IGFBP7 is not required for B-RAF-induced melanocyte senescence.

Induction of senescence permanently restricts cellular proliferation after oncogenic stimulation thereby acting as a potent barrier to tumor development. The relevant effector proteins may therefore be fundamental to cancer development. A recent study identified IGFBP7 as a secreted factor mediating melanocyte senescence induced by oncogenic B-RAF, which is found commonly in cutaneous nevi. In contrast to the previous report, we demonstrate that B-RAF signaling does not induce IGFBP7 expression, nor the expression of the IGFBP7 targets, BNIP3L, SMARCB1, or PEA15, in human melanocytes or fibroblasts. We also found no correlation between B-RAF mutational status and IGFBP7 protein expression levels in 22 melanoma cell lines, 90 melanomas, and 46 benign nevi. Furthermore, using a lentiviral silencing strategy we show that B-RAF induces senescence in melanocytes and fibroblasts, irrespective of the presence of IGFBP7. Therefore, we conclude that the secreted protein IGFBP7 is dispensable for B-RAF(V600E)-induced senescence in human melanocytes.

Merkel cell polyomavirus pan-T antigen knockdown reduces cancer cell stemness and promotes neural differentiation independent of RB1.

Merkel cell carcinoma (MCC) is a highly aggressive skin cancer associated with integration of Merkel cell polyomavirus (MCPyV). MCPyV-encoded T-antigens (TAs) are pivotal for sustaining MCC's oncogenic phenotype, i.e., repression of TAs results in reactivation of the RB pathway and subsequent cell cycle arrest. However, the MCC cell line LoKe, characterized by a homozygous loss of the RB1 gene, exhibits uninterrupted cell cycle progression after shRNA-mediated TA repression. This unique feature allows an in-depth analysis of the effects of TAs beyond inhibition of the RB pathway, revealing the decrease in expression of stem cell-related genes upon panTA-knockdown. Analysis of gene regulatory networks identified members of the E2F family (E2F1, E2F8, TFDP1) as key transcriptional regulators that maintain stem cell properties in TA-expressing MCC cells. Furthermore, minichromosome maintenance (MCM) genes, which encodes DNA-binding licensing proteins essential for stem cell maintenance, were suppressed upon panTA-knockdown. The decline in stemness occurred simultaneously with neural differentiation, marked by the increased expression of neurogenesis-related genes such as neurexins, BTG2, and MYT1L. This upregulation can be attributed to heightened activity of PBX1 and BPTF, crucial regulators of neurogenesis pathways. The observations in LoKe were confirmed in an additional MCPyV-positive MCC cell line in which RB1 was silenced before panTA-knockdown. Moreover, spatially resolved transcriptomics demonstrated reduced TA expression in situ in a part of a MCC tumor characterized by neural differentiation. In summary, TAs are critical for maintaining stemness of MCC cells and suppressing neural differentiation, irrespective of their impact on the RB-signaling pathway.

Gene fusions in poroma, porocarcinoma and related adnexal skin tumours: An update.

Poroma is a benign sweat gland tumour showing morphological features recapitulating the superficial portion of the eccrine sweat coil. A subset of poromas may transform into porocarcinoma, its malignant counterpart. Poroma and porocarcinoma are characterised by recurrent gene fusions involving YAP1, a transcriptional co-activator, which is controlled by the Hippo signalling pathway. The fusion genes frequently involve MAML2 and NUTM1, which are also rearranged in other cutaneous and extracutaneous neoplasms. We aimed to review the clinical, morphological and molecular features of this category of adnexal neoplasms with a special focus upon emerging differential diagnoses, and discuss how their systematic molecular characterisation may contribute to a standardisation of diagnosis, more accurate classification and, ultimately, refinement of their prognosis and therapeutic modalities.

Detection of wildtype Merkel cell polyomavirus genomic sequence and VP1 transcription in a subset of Merkel cell carcinoma.

