Imipridones inhibit tumor growth and improve survival in an orthotopic liver metastasis mouse model of human uveal melanoma.

BACKGROUND: Uveal melanoma (UM) is a highly aggressive disease with very few treatment options. We previously demonstrated that mUM is characterized by high oxidative phosphorylation (OXPHOS). Here we tested the anti-tumor, signaling and metabolic effects of imipridones, which are CLPP activators, which inhibit OXPHOS indirectly and have demonstrated safety in patients. METHODS: We assessed CLPP expression in UM patient samples. We tested the effects of imipridones (ONC201 and ONC212) on the growth, survival, signaling and metabolism of UM cell lines in vitro, and for therapeutic efficacy in vivo in UM liver metastasis models. RESULTS: CLPP expression was detected in primary and mUM patient samples. ONC201 and 212 decreased OXPHOS effectors, inhibited cell growth and migration, and induced apoptosis in human UM cell lines in vitro. ONC212 inhibited OXPHOS, increased metabolic stress and apoptotic pathways, inhibited amino acid metabolism, and induced cell death-related lipids. ONC212 also decreased tumor burden and increased survival in vivo in two UM liver metastasis models. CONCLUSIONS: Imipridones are a promising strategy for further testing and development in mUM.

Mitochondrial dynamic alterations regulate melanoma cell progression.

Research on mitochondrial fusion and fission (mitochondrial dynamics) has gained much attention in recent years, as it is important for understanding many biological processes, including the maintenance of mitochondrial functions, apoptosis, and cancer. The rate of mitochondrial biosynthesis and degradation can affect various aspects of tumor progression. However, the role of mitochondrial dynamics in melanoma progression remains controversial and requires a mechanistic understanding to target the altered metabolism of cancer cells. Therefore, in our study, we disrupted mitochondrial fission with mdivi-1, the reported inhibitor of dynamin related protein 1 (Drp1), and knocked down Drp1 and Mfn2 to evaluate the effects of mitochondrial dynamic alterations on melanoma cell progression. Our confocal study results showed that mitochondrial fission was inhibited both in mdivi-1 and in Drp1 knockdown cells and, in parallel, mitochondrial fusion was induced. We also found that mitochondrial fission inhibition by mdivi-1 induced cell death in melanoma cells. However, silencing Drp1 and Mfn2 did not affect cell viability, but enhanced melanoma cell migration. We further show that dysregulated mitochondrial fusion by Mfn2 knockdowns suppressed the oxygen consumption rate of melanoma cells. Together, our findings suggest that mitochondrial dynamic alterations regulate melanoma cell migration and progression.

TNFSF4 (OX40L) expression and survival in locally advanced and metastatic melanoma.

Immunotherapy with checkpoint inhibitors revolutionized melanoma treatment in both the adjuvant and metastatic setting, yet not all metastatic patients respond, and metastatic disease still often recurs among immunotherapy-treated patients with locally advanced disease. TNFSF4 is a co-stimulatory checkpoint protein expressed by several types of immune and non-immune cells, and was shown in the past to enhance the anti-neoplastic activity of T cells. Here, we assessed its expression in melanoma and its association with outcome in locally advanced and metastatic disease. We used publicly available data from The Cancer Genome Atlas (TCGA) and the Cancer Cell Line Encyclopedia (CCLE), and RNA sequencing data from anti-PD1-treated patients at Sheba medical center. TNFSF4 mRNA is expressed in melanoma cell lines and melanoma samples, including those with low lymphocytic infiltrates, and is not associated with the ulceration status of the primary tumor. Low expression of TNFSF4 mRNA is associated with worse prognosis in all melanoma patients and in the cohorts of stage III and stage IIIc-IV patients. Low expression of TNFSF4 mRNAs is also associated with worse prognosis in the subgroup of patients with low lymphocytic infiltrates, suggesting that tumoral TNFSF4 is associated with outcome. TNFSF4 expression was not correlated with the expression of other known checkpoint mRNAs. Last, metastatic patients with TNFSF4 mRNA expression within the lowest quartile have significantly worse outcome on anti-PD1 treatment, and a significantly lower response rate to these agents. Our current work points to TNFSF4 expression in melanoma as a potential determinant of prognosis, and warrants further translational and clinical research.

