Immune checkpoint inhibition and in particular anti-PD-1 immunotherapy have revolutionized the treatment of advanced melanoma. In this regard, higher tumoral PD-L1 protein (gene name: CD274) ...expression is associated with better clinical response and increased survival to anti-PD-1 therapy. Moreover, there is increasing evidence that tumor suppressor proteins are involved in immune regulation and are capable of modulating the expression of immune checkpoint proteins. Here, we determined the role of p53 protein (gene name: TP53) in the regulation of PD-L1 expression in melanoma.
We analyzed publicly available mRNA and protein expression data from the cancer genome/proteome atlas and performed immunohistochemistry on tumors with known TP53 status. Constitutive and IFN-ɣ-induced PD-L1 expression upon p53 knockdown in wildtype, TP53-mutated or JAK2-overexpressing melanoma cells or in cells, in which p53 was rendered transcriptionally inactive by CRISPR/Cas9, was determined by immunoblot or flow cytometry. Similarly, PD-L1 expression was investigated after overexpression of a transcriptionally-impaired p53 (L22Q, W23S) in TP53-wt or a TP53-knockout melanoma cell line. Immunoblot was applied to analyze the IFN-ɣ signaling pathway.
For TP53-mutated tumors, an increased CD274 mRNA expression and a higher frequency of PD-L1 positivity was observed. Interestingly, positive correlations of IFNG mRNA and PD-L1 protein in both TP53-wt and -mutated samples and of p53 and PD-L1 protein suggest a non-transcriptional mode of action of p53. Indeed, cell line experiments revealed a diminished IFN-ɣ-induced PD-L1 expression upon p53 knockdown in both wildtype and TP53-mutated melanoma cells, which was not the case when p53 wildtype protein was rendered transcriptionally inactive or by ectopic expression of p53
, a transcriptionally-impaired variant, in TP53-wt cells. Accordingly, expression of p53
in a TP53-knockout melanoma cell line boosted IFN-ɣ-induced PD-L1 expression. The impaired PD-L1-inducibility after p53 knockdown was associated with a reduced JAK2 expression in the cells and was almost abrogated by JAK2 overexpression.
While having only a small impact on basal PD-L1 expression, both wildtype and mutated p53 play an important positive role for IFN-ɣ-induced PD-L1 expression in melanoma cells by supporting JAK2 expression. Future studies should address, whether p53 expression levels might influence response to anti-PD-1 immunotherapy.
Merkel cell carcinoma (MCC) is a rare and aggressive, yet highly immunogenic skin cancer. The latter is due to its viral or UV-associated carcinogenesis. For tumor progression MCC has to escape the ...host's immuno-surveillance, e.g. by loss of HLA class-I expression. Indeed, a reduced HLA class-I expression was observed in MCC tumor tissues and MCC cell lines. This reduced HLA class-I surface expression is caused by an impaired expression of key components of the antigen processing machinery (APM), including LMP2 and LMP7 as well as TAP1 and TAP2. Notably, experimental provisions of HLA class-I binding peptides restored HLA class-I surface expression on MCC cells. Silencing of the HLA class-I APM is due to histone deacetylation as inhibition of histone deacetylases (HDACs) not only induced acetylation of histones in the respective promoter regions but also re-expression of APM components. Thus, HDAC inhibition restored HLA class-I surface expression in vitro and in a mouse xenotransplantation model. In contrast to re-induction of HLA class-I by interferons, HDAC inhibitors did not interfere with the expression of immuno-dominant viral proteins. In summary, restoration of HLA class-I expression on MCC cells by epigenetic priming is an attractive approach to enhance therapies boosting adaptive immune responses.
The best characterized polyomavirus family member, i.e., simian virus 40 (SV40), can cause different tumors in hamsters and can transform murine and human cells in vitro. Hence, the SV40 ...contamination of millions of polio vaccine doses administered from 1955-1963 raised fears that this may cause increased tumor incidence in the vaccinated population. This is, however, not the case. Indeed, up to now, the only polyomavirus family member known to be the most important cause of a specific human tumor entity is Merkel cell polyomavirus (MCPyV) in Merkel cell carcinoma (MCC). MCC is a highly deadly form of skin cancer for which the cellular origin is still uncertain, and which appears as two clinically very similar but molecularly highly different variants. While approximately 80% of cases are found to be associated with MCPyV the remaining MCCs carry a high mutational load. Here, we present an overview of the multitude of molecular functions described for the MCPyV encoded oncoproteins and non-coding RNAs, present the available MCC mouse models and discuss the increasing evidence that both, virus-negative and -positive MCC constitute epithelial tumors.