AIMS: Merkel cell carcinoma (MCC) is frequently caused by the Merkel cell polyomavirus (MCPyV). Characteristic for these virus-positive (VP) MCC is MCPyV integration into the host genome and truncation of the viral oncogene Large T antigen (LT), with full-length LT expression considered as incompatible with MCC growth. Genetic analysis of a VP-MCC/trichoblastoma combined tumour demonstrated that virus-driven MCC can arise from an epithelial cell. Here we describe two further cases of VP-MCC combined with an adnexal tumour, i.e. one trichoblastoma and one poroma. METHODS AND RESULTS: Whole-genome sequencing of MCC/trichoblastoma again provided evidence of a trichoblastoma-derived MCC. Although an MCC-typical LT-truncating mutation was detected, we could not determine an integration site and we additionally detected a wildtype sequence encoding full-length LT. Similarly, Sanger sequencing of the combined MCC/poroma revealed coding sequences for both truncated and full-length LT. Moreover, in situ RNA hybridization demonstrated expression of a late region mRNA encoding the viral capsid protein VP1 in both combined as well as in a few cases of pure MCC. CONCLUSION: The data presented here suggest the presence of wildtype MCPyV genomes and VP1 transcription in a subset of MCC.

Interaction of transcription factor FoxO3 with histone acetyltransferase complex subunit TRRAP modulates gene expression and apoptosis.

Forkhead box O (FoxO) transcription factors are conserved proteins involved in the regulation of life span and age-related diseases, such as diabetes and cancer. Stress stimuli or growth factor deprivation promotes nuclear localization and activation of FoxO proteins, which-depending on the cellular context-can lead to cell cycle arrest or apoptosis. In endothelial cells (ECs), they further regulate angiogenesis and may promote inflammation and vessel destabilization implicating a role of FoxOs in vascular diseases. In several cancers, FoxOs exert a tumor-suppressive function by regulating proliferation and survival. We and others have previously shown that FoxOs can regulate these processes via two different mechanisms: by direct binding to forkhead-responsive elements at the promoter of target genes or by a poorly understood alternative process that does not require direct DNA binding and regulates key targets in primary human ECs. Here, we performed an interaction study in ECs to identify new nuclear FoxO3 interaction partners that might contribute to FoxO-dependent gene regulation. Mass spectrometry analysis of FoxO3-interacting proteins revealed transformation/transcription domain-associated protein (TRRAP), a member of multiple histone acetyltransferase complexes, as a novel binding partner of FoxO family proteins. We demonstrate that TRRAP is required to support FoxO3 transactivation and FoxO3-dependent G1 arrest and apoptosis in ECs via transcriptional activation of the cyclin-dependent kinase inhibitor p27kip1 and the proapoptotic B-cell lymphoma 2 family member, BIM. Moreover, FoxO-TRRAP interaction could explain FoxO-induced alternative gene regulation via TRRAP-dependent recruitment to target promoters lacking forkhead-responsive element sequences.

Distinct regulations driving YAP1 expression loss in poroma, porocarcinoma and RB1-deficient skin carcinoma.

AIMS: Recently, YAP1 fusion genes have been demonstrated in eccrine poroma and porocarcinoma, and the diagnostic use of YAP1 immunohistochemistry has been highlighted in this setting. In other organs, loss of YAP1 expression can reflect YAP1 rearrangement or transcriptional repression, notably through RB1 inactivation. In this context, our objective was to re-evaluate the performance of YAP1 immunohistochemistry for the diagnosis of poroma and porocarcinoma. METHODS AND RESULTS: The expression of the C-terminal part of the YAP1 protein was evaluated by immunohistochemistry in 543 cutaneous epithelial tumours, including 27 poromas, 14 porocarcinomas and 502 other cutaneous tumours. Tumours that showed a lack of expression of YAP1 were further investigated for Rb by immunohistochemistry and for fusion transcripts by real-time PCR (YAP1::MAML2 and YAP1::NUTM1). The absence of YAP1 expression was observed in 24 cases of poroma (89%), 10 porocarcinoma (72%), 162 Merkel cell carcinoma (98%), 14 squamous cell carcinoma (SCC) (15%), one trichoblastoma and one sebaceoma. Fusions of YAP1 were detected in only 16 cases of poroma (n = 66%), 10 porocarcinoma (71%) all lacking YAP1 expression, and in one sebaceoma. The loss of Rb expression was detected in all cases except one of YAP1-deficient SCC (n = 14), such tumours showing significant morphological overlap with porocarcinoma. In-vitro experiments in HaCat cells showed that RB1 knockdown resulted in repression of YAP1 protein expression. CONCLUSION: In addition to gene fusion, we report that transcriptional repression of YAP1 can be observed in skin tumours with RB1 inactivation, including MCC and a subset of SCC.

