Temporal activation of WNT/ß-catenin signaling is sufficient to inhibit SOX10 expression and block melanoma growth.

Despite advances in the systemic treatment of patients with metastatic melanoma using immune checkpoint and tyrosine kinase inhibitors (TKI), the majority of stage IV melanoma patients eventually succumb to the disease. We have previously identified the transcription factor Sox10 as a crucial player in melanoma, yet the underlying molecular mechanisms mediating Sox10-dependent tumorigenesis remain largely uncharacterized. Here, we show that MEK and RAF inhibitors do not suppress levels of SOX10 protein in patient-derived cells in vitro, as well as in melanoma patients in vivo. In a search for pharmacological inhibitors of SOX10, we performed a mass spectrometry-based screen in human melanoma cells. Subsequent analysis revealed that SOX10 directly interacts with ß-catenin, which is a key mediator of canonical Wnt/ß-catenin signaling. We demonstrate that inhibitors of glycogen synthase kinase 3 alpha/beta (GSK3α/ß) efficiently abrogate SOX10 protein in human melanoma cells in vitro and in melanoma mouse models in vivo. The mechanism of action of GSK3-mediated SOX10 suppression is transcription-independent and relies on the presence of a proteasome degradable form of ß-catenin. Taken together, we provide evidence that activation of canonical Wnt signaling has a profound effect on melanoma growth and is able to counteract Sox10-dependent melanoma maintenance both in vitro and in vivo.

Antagonistic cross-regulation between Sox9 and Sox10 controls an anti-tumorigenic program in melanoma.

Melanoma is the most fatal skin cancer, but the etiology of this devastating disease is still poorly understood. Recently, the transcription factor Sox10 has been shown to promote both melanoma initiation and progression. Reducing SOX10 expression levels in human melanoma cells and in a genetic melanoma mouse model, efficiently abolishes tumorigenesis by inducing cell cycle exit and apoptosis. Here, we show that this anti-tumorigenic effect functionally involves SOX9, a factor related to SOX10 and upregulated in melanoma cells upon loss of SOX10. Unlike SOX10, SOX9 is not required for normal melanocyte stem cell function, the formation of hyperplastic lesions, and melanoma initiation. To the contrary, SOX9 overexpression results in cell cycle arrest, apoptosis, and a gene expression profile shared by melanoma cells with reduced SOX10 expression. Moreover, SOX9 binds to the SOX10 promoter and induces downregulation of SOX10 expression, revealing a feedback loop reinforcing the SOX10 low/SOX9 high ant,m/ii-tumorigenic program. Finally, SOX9 is required in vitro and in vivo for the anti-tumorigenic effect achieved by reducing SOX10 expression. Thus, SOX10 and SOX9 are functionally antagonistic regulators of melanoma development.

Tumor-associated macrophages subvert T-cell function and correlate with reduced survival in clear cell renal cell carcinoma.

Although malignant cells can be recognized and controlled by the immune system, in patients with clinically apparent cancer immunosurveillance has failed. To better understand local immunoregulatory processes that impact on cancer progression, we correlated intratumoral immunological profiles with the survival of patients affected by primary clear cell renal cell carcinoma (ccRCC). A retrospective analysis of 54 primary ccRCC samples for 31 different immune response-related transcripts, revealed a negative correlation of CD68 (a marker of tumor-associated macrophages, TAMs) and FOXP3 (a marker of regulatory T cells, Tregs) with survival. The subsequent analysis of 12 TAM-related transcripts revealed an association between the genes coding for CD163, interferon regulatory factor 4 (IRF4) and fibronectin 1 (FN1), all of which have been linked to the M2 TAM phenotype, with reduced survival and increased tumor stage, whereas the opposite was the case for the M1-associated gene coding for inducible nitric oxide synthetase (iNOS). The M2 signature of (CD68+) TAMs was found to correlate with CD163 expression, as determined in prospectively collected fresh ccRCC tissue samples. Upon co-culture with autologous tumor cells, CD11b+ cells isolated from paired blood samples expressed CD163 and other M2-associated proteins, suggesting that the malignant cells promote the accumulation of M2 TAMs. Furthermore, the tumor-associated milieu as well as isolated TAMs induced the skewing of autologous, blood-derived CD4+ T cells toward a more immunosuppressive phenotype, as shown by decreased production of effector cytokines, increased production of interleukin-10 (IL-10) and enhanced expression of the co-inhibitory molecules programmed death 1 (PD-1) and T-cell immunoglobulin mucin 3 (TIM-3). Taken together, our data suggest that ccRCC progressively attracts macrophages and induces their skewing into M2 TAMs, in turn subverting tumor-infiltrating T cells such that immunoregulatory functions are increased at the expense of effector functions.