Uveal melanoma: From diagnosis to treatment and the science in between.

Melanomas of the choroid, ciliary body, and iris of the eye are collectively known as uveal melanomas. These cancers represent 5% of all melanoma diagnoses in the United States, and their age-adjusted risk is 5 per 1 million population. These less frequent melanomas are dissimilar to their more common cutaneous melanoma relative, with differing risk factors, primary treatment, anatomic spread, molecular changes, and responses to systemic therapy. Once uveal melanoma becomes metastatic, therapy options are limited and are often extrapolated from cutaneous melanoma therapies despite the routine exclusion of patients with uveal melanoma from clinical trials. Clinical trials directed at uveal melanoma have been completed or are in progress, and data from these well designed investigations will help guide future directions in this orphan disease. Cancer 2016;122:2299-2312. © 2016 American Cancer Society.

Imipridones inhibit tumor growth and improve survival in an orthotopic liver metastasis mouse model of human uveal melanoma.

Purpose: Uveal melanoma (UM) is a highly aggressive disease with very few treatment options. We previously demonstrated that mUM is characterized by high oxidative phosphorylation (OXPHOS). Here we tested the anti-tumor, signaling and metabolic effects of imipridones, CLPP activators which reduce OXPHOS indirectly and have demonstrated safety in patients. Experimental Design: We assessed CLPP expression in UM patient samples. We tested the effects of imipridones (ONC201, ONC212) on the growth, survival, signaling and metabolism of UM cell lines in vitro, and for therapeutic effects in vivo in UM liver metastasis models. Results: CLPP expression was confirmed in primary and mUM patient samples. ONC201/212 treatment of UM cell lines in vitro decreased OXPHOS effectors, inhibited cell growth and migration, and induced apoptosis. ONC212 increased metabolic stress and apoptotic pathways, inhibited amino acid metabolism, and induced cell death-related lipids. ONC212 also decreased tumor burden and increased survival in vivo in two UM liver metastasis models. Conclusion: Imipridones are a promising strategy for further testing and development in mUM.

Genome-wide association study identifies novel loci predisposing to cutaneous melanoma.

We performed a multistage genome-wide association study of melanoma. In a discovery cohort of 1804 melanoma cases and 1026 controls, we identified loci at chromosomes 15q13.1 (HERC2/OCA2 region) and 16q24.3 (MC1R) regions that reached genome-wide significance within this study and also found strong evidence for genetic effects on susceptibility to melanoma from markers on chromosome 9p21.3 in the p16/ARF region and on chromosome 1q21.3 (ARNT/LASS2/ANXA9 region). The most significant single-nucleotide polymorphisms (SNPs) in the 15q13.1 locus (rs1129038 and rs12913832) lie within a genomic region that has profound effects on eye and skin color; notably, 50% of variability in eye color is associated with variation in the SNP rs12913832. Because eye and skin colors vary across European populations, we further evaluated the associations of the significant SNPs after carefully adjusting for European substructure. We also evaluated the top 10 most significant SNPs by using data from three other genome-wide scans. Additional in silico data provided replication of the findings from the most significant region on chromosome 1q21.3 rs7412746 (P = 6 × 10(-10)). Together, these data identified several candidate genes for additional studies to identify causal variants predisposing to increased risk for developing melanoma.

Slug expression during melanoma progression.