Recurrent FOXK1::GRHL and GPS2::GRHL fusions in trichogerminoma.

We report 21 cases of trichogerminoma harbouring previously undescribed FOXK1::GRHL1/2 or GPS2::GRHL1/2/3 in-frame fusion transcripts. Microscopic examination of a preliminary set of five cases revealed well-delimitated tumours located in the dermis with frequent extension to the subcutaneous tissue. Tumours presented a massive and nodular architecture and consisted of a proliferation of basaloid cells. A biphasic pattern sometime resulting in tumour cell nests ('cell balls') was present. Immunohistochemistry demonstrated the expression of cytokeratins (CKs) 15, 17, and PHLDA1. In addition, numerous CK20-positive Merkel cells were detected. RNA sequencing (RNA-seq) revealed a FOXK1::GRHL1 chimeric transcript in three cases and a FOXK1::GRHL2 fusion in two cases. In a second series for validation (n = 88), FOXK1::GRHL1/2 fusion transcripts were detected by RT-qPCR or FISH in an additional 12 trichogerminomas and not in any other follicular tumour entities or basal cell carcinoma cases (n = 66). Additional RNA-seq analysis in trichogerminoma cases without detected FOXK1::GRHL1/2 rearrangements revealed GPS2::GRHL1 fusion transcripts in two cases, GPS2::GRHL2 in one case, and GPS2::GRHL3 fusion transcript in one case. Therefore, our study strongly suggests that GRHL1/2/3 gene rearrangements might represent the oncogenic driver in trichogerminoma, a subset of follicular tumours characterized by immature features and numerous Merkel cells. © 2022 The Pathological Society of Great Britain and Ireland.

Homozygous CRISPR/Cas9 Knockout Generated a Novel Functionally Active Exon 1 Skipping XPA Variant in Melanoma Cells.

Defects in DNA repair pathways have been associated with an improved response to immune checkpoint inhibition (ICI). In particular, patients with the nucleotide excision repair (NER) defect disease Xeroderma pigmentosum (XP) responded impressively well to ICI treatment. Recently, in melanoma patients, pretherapeutic XP gene expression was predictive for anti-programmed cell death-1 (PD-1) ICI response. The underlying mechanisms of this finding are still to be revealed. Therefore, we used CRISPR/Cas9 to disrupt XPA in A375 melanoma cells. The resulting subclonal cell lines were investigated by Sanger sequencing. Based on their genetic sequence, candidates from XPA exon 1 and 2 were selected and further analyzed by immunoblotting, immunofluorescence, HCR and MTT assays. In XPA exon 1, we established a homozygous (c.19delG; p.A7Lfs*8) and a compound heterozygous (c.19delG/c.19_20insG; p.A7Lfs*8/p.A7Gfs*55) cell line. In XPA exon 2, we generated a compound heterozygous mutated cell line (c.206_208delTTG/c.208_209delGA; p.I69_D70delinsN/p.D70Hfs*31). The better performance of the homozygous than the heterozygous mutated exon 1 cells in DNA damage repair (HCR) and post-UV-C cell survival (MTT), was associated with the expression of a novel XPA protein variant. The results of our study serve as the fundamental basis for the investigation of the immunological consequences of XPA disruption in melanoma.

Characterization of six Merkel cell polyomavirus-positive Merkel cell carcinoma cell lines: Integration pattern suggest that large T antigen truncating events occur before or during integration.

Merkel cell carcinoma (MCC), an aggressive neuroendocrine skin tumor, is a polyomavirus-induced human cancer. To study the causal relationship of MCC carcinogenesis with the integrated Merkel cell polyomavirus (MCPyV) in detail, well-characterized MCC cell lines are needed. Consequently, in the current study, we established and characterized six MCPyV-positive MCC cell lines. Microarray-based comparative genomic hybridization revealed a stable genome carrying only a limited number of chromosomal gains and deletions. All cell lines expressed MCC markers Keratin-20 and neuron-specific enolase as well as truncated MCPyV-encoded large T antigen (LT). For five cell lines, we were able to identify the MCPyV-integration sites in introns of different genes. The LT-truncating stop codon mutations and integration sites were affirmed in the respective clinical patient samples. Inverse PCR suggested that three of the cell lines contained MCPyV genomes as concatemers. This notion was confirmed for the two cell lines with known integration sites. Importantly, our observation of distinct stop codon mutations in cell lines with concatemeric MCPyV integration indicates that these LT-truncating mutations occur before integration. In summary, we provide the detailed characterization of six MCPyV-positive MCC cell lines, which are likely to serve as valuable tools in future MCC research.

