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.

Purpose A CTEP-sponsored phase II trial was performed to evaluate safety and clinical activity of combination therapy with CCI-779 (temsirolimus) and bevacizumab in patients with advanced melanoma. Experimental Design Patients with unresectable stage III to IV melanoma were treated intravenously with temsirolimus 25mg weekly and bevacizumab 10mg every 2 weeks. Adverse events were recorded using CTCAE v3.0. Tumor response was assessed by Response Evaluation Criteria in Solid Tumors, and overall survival was recorded. Correlative studies measured protein kinases and histology of tumor biopsies, and immune function in peripheral blood. Results Seventeen patients were treated. Most patients tolerated treatment well, but two had grade 4 lymphopenia and one developed reversible grade 2 leukoencephalopathy. Best clinical response was partial response (PR) in three patients (17.7%, 90%CI 5,0–39.6), stable disease at 8 weeks (SD) in 9 patients, progressive disease (PD) in 4 patients, and not evaluable in 1 patient. Maximal response duration for PR was 35 months. Ten evaluable patients had BRAFWT tumors, among whom 3 had PRs, 5 had SD, and 2 had PD. Correlative studies of tumor biopsies revealed decreased phospho-S6K (d2 and d23 vs d1, p<0.001), and decreased mitotic rate (Ki67+) among melanoma cells by d23 (p=0.007). Effects on immune functions were mixed, with decreased alloreactive T cell responses and decreased circulating CD4+FoxP3+ cells. Conclusion These data provide preliminary evidence for clinical activity of combination therapy with temsirolimus and bevacizumab, which may be greater in patients with BRAFwt melanoma. Mixed effects on immunologic function also support combination with immune therapies.

BackgroundTargeted therapies directed at commonly overexpressed pathways in melanoma have clinical activity in numerous trials. Little is known about how these therapies influence microRNA (miRNA) expression, particularly with combination regimens. Knowledge of miRNAs altered with treatment may contribute to understanding mechanisms of therapeutic effects, as well as mechanisms of tumor escape from therapy. We analyzed miRNA expression in metastatic melanoma tissue samples treated with a novel combination regimen of Temsirolimus and Bevacizumab. Given the preliminary clinical activity observed with this combination regimen, we hypothesized that we would see significant changes in miRNA expression with combination treatment.MethodsUsing microarray analysis we analyzed miRNA expression levels in melanoma samples from a Cancer Therapy Evaluation Program-sponsored phase II trial of combination Temsirolimus and Bevacizumab in advanced melanoma, which elicited clinical benefit in a subset of patients. Pre-treatment and post-treatment miRNA levels were compared using paired t-tests between sample groups (patients), using a p-value < 0.01 for significance.ResultsmicroRNA expression remained unchanged with Temsirolimus alone; however, expression of 15 microRNAs was significantly upregulated (1.4 to 2.5-fold) with combination treatment, compared to pre-treatment levels. Interestingly, twelve of these fifteen miRNAs possess tumor suppressor capabilities. We identified 15 putative oncogenes as potential targets of the 12 tumor suppressor miRNAs, based on published experimental evidence. For 15 of 25 miRNA-target mRNA pairings, changes in gene expression from pre-treatment to post-combination treatment samples were inversely correlated with changes in miRNA expression, supporting a functional effect of those miRNA changes. Clustering analyses based on selected miRNAs suggest preliminary signatures characteristic of clinical response to combination treatment and of tumor BRAF mutational status.ConclusionsTo our knowledge, this is the first study analyzing miRNA expression in pre-treatment and post-treatment human metastatic melanoma tissue samples. This preliminary investigation suggests miRNAs that may be involved in the mechanism of action of combination Temsirolimus and Bevacizumab in metastatic melanoma, possibly through inhibition of oncogenic pathways, and provides the preliminary basis for further functional studies of these miRNAs.

Targeted molecular therapies inhibit cancer cell proliferation and survival but may interfere with proliferation and survival of lymphoid cells. Treatment with Sirolimus or Sorafenib was cytotoxic to CD4 + CD25 high T cells, and was growth inhibitory for CD4 + and CD8 + T cells. The cytotoxicity depended on CD3/CD28 stimulation and was detectable as early as 12 hours post-treatment, with 80 −90% of the CD4 + CD25 high T cells killed by 72 hours. Cell death was due to apoptosis, based on time-dependent increase in Annexin V and 7AAD staining. Addition of IL-2 prevented the apoptotic response to Sirolimus, potentially accounting for reports that Sirolimus stimulates T cell proliferation. These results predict that Sirolimus or Sorafenib reduce the CD4 + CD25 high T cells if administered prior to antigenic stimulation in an immunotherapy protocol. However, administration of IL-2 protects these CD4 + CD25 high T cells from the cytotoxic effects of Sirolimus, a response that needs to be taken into consideration in therapeutic protocols.