Slug (Snai2), a member of the Snail family of zinc finger transcription factors, plays a role in the epithelial-to-mesenchymal transformation (EMT) that occurs during melanocyte emigration from the neural crest. A role for Slug in the EMT-like loss of cell adhesion and increased cell motility exhibited during melanoma progression has also been proposed. Our immunohistochemical studies of melanoma arrays, however, revealed that Slug expression was actually higher in nevi than in primary or metastatic melanomas. Moreover, Slug expression in melanomas was not associated with decreased expression of E-cadherin, the canonical Slug target in EMT. Comparisons of endogenous Slug and E-cadherin expression in cultured normal human melanocytes and melanoma cell lines supported our immunohistochemical findings. Expression of exogenous Slug in melanocytes and melanoma cells in vitro, however, suppressed E-cadherin expression, enhanced N-cadherin expression, and stimulated cell migration and invasion. Interestingly, both in tumors and cultured cell lines, there was a clear relationship between expression of Slug and MITF, a transcription factor known to regulate Slug expression during development. Taken together, our findings suggest that Slug expression during melanomagenesis is highest early in the process and that persistent Slug expression is not required for melanoma progression. The precise role of Slug in melanomagenesis remains to be elucidated and may be related to its interactions with other drivers of EMT, such as Snail.

Inhibition of Microsomal Prostaglandin E2 Synthase Reduces Collagen Deposition in Melanoma Tumors and May Improve Immunotherapy Efficacy by Reducing T-cell Exhaustion.

The arachidonic acid pathway participates in immunosuppression in various types of cancer. Our previous observation detailed that microsomal prostaglandin E2 synthase 1 (mPGES-1), an enzyme downstream of cyclooxygenase 2 (COX-2), limited antitumor immunity in melanoma; in addition, genetic depletion of mPGES-1 specifically enhanced immune checkpoint blockade therapy. The current study set out to distinguish the roles of mPGES-1 from those of COX-2 in tumor immunity and determine the potential of mPGES-1 inhibitors for reinforcing immunotherapy in melanoma. Genetic deletion of mPGES-1 showed different profiles of prostaglandin metabolites from that of COX-2 deletion. In our syngeneic mouse model, mPGES-1-deficient cells exhibited similar tumorigenicity to that of COX-2-deficient cells, despite a lower ability to suppress PGE2 synthesis by mPGES-1 depletion, indicating the presence of factors other than PGE2 that are likely to regulate tumor immunity. RNA-sequencing analysis revealed that mPGES-1 depletion reduced the expressions of collagen-related genes, which have been found to be associated with immunosuppressive signatures. In our mouse model, collagen was reduced in mPGES-1-deficient tumors, and phenotypic analysis of tumor-infiltrating lymphocytes indicated that mPGES-1-deficient tumors had fewer TIM3+ exhausted CD8+ T cells compared with COX-2-deficient tumors. CAY10678, an mPGES-1 inhibitor, was equivalent to celecoxib, a selective COX-2 inhibitor, in reinforcing anti-PD-1 treatment. Our study indicates that mPGES-1 inhibitors represent a promising adjuvant for immunotherapies in melanoma by reducing collagen deposition and T-cell exhaustion. Significance: Collagen is a predominant component of the extracellular matrix that may influence the tumor immune microenvironment for cancer progression. We present here that mPGES-1 has specific roles in regulating tumor immunity, associated with several collagen-related genes and propose that pharmacologic inhibition of mPGES-1 may hold therapeutic promise for improving immune checkpoint-based therapies.

The role of melanoma tumor-derived nitric oxide in the tumor inflammatory microenvironment: its impact on the chemokine expression profile, including suppression of CXCL10.

Melanoma appears to be heterogeneous in terms of its molecular biology, etiology and epidemiology. We previously reported that the expression of inducible nitric-oxide synthase (iNOS) in melanoma tumor cells is strongly correlated with poor patient survival. Therefore, we hypothesized that nitric oxide (NO) produced by iNOS promotes the melanoma inflammatory tumor microenvironment associated with poor outcome. To understand the role of NO and iNOS in the melanoma inflammatory tumor microenvironment, polymerase chain reaction arrays of inflammatory and autoimmunity genes were performed on a series of stage III melanoma lymph node metastasis samples to compare the gene expression profiles of iNOS-expressing and nonexpressing tumor samples. The results indicate that expression of CXC chemokine ligand 10 (CXCL10) was inversely correlated with iNOS expression, and the high CXCL10-expressing cases had more favorable prognoses than the low CXCL10-expressing cases. Functional studies revealed that treating iNOS-negative/CXCL10-positive melanoma cell lines with a NO donor suppressed the expression of CXCL10. Furthermore, scavenging NO from iNOS-expressing cell lines significantly affected the chemokine expression profile. Culture supernatants from NO scavenger-treated melanoma cells promoted the migration of plasmacytoid dendritic cells, which was diminished when the cells were treated with a CXCL10-neutralizing antibody. CXCL10 has been reported to be an antitumorigenic chemokine. Our study suggests that the production of NO by iNOS inhibits the expression of CXCL10 in melanoma cells and leads to a protumorigenic tumor microenvironment. Inhibiting NO induces an antitumorigenic environment, and thus, iNOS should be considered to be an important therapeutic target in melanoma.