Aldara activates TLR7-independent immune defence.

Aldara is a cream used for topical treatment of non-melanoma skin cancer, and is thought to act through stimulation of anti-tumour immunity. The active ingredient, imiquimod, has been shown to stimulate toll-like receptor 7. Aldara also induces psoriasis-like lesions when applied to naive murine skin, and as such is used as a mouse model for psoriasis. Here we find that in naive murine skin, Aldara induces inflammation largely independently of toll-like receptor 7. Surprisingly, inflammasome activation, keratinocyte death and interleukin 1 release also occur in response to the vehicle cream in the absence of imiquimod. We show that isostearic acid, a major component of the vehicle, promotes inflammasome activation in cultured keratinocytes, and so may contribute to the observed effects of Aldara on murine skin. Aldara therefore stimulates at least two immune pathways independently, and both imiquimod and vehicle are required for a full inflammatory response. Although it remains to be tested, it is possible that imiquimod-independent effects also contribute to the therapeutic efficacy of Aldara.

Spontaneous peripheral T-cell responses toward the tumor-associated antigen cyclin D1 in patients with clear cell renal cell carcinoma.

Renal cell carcinoma (RCC) is a heterogeneous group of kidney cancers with clear cell RCC (ccRCC) as the major subgroup. To expand the number of clinically relevant tumor-associated antigens (TAA) that can be targeted by immunotherapy, we analyzed samples from 23 patients with primary ccRCC for the expression and immunogenicity of various TAAs. We found high-frequency expression of MAGE-A9 and NY-ESO-1 in 36% and 55% of samples, respectively, and overexpression of PRAME, RAGE-1, CA-IX, Cyclin D1, ADFP, C-MET, and RGS-5 in many of the tumor samples. We analyzed the blood of patients with HLA-A2(+) ccRCC for the presence of CD8(+) T cells specific for TAA-derived HLA-A2-restricted peptides and found spontaneous responses to cyclin D1 in 5 of 6 patients with Cyclin D1-positive tumors. Cyclin D1-specific CD8(+) T cells secreted TNF-α, IFN-γ, and interleukin-2 (IL-2), and degranulated, indicating the presence of polyfunctional tumor-specific CD8(+) T cells in the blood of these patients with ccRCC. The high frequency (43%) of Cyclin D1 overexpression and the presence of functional cyclin D1-specific T cells in 83% of these patients with ccRCC suggest that cyclin D1 may be a target for immunotherapeutic strategies.

Fine analysis of spontaneous MAGE-C1/CT7-specific immunity in melanoma patients.

Cancer/testis (CT) antigens represent prime candidates for immunotherapy in cancer patients, because their expression is restricted to cancer cells and germ cells of the testis. MAGE-C1/CT7 is a CT antigen that is highly expressed in several types of cancers. Spontaneous occurrence of CT7-specific antibodies was previously detected by SEREX screen in a melanoma patient. However, naturally occurring CT7-specific T-cell responses have thus far not been detected. Peripheral blood mononuclear cells (PBMCs) from 26 metastatic melanoma patients expressing CT7 in their tumor lesions (CT7(+)) were analyzed for CT7-specific T-cell responses using overlapping peptides. CT7-specific CD4(+) T-cell responses were detected in three patients (11.5%). These CT7-specific CD4(+) T-cell responses were detectable in melanoma patients' PBMCs exclusively from preexisting CD45RA(-) memory CD4(+) T-cell pool. Additional CT7-specific memory CD4(+) T-cell responses were detected in CT7(+) melanoma patients after depletion of CD4(+)CD25high Treg cells showing that Treg cells impact on CT7-specific CD4(+) T cells in melanoma patients. CT7-specific CD4(+) T-cell clones were generated and used to define minimal epitopes, restriction elements, and confirm the recognition of naturally processed antigen. Surprisingly, these clones were able to secrete perforin and exert cytotoxicity. This study shows that CT7 can induce specific cellular immunity in melanoma patients. Based on these findings, CT7 will be further explored as a potential vaccine for melanoma immunotherapy.