Merkel cell carcinoma is a rare neuroendocrine carcinoma of the skin mostly induced by Merkel cell polyomavirus integration. Cytokeratin 20 (CK20) positivity is currently used to distinguish Merkel ...cell carcinomas from other neuroendocrine carcinomas. However, this distinction may be challenging in CK20-negative cases and in cases without a primary skin tumor. The objectives of this study were first to evaluate the diagnostic accuracy of previously described markers for the diagnosis of Merkel cell carcinoma and second to validate these markers in the setting of difficult-to-diagnose Merkel cell carcinoma variants. In a preliminary set (n = 30), we assessed optimal immunohistochemical patterns (CK20, thyroid transcription factor 1 TTF-1, atonal homolog 1 ATOH1, neurofilament NF, special AT-rich sequence-binding protein 2 SATB2, paired box protein 5, terminal desoxynucleotidyl transferase, CD99, mucin 1, and Merkel cell polyomavirus-large T antigen) and Merkel cell polyomavirus load thresholds (real-time PCR). The diagnostic accuracy of each marker was then assessed in a validation set of 103 Merkel cell carcinomas (9 CK20-negative cases and 15 cases without a primary skin tumor) and 70 extracutaneous neuroendocrine carcinoma cases. The most discriminant markers for a diagnosis of Merkel cell carcinoma were SATB2, NF expression, and Merkel cell polyomavirus DNA detection (positive likelihood ratios: 36.6, 44.4, and 28.2, respectively). Regarding Merkel cell carcinoma variants, cases without a primary skin tumor retained a similar immunohistochemical profile and CK20-negative tumors displayed a different profile (decrease frequency of NF and SATB2 expression), but Merkel cell polyomavirus DNA remained detected (78% of cases by qPCR). Moreover, 8/9 (89%) CK20-negative Merkel cell carcinoma cases but only 3/61 (5%) CK20-negative extracutaneous neuroendocrine cases were positive for at least one of these markers. In conclusion, detection of SATB2 and NF expression and Merkel cell polyomavirus DNA helps distinguish between Merkel cell carcinoma classical and variant cases and extracutaneous neuroendocrine carcinomas.
Merkel cell carcinoma (MCC) is a highly invasive and metastatic skin cancer. While high expression of miR-375 is a characteristic of MCC, it seems not to contribute to the malignant phenotype of MCC ...cells. miR-375 enrichment in MCC-derived extracellular vesicles suggests its intercellular signaling function. Here, we demonstrate that horizontally transferred miR-375 causes fibroblast polarization toward cancer-associated fibroblasts (CAFs). The polarization is evidenced by phenotypic changes and induction of α-SMA, CXCL2, and IL-1β. Fibroblast polarization is inhibited by specific antagomirs and mimicked by experimental miR-375 expression. Mechanistically, miR-375 downregulates RBPJ and p53, two key players regulating fibroblast polarization. In clinical MCC samples, in situ hybridization located miR-375 in CAFs, which correlated with high α-SMA protein and low RBPJ and TP53 expression; single-cell RNAseq revealed a disparate fibroblast polarization negatively correlating with p53 pathway-related gene expression. Thus, the functional role of miR-375 in MCC is to generate a pro-tumorigenic microenvironment by inducing fibroblast polarization.
Merkel cell carcinoma (MCC) is a highly aggressive, often lethal neuroendocrine cancer. Its carcinogenesis may be either caused by the clonal integration of the Merkel cell polyomavirus into the host ...genome or by UV-induced mutations. Notably, virally-encoded oncoproteins and UV-induced mutations affect comparable signaling pathways such as RB restriction of cell cycle progression or p53 inactivation. Despite its low incidence, MCC recently received much attention based on its exquisite immunogenicity and the resulting major success of immune modulating therapies. Here, we summarize current knowledge on epidemiology, biology and therapy of MCC as conclusion of the project ‘Immune Modulating strategies for treatment of Merkel Cell Carcinoma’, which was funded over a 5-year period by the European Commission to investigate innovative immunotherapies for MCC.
Merkel cell carcinoma (MCC) is a polyomavirus-associated skin cancer that is frequently lethal and lacks established prognostic biomarkers. This study sought to identify biomarkers that improve ...prognostic accuracy and provide insight into MCC biology.
Gene expression profiles of 35 MCC tumors were clustered based on prognosis. The cluster of genes overexpressed in good-prognosis tumors was tested for biologic process enrichment. Relevant mRNA expression differences were confirmed by quantitative polymerase chain reaction and immunohistochemistry. An independent set of 146 nonoverlapping MCC tumors (median follow-up, 25 months among 116 living patients) was employed for biomarker validation. Univariate and multivariate Cox regression analyses were performed.
Immune response gene signatures were prominent in patients with good prognoses. In particular, genes associated with cytotoxic CD8+ lymphocytes were overexpressed in tumors from patients with favorable prognoses. In the independent validation set, cases with robust intratumoral CD8+ lymphocyte infiltration had improved outcomes (100% MCC-specific survival, n = 26) compared with instances characterized by sparse infiltration (60% survival, n = 120). Only stage and intratumoral CD8 infiltration (but not age, sex, or CD8+ lymphocytes localized to the tumor-stroma interface) were significant in both univariate and multivariate Cox regression analyses. Notably, traditional histologic identification of tumor-infiltrating lymphocytes was not a significant independent predictor of survival.
Intratumoral CD8+ lymphocyte infiltration can be readily assessed on paraffin-embedded tissue, is independently associated with improved MCC-specific survival, and therefore, may provide prognostic information that enhances established MCC staging protocols.
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