Background: Targeted inhibition of protein kinases is now acknowledged as an effective approach for cancer therapy. However, targeted therapies probably have limited success because cancer cells have alternate pathways for survival and proliferation thereby avoiding inhibition. We tested the hypothesis that combination of targeted agents would be more effective than single agents in arresting melanoma cell proliferation.

Vascular endothelial growth factor

Melanomas depend on autocrine signals for proliferation and survival; however, no systematic screen of known RTKs has been performed to identify which autocrine signaling pathways are activated in melanoma. Here we performed a comprehensive analysis of 42 receptor tyrosine kinases (RTKs) in 6 individual human melanoma tumor specimens as well as 17 melanoma cell lines, some of which were derived from the tumor specimens. We identified 5 RTKs that were active in almost every one of the melanoma tissue specimens and cell lines, including two previously unreported receptors, IGF1R and MSPR, in addition to three receptors (VEGFR, FGFR and HGFR) known to be autocrine activated in melanoma. We show by real time quantitative PCR that all melanoma cell lines expressed genes for the RTK ligands HGF, IGF1 and MSP. Addition of antibodies to either IGF1 or HGF, but not to MSP, to the culture medium blocked melanoma cell proliferation, and even caused net loss of melanoma cells. Antibody addition deactivated IGF1R and HGFR receptors, as well as MAPK signaling. Thus, IGF1 is a new growth factor for autocrine driven proliferation of human melanoma in vitro. Our results suggest that IGF1-IGF1R autocrine pathway in melanoma is a possible target for therapy in human melanomas.

Epithelial to mesenchymal transition is a developmental process allowing epithelial cells to dedifferentiate into cells displaying mesenchymal phenotypes. The pathological role of EMT has been implicated in invasion and metastasis for numerous carcinomas, yet limited data exist addressing whether mesenchymal transition (MT) occurs in malignant melanoma cells. Our group developed an in vitro 3D culture system to address MT in melanoma cells upon TGF-β/TNF-α treatment. Loss of E-cadherin is one of the best indicators of MT in epithelial cells. Not surprisingly, E-cadherin was expressed in only three of twelve (25%) melanoma cell lines and all three mesenchymal proteins, N-cadherin, vimentin, and fibronectin, were expressed by seven (58%) lines. However, following cytokine treatment, two or more mesenchymal proteins were elevated in nine (75%) lines. Data support the TGF-β production by melanoma cells which may induce/support MT. Evaluation of E-cadherin, N-cadherin, and Snail expression in melanoma tissue samples are consistent with an inverse coupling of E-cadherin and N-cadherin expression, however, there are also examples suggesting a more complex control of their expression. These results indicate that malignant melanoma cell lines are susceptible to MT following cytokine treatment and highlight the importance of understanding the effects of cytokines on melanoma to undergo MT.

Cancer vaccines have not been optimized. They depend on adjuvants to create an immunogenic microenvironment for antigen presentation. However, remarkably little is understood about cellular and molecular changes induced by these adjuvants in the vaccine microenvironment. We hypothesized that vaccination induces dendritic cell activation in the dermal vaccination microenvironment but that regulatory processes may also limit the effectiveness of repeated vaccination. We evaluated biopsies from immunization sites in two clinical trials of melanoma patients. In one study (Mel38), patients received one injection with an adjuvant mixture alone, comprised of incomplete Freund's adjuvant (IFA) plus granulocyte-macrophage colony stimulating factor (GM-CSF). In a second study, patients received multiple vaccinations with melanoma peptide antigens plus IFA. Single injections with adjuvant alone induced dermal inflammatory infiltrates consisting of B cells, T cells, mature dendritic cells (DC) and vessels resembling high endothelial venules (HEV). These cellular aggregates usually lacked organization and were transient. In contrast, multiple repeated vaccinations with peptides in adjuvant induced more organized and persistent lymphoid aggregates containing separate B and T cell areas, mature DC, HEV-like vessels, and lymphoid chemokines. Within these structures, there are proliferating CD4+ and CD8+ T lymphocytes, as well as FoxP3+CD4+ lymphocytes, suggesting a complex interplay of lymphoid expansion and regulation within the dermal immunization microenvironment. Further study of the physiology of the vaccine site microenvironment promises to identify opportunities for enhancing cancer vaccine efficacy by modulating immune activation and regulation at the site of vaccination.