Association of activated c-Met with NRAS-mutated human melanomas.

Cutaneous melanomas can be divided into three mutually exclusive genetic subsets: tumors with mutated BRAF, tumors with mutated NRAS and tumors wild type at both loci (wt/wt). Targeted therapy for melanoma has been advancing with agents directed to mutated BRAF, accounting for 50% of melanoma patients. The c-Met pathway is known to play a role in melanoma tumorigenesis and preliminary data from our laboratory suggested that this pathway is preferentially activated in NRAS-mutated tumors. The objective of this study was to test the hypothesis that melanomas carrying the mutated NRAS genotype are uniquely sensitively to c-Met inhibition, thus providing rationale for therapeutic targeting of c-Met in this patient cohort. Using primary human melanomas with known BRAF/NRAS genotypes, we observed greater immunostaining for phosphorylated (activated) c-Met in NRAS-mutated and wt/wt tumors, compared to BRAF-mutated tumors. NRAS-mutated and wt/wt cell lines also demonstrated more robust c-Met activation in response to hepatocyte growth factor (HGF). Knock-down of mutated N-Ras, but not wild type N-Ras, by RNA interference resulted in decreased c-Met phosphorylation. Compared to BRAF mutants, NRAS-mutated melanoma cells were more sensitive to pharmacologic c-Met inhibition in terms of c-Met activation, Akt phosphorylation, tumor cell proliferation, migration and apoptosis. This enhanced sensitivity was observed in wt/wt cells as well, but was a less consistent finding. On the basis of these experimental results, we propose that c-Met inhibition may be a useful therapeutic strategy for melanomas with NRAS mutations, as well as some tumors with a wt/wt genotype.

Comparison of the IPAQ-Short Form and accelerometry predictions of physical activity in older adults.

Older adult physical activity (PA) levels obtained from the International Physical Activity Questionnaire-Short Form (IPAQ) and accelerometry (ACC) were compared. Mean difference scores between accumulated or bout ACC PA and the IPAQ were computed. Spearman rank-order correlations were used to assess relations between time spent in PA measured from ACC and self-reported form of the IPAQ, and percentage agreement across measures was used to classify meeting or not meeting PA recommendations. The IPAQ significantly underestimated sitting and overestimated time spent in almost all PA intensities. Group associations across measures revealed significant relations in walking, total PA, and sitting for the whole group (r = .29-.36, p < .05). Significant relationships between bout ACC and IPAQ walking (r = .28-.39, p < .05) were found. There was 40-46% agreement between measures for meeting PA recommendations. The IPAQ appears not to be a good indicator of individual older adult PA behavior but is better suited for larger population-based samples.

Prediction of body fat in older adults by time spent in sedentary behavior.

The purpose of the study was to determine the relationship between sedentary behavior (SB), physical activity (PA), and body fat (total, abdominal) or body size (body-mass index [BMI], waist circumference [WC]) in community-dwelling adults 50 yr old and over. This study included 232 ambulatory adults (50-87 yr, 37.4% ± 9.6% body fat [BF]). Average daily time spent in SB (<100 counts/min) and light (100-759 counts/min), lifestyle-moderate (760-1,951 counts/min), walking-moderate (1,952-5,724 cts/min), and vigorous-intensity (≥ 5,725 counts/min) PA were determined by accelerometer and corrected for wear time. BF was measured with dual-energy X-ray absorptiometry. SB was positively related to measures of BF. Measures of SB, PA, and gender accounted for 55.6% of the variance in total BF, 32.4% of the variance in abdominal fat, and 28.0% of the variance in WC. SB, PA, and age accounted for 27.1% of the variance in BMI. Time spent in SB should be considered when designing obesity interventions for adults 50 yr old and over.