The identification of patient-specific mutations reveals dual pathway activation in most patients with melanoma and activated receptor tyrosine kinases in BRAF/NRAS wild-type melanomas.

BACKGROUND: Increasing knowledge of cancer genomes has triggered the development of specific targeted inhibitors, thus providing a valuable therapeutic pool. METHODS: In this report, the authors analyze the presence of targetable alterations in 136 tumor samples from 92 patients with melanoma using a comprehensive approach based on targeted DNA sequencing and supported by RNA and protein analysis. Three topics of high clinical relevance are addressed: the identification of rare, activating alterations; the detection of patient-specific, co-occurring single nucleotide variants (SNVs) and copy number variations (CNVs) in parallel pathways; and the presence of cancer-relevant germline mutations. RESULTS: The analysis of patient-matched blood and tumor samples was done with a custom-designed gene panel that was enriched for genes from clinically targetable pathways. To detect alterations with high therapeutic relevance for patients with unknown driver mutations, genes that are untypical for melanoma also were included. Among all patients, CNVs were identified in one-third of samples and contained amplifications of druggable kinases, such as CDK4, ERBB2, and KIT. Considering SNVs and CNVs, 60% of patients with metastases exhibited co-occurring activations of at least 2 pathways, thus providing a rationale for individualized combination therapies. Unexpectedly, 9% of patients carry potentially protumorigenic germline mutations frequently affecting receptor tyrosine kinases. Remarkably two-thirds of BRAF/NRAS wild-type melanomas harbor activating mutations or CNVs in receptor tyrosine kinases. CONCLUSIONS: The results indicate that the integrated analysis of SNVs, CNVs, and germline mutations reveals new druggable targets for combination tumor therapy.

Diagnostic accuracy of a panel of immunohistochemical and molecular markers to distinguish Merkel cell carcinoma from other neuroendocrine carcinomas.

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.

Morphologic and immunophenotypical features distinguishing Merkel cell polyomavirus-positive and negative Merkel cell carcinoma.

In 2008, Feng et al. identified Merkel cell polyomavirus integration as the primary oncogenic event in ~80% of Merkel cell carcinoma cases. The remaining virus-negative Merkel cell carcinoma cases associated with a high mutational load are most likely caused by UV radiation. The current study aimed to compare the morphological and immunohistochemical features of 80 virus-positive and 21 virus-negative Merkel cell carcinoma cases. Microscopic evaluation revealed that elongated nuclei-similar to the spindle-shape variant of small cell lung cancer-were less frequent in Merkel cell polyomavirus-positive Merkel cell carcinoma compared to the virus-negative subset (p = 0.005). Moreover, virus-negative cases more frequently displayed a "large-cell neuroendocrine carcinoma" phenotype with larger cell size (p = 0.0026), abundant cytoplasm (p = 4×10-7) and prominent nucleoli (p = 0.002). Analysis of immunohistochemical data revealed frequent positivity for thyroid transcription factor 1 and cytokeratin 7, either absence or overexpression of p53, as well as frequent lack of neurofilament expression in virus-negative cases. By contrast, cytokeratin 8, 18 and 20 and a CD99 with a dot pattern as well as high EMA expression were identified as characteristic features of virus-positive Merkel cell carcinoma. In particular, the CD99 dot-like expression pattern was strongly associated with presence of the Merkel cell polyomavirus in Merkel cell carcinoma (sensitivity = 81%, specificity = 90%, positive likelihood ratio = 8.08). To conclude, virus-positive and -negative Merkel cell carcinoma are characterized by distinct morphological and immunohistochemical features, which implies a significant difference in tumor biology and behavior. Importantly, we identified the CD99 staining pattern as a marker indicating the virus status of this skin cancer.

Epidemiology, biology and therapy of Merkel cell carcinoma: conclusions from the EU project IMMOMEC.

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 carcinomas infiltrated with CD33+ myeloid cells and CD8+ T cells are associated with improved outcome.

