM1 binds to CD7 and significantly increases monocyte migration by activation of the PI3K (phosphatidylinositol 3′-kinase) pathway. In human melanoma tissues, tumor-infiltrating macrophages expressing CD7 are present. These melanomas, with CD7-positive inflammatory cell infiltrations, frequently highly express SECTM1, including an N-terminal, soluble form, which can be detected in the sera of metastatic melanoma patients but not in normal sera. Taken together, our data demonstrate that CD7 is present on monocytes and tumor macrophages and that its ligand, SECTM1, is frequently expressed in corresponding melanoma tissues, possibly acting as a chemoattractant for monocytes to modulate the melanoma microenvironment.

Recent studies report that the polarity gene myelin and lymphocyte protein 2 (MAL2), is overexpressed in multiple human carcinomas largely at the transcript level. Because chromosome 8q24 amplification (where MAL2 resides) is associated with hepatocellular- and cholangio-carcinomas, we examined MAL2 protein expression in these human carcinoma lesions and adjacent benign tissue using immunohistochemistry. For comparison, we analyzed renal cell carcinomas that are not associated with chromosome 8q24 amplification. Surprisingly, we found that MAL2 protein levels were decreased in the malignant tissues compared to benign in all three carcinomas, suggesting MAL2 expression may be anti-oncogenic. Consistent with this conclusion, we determined that endogenously overexpressed MAL2 in HCC-derived Hep3B cells or exogenously expressed MAL2 in hepatoma-derived Clone 9 cells (that lack endogenous MAL2) promoted actin-based protrusion formation with a reciprocal decrease in invadopodia. MAL2 overexpression also led to decreased cell migration, invasion and proliferation (to a more modest extent) while loss of MAL2 expression reversed the phenotypes. Mutational analysis revealed that a putative Ena/VASP homology 1 recognition site confers the MAL2-phenotype suggesting its role in tumor suppression involves actin remodeling. To reconcile decreased MAL2 protein expression in human carcinomas and its anti-oncogenic phenotypes with increased transcript levels, we propose a transcriptional regulatory model for MAL2 transient overexpression.

The plasma membrane of polarized hepatocytes is functionally divided into two domains: the apical and basolateral. Our focus is to define the molecular basis of polarized protein sorting of newly-synthesized membrane and secretory proteins in WIF-B cells, an excellent model system for polarized hepatocytes. We determined that MAL2 (myelin and lymphocyte protein 2) and its binding partner, serine/threonine kinase 16 (STK16) regulate basolateral constitutive secretion. Because STK16 is a constitutively active kinase, we reasoned that constitutively phosphorylated substrates must participate in constitutive secretion. To identify either STK16 substrates or other proteins that regulate constitutive secretion, we took a proteomics approach. Post-nuclear supernatants from cells expressing wild type or a kinase-dead (E202A) STK16 were separated on 2D gels and immunoblotted with antibodies against phospho-serine/threonine residues. Sixteen spots were identified from E202A-expressing cells that reproducibly displayed decreased immunoreactivity. From these spots, 28 proteins were identified as possible STK16 substrates. Out of these 28 possible substrates, 25% of them encode predicted STK16 phosphorylation consensus sites, with WD repeat containing protein-1 (WDR1) encoding two such sites. Based on this finding and on the finding that actin remodeling is required for hepatic secretion, we further confirmed that WDR1 is a phosphoprotein that regulates secretion.