Targeting IRS-1/2 in Uveal Melanoma Inhibits In Vitro Cell Growth, Survival and Migration, and In Vivo Tumor Growth.

Uveal melanoma originating in the eye and metastasizing to the liver is associated with poor prognosis and has only one approved therapeutic option. We hypothesized that liver-borne growth factors may contribute to UM growth. Therefore, we investigated the role of IGF-1/IGF-1R signaling in UM. Here, we found that IRS-1, the insulin receptor substrate, is overexpressed in both UM cells and tumors. Since we previously observed that IGF-1R antibody therapy was not clinically effective in UM, we investigated the potential of NT157, a small molecule inhibitor of IRS-1/2, in blocking this pathway in UM. NT157 treatment of multiple UM cell lines resulted in reduced cell growth and migration and increased apoptosis. This treatment also significantly inhibited UM tumor growth in vivo, in the chicken egg chorioallantoic membrane (CAM) and subcutaneous mouse models, validating the in vitro effect. Mechanistically, through reverse phase protein array (RPPA), we identified significant proteomic changes in the PI3K/AKT pathway, a downstream mediator of IGF-1 signaling, with NT157 treatment. Together, these results suggest that NT157 inhibits cell growth, survival, and migration in vitro, and tumor growth in vivo via inhibiting IGF-1 signaling in UM.

Interplay between soluble CD74 and macrophage-migration inhibitory factor drives tumor growth and influences patient survival in melanoma.

Soluble forms of receptors play distinctive roles in modulating signal-transduction pathways. Soluble CD74 (sCD74) has been identified in sera of inflammatory diseases and implicated in their pathophysiology; however, few relevant data are available in the context of cancer. Here we assessed the composition and production mechanisms, as well as the clinical significance and biological properties, of sCD74 in melanoma. Serum sCD74 levels were significantly elevated in advanced melanoma patients compared with normal healthy donors, and the high ratio of sCD74 to macrophage-migration inhibitory factor (MIF) conferred significant predictive value for prolonged survival in these patients (p = 0.0035). Secretion of sCD74 was observed primarily in melanoma cell lines as well as a THP-1 line of macrophages from monocytes and primary macrophages, especially in response to interferon-γ (IFN-γ). A predominant form that showed clinical relevance was the 25-KDa sCD74, which originated from the 33-KDa isoform of CD74. The release of this sCD74 was regulated by either a disintegrin and metalloproteinase-mediated cell-surface cleavage or cysteine-protease-mediated lysosomal cleavage, depending on cell types. Both recombinant and THP-1 macrophage-released endogenous sCD74 suppressed melanoma cell growth and induced apoptosis under IFN-γ stimulatory conditions via inhibiting the MIF/CD74/AKT-survival pathway. Our findings demonstrate that the interplay between sCD74 and MIF regulates tumor progression and determines patient outcomes in advanced melanoma.

Multi-modal molecular programs regulate melanoma cell state.

Melanoma cells display distinct intrinsic phenotypic states. Here, we seek to characterize the molecular regulation of these states using multi-omic analyses of whole exome, transcriptome, microRNA, long non-coding RNA and DNA methylation data together with reverse-phase protein array data on a panel of 68 highly annotated early passage melanoma cell lines. We demonstrate that clearly defined cancer cell intrinsic transcriptomic programs are maintained in melanoma cells ex vivo and remain highly conserved within melanoma tumors, are associated with distinct immune features within tumors, and differentially correlate with checkpoint inhibitor and adoptive T cell therapy efficacy. Through integrative analyses we demonstrate highly complex multi-omic regulation of melanoma cell intrinsic programs that provide key insights into the molecular maintenance of phenotypic states. These findings have implications for cancer biology and the identification of new therapeutic strategies. Further, these deeply characterized cell lines will serve as an invaluable resource for future research in the field.