BACKGROUND: Merkel cell carcinoma (MCC) is a rare tumor of the skin that has an aggressive behavior. Immunity is the main regulator of MCC development, and many interactions between lymphocytes and tumor cells have been proven. However, the impact of tumor-infiltrating myeloid cells needs better characterization. OBJECTIVE: To characterize tumor-infiltrating myeloid cells in MCC and their association with other immune effectors and patient outcome. METHODS: MCC cases were reviewed from an ongoing prospective cohort study. In all, 103 triplicate tumor samples were included in a tissue microarray. Macrophages, neutrophils, and myeloid-derived suppressor cells were characterized by the following markers: CD68, CD33, CD163, CD15, CD33, and human leukocyte antigen-DR. Associations of these cell populations with programmed cell death ligand 1 expression, CD8 infiltrates, and vascular density were assessed. Impact on survival was analyzed by log-rank tests and a Cox multivariate model. RESULTS: The median density of macrophages was 216 cells/mm2. CD68+ and CD33+ macrophage densities were associated with CD8+ T-cell infiltrates and programmed cell death ligand 1 expression. In addition, MCC harboring CD8+ T cell infiltrates and brisk CD33+ myeloid cell infiltrates were significantly and independently associated with improved outcomes (recurrence-free and overall survival). LIMITATIONS: Sampling bias and the retrospective design were potential study limitations. CONCLUSION: Infiltration of CD33+ myeloid cells and CD8+ T lymphocytes defines a subset of MCC associated with improved outcome.

Serine 220 phosphorylation of the Merkel cell polyomavirus large T antigen crucially supports growth of Merkel cell carcinoma cells.

Merkel cell polyomavirus (MCPyV) is regarded as a major causal factor for Merkel cell carcinoma (MCC). Indeed, tumor cell growth of MCPyV-positive MCC cells is dependent on the expression of a truncated viral Large T antigen (LT) with an intact retinoblastoma protein (RB)-binding site. Here we determined the phosphorylation pattern of a truncated MCPyV-LT characteristically for MCC by mass spectrometry revealing MCPyV-LT as multi-phospho-protein phosphorylated at several serine and threonine residues. Remarkably, disruption of most of these phosphorylation sites did not affect its ability to rescue knockdown of endogenous T antigens in MCC cells indicating that phosphorylation of the respective amino acids is not essential for the growth promoting function of MCPyV-LT. However, alteration of serine 220 to alanine completely abolished the ability of MCPyV-LT to support proliferation of MCC cells. Conversely, mimicking the phosphorylated state by mutation of serine 220 to glutamic acid resulted in a fully functional LT. Moreover, MCPyV-LT(S220A) demonstrated reduced binding to RB in co-immunoprecipitation experiments as well as weaker induction of RB target genes in MCC cells. In conclusion, we provide evidence that phosphorylation of serine 220 is required for efficient RB inactivation in MCC and may therefore be a potential target for future therapeutic approaches.

Characterization of functional domains in the Merkel cell polyoma virus Large T antigen.

Merkel cell polyomavirus (MCPyV)--positive Merkel cell carcinoma (MCC) tumor cell growth is dependent on the expression of a viral Large T antigen (LT) with an intact retinoblastoma protein (RB)-binding site. This RB-binding domain in MCPyV-LT is--in contrast to other polyomavirus LTs (e.g., SV40)--embedded between two large MCPyV unique regions (MUR1 and MUR2). To identify elements of the MCPyV-LT necessary for tumor cell growth, we analyzed the rescue activity of LT variants following knockdown of the endogenous LT in MCC cells. These experiments demonstrate that nuclear localization is essential for LT function, but that a motif previously described to be a nuclear localization sequence is neither required for nuclear accumulation of truncated MCPyV-LT nor for promotion of MCC cell proliferation. Furthermore, large parts of the MURs distal to the RB binding domain as well as ALTO--a second protein encoded by an alternative reading frame in the MCPyV-LT mRNA--are completely dispensable for MCPyV-driven tumor cell proliferation. Notably, even MCPyV-LTs in which the entire MURs have been removed are still able to promote MCC cellular growth although rescue activity is reduced which may be due to MUR1 being required for stable LT expression in MCC cells. Finally, we provide evidence implying that--while binding to Vam6p is not essential--HSC-70 interaction is significantly involved in mediating MCPyV-LT function in MCC cells including growth promotion and induction of E2F target genes.