Zyflamend mediates therapeutic induction of autophagy to apoptosis in melanoma cells.

Melanoma is the most aggressive form of skin cancer. The rising incidence of melanoma and its poor prognosis in advanced stages are compelling reasons to identify novel therapeutic agents. Though isolated dietary components such as lycopene, resveratrol, and isothiocyanate compounds have been shown to provide limited protection against cancer development, the use of whole herbs and herbal extracts for the treatment of cancer remains of great interest. As suggested by earlier studies, the antiinflammatory activity of many plants available as intact products or as extracts has long been considered for supplemental therapeutics for cancer. Zyflamend, a unique multiherbal extract preparation, is a promising antiinflammatory agent that has also been suggested to regulate multiple pathways in cancer progression. As Zyflamend contains ingredients that can suppress tumor cell proliferation, invasion, angiogenesis, and metastasis through regulation of inflammatory pathway products, we hypothesized that this preparation might inhibit melanoma proliferation. To test this hypothesis, we studied the effect of Zyflamend on melanoma proliferation. Here, we present that Zyflamend inhibits melanoma growth by regulating the autophagy-apoptosis switch. Based on the responsible molecular mechanisms of Zyflamend, our study highlights the importance of the use of herbal preparations for the prevention and treatment of cancer.

iNOS Associates With Poor Survival in Melanoma: A Role for Nitric Oxide in the PI3K-AKT Pathway Stimulation and PTEN S-Nitrosylation.

We previously showed that inducible nitric oxide synthase (iNOS) protein expression in melanoma tumor cells is associated with poor patient prognosis. Here, we analyzed the association between iNOS and the oncogenic PI3K-AKT pathway. TCGA data show that iNOS and phospho-Akt Ser473 expression were associated significantly only in the subset of tumors with genetically intact PTEN. Employing a stage III melanoma TMA, we showed that iNOS protein presence is significantly associated with shorter survival only in tumors with PTEN protein expression. These findings led to our hypothesis that the iNOS product, nitric oxide (NO), suppresses the function of PTEN and stimulates PI3K-Akt activation. Melanoma cells in response to NO exposure in vitro exhibited enhanced AKT kinase activity and substrate phosphorylation, as well as attenuated PTEN phosphatase activity. Biochemical analysis showed that NO exposure resulted in a post-translationally modified S-Nitrosylation (SNO) PTEN, which was also found in cells expressing iNOS. Our findings provide evidence that NO-rich cancers may exhibit AKT activation due to post-translational inactivation of PTEN. This unique activation of oncogenic pathway under nitrosative stress may contribute to the pathogenesis of iNOS in melanoma. Significance: Our study shows that iNOS expression is associated with increased PI3K-AKT signaling and worse clinical outcomes in melanoma patients with wt (intact) PTEN. Mutated PTEN is already inactivated. We also demonstrate that NO activates the PI3K-AKT pathway by suppressing PTEN suppressor function concurrent with the formation of PTEN-SNO. This discovery provides insight into the consequences of inflammatory NO produced in human melanoma and microenvironmental cells. It suggests that NO-driven modification provides a marker of PTEN inactivation, and represents a plausible mechanism of tumor suppressor inactivation in iNOS expressing subset of cancers.

IL-24 is expressed during wound repair and inhibits TGFalpha-induced migration and proliferation of keratinocytes.

Interleukin (IL)-24 is the protein product of melanoma differentiation-associated gene 7 (MDA-7). Originally identified as a tumor suppressor molecule, MDA-7 was renamed IL-24 and classified as a cytokine because of its chromosomal location in the IL-10 locus, its mRNA expression in leukocytes, and its secretory sequence elements. We previously reported that IL-24 is expressed by cytokine-activated monocytes and T lymphocytes. Here, we show that IL-24 is expressed in keratinocytes during wound repair. Paraffin-embedded tissues prepared from human skin sampled at days 2, 6, and 10 after wounding were examined by immunohistochemistry for the expression of IL-24. Protein expression was detected in the keratinocyte population with maximum expression at days 2 and 6, and no expression by day 10 (four of four subjects). In vitro studies showed that cytokines involved in wound repair, most notably transforming growth factor alpha (TGFalpha), TGFbeta, IFNgamma, and IFNbeta, upregulated IL-24 protein expression in normal human epidermal keratinocytes (NHEKs). Examination of the function of IL-24 in both in vitro wound repair and migration assays demonstrated that IL-24 inhibits TGFalpha-induced proliferation and migration of NHEKs. These data support the hypothesis that IL-24 functions during an inflammatory response in the skin by inhibiting the proliferation and migration of keratinocytes.

The Expression of CD74-Regulated Inflammatory Markers in Stage IV Melanoma: Risk of CNS Metastasis and Patient Survival.

Innate inflammatory features have been found in melanoma tumors from patients at all stages, and molecular analysis has identified definitive inflammatory proteins expressed by tumors cells in patients who presents the worst prognosis. We have previously observed weakened outcomes in patients with constitutive expression of inducible nitric oxide synthase (iNOS), macrophage migration inhibitory factor (MIF) and improved outcomes with CD74 expression in stage III melanoma. In our current study, we tested our hypothesis on CD74-regulated inflammatory markers' expression in stage IV melanoma tumors whether the signature is associated with survival outcome and/or risk of developing CNS metastasis. We retrospectively identified 315 patients with stage IV melanoma. In a tissue microarray (TMA), we examined the expression of cells with CD74, its receptor MIF, and downstream inflammatory markers iNOS, nitrotyrosine (NT), cyclooxygenase (COX)-2 and microsomal prostaglandin E synthase-1 (mPGES1). We analyzed the association of those inflammatory markers with overall survival time (OS) and time to CNS metastasis using Kaplan-Meier survival analyses. Our data validates CD74 as a useful prognostic tumor cell protein marker associated with favorable OS as in stage III melanomas, while the tumor NT expression strongly predicts an increased risk of developing CNS metastasis (p = 0.0008) in those patients.

A novel phosphorylated STAT3 inhibitor enhances T cell cytotoxicity against melanoma through inhibition of regulatory T cells.

The activation of signal transducer and activator of transcription 3 (STAT3) has been identified as a key mediator that drives the fundamental components of melanoma malignancy, including immune suppression in melanoma patients. Increasing evidence also suggests that regulatory T cells (Tregs) are important in suppressing anti-tumor immunity and play a dominant role in negating efficacious immunotherapy approaches. We hypothesized that WP1066, a novel inhibitor of STAT3 signaling, reverses immune suppression through the inhibition of Tregs and that this contributes to the antitumor activity of this agent against melanoma brain metastases. We found that the mean percentage of peripheral blood mononuclear cells expressing phosphorylated STAT3 (p-STAT3) was significantly elevated in samples from patients with melanoma brain metastases compared to healthy donors, 16.13 +/- 2.48% versus 4.17 +/- 1.79%. The p-STAT3 inhibitor WP1066 enhanced CD3+ (which contained Tregs) but not CD8+ T cell cytotoxicity against human A375 melanoma cells, indicating that this p-STAT3 blockade agent did not directly activate CD8+ T cells. Furthermore, the p-STAT3 inhibitor did not enhance the cytotoxicity of CD3+CD25- T cells (from which Tregs were excluded), indicating that the enhanced cytotoxicity of WP1066 is secondary to its inhibition of Tregs. This was confirmed by demonstrating that WP1066 inhibited FoxP3+ Treg induction in a dose-dependent manner. Moreover, CD3+ T cells exhibited markedly enhanced levels of phosphorylated ZAP-70, a critical proximal signal in T cell activation, after exposure to WP1066. Similar effects were not observed in Treg-depleted CD3+CD25- T cell populations, confirming that the T cell activation by WP compounds is secondary to their inhibition of the Tregs. These results suggest that WP1066 enhances T cell cytotoxicity against melanoma through inhibition of Tregs.