Merkel Cell Carcinoma Cell Research Articles

Clinical significance of the expression of FOXP3 and TIGIT in Merkel cell carcinoma

The pathogenesis of 80% of Merkel cell carcinoma (MCC) cases is associated with Merkel cell polyomavirus (MCPyV). Forkhead helix transcription factor P3 (FOXP3) and the T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibition motif domains (TIGIT)–CD155 pathway, which are targets for immunotherapy, were assessed as prognostic factors of MCC. We analyzed mRNA expression data of 111 patients with MCC and performed immunohistochemical analysis to detect the expression of programmed death ligand 1 (PD-L1), CD8, FOXP3, TIGIT, and CD155 in 65 cases of MCC. In CD8 and FOXP3 immunostaining, the number of expressing-infiltrating cells was determined by dividing the region into tumor center and invasive front areas. FOXP3 expression was evaluated separately in cells with high and low intensities. Aberrant TIGIT expression and weak CD155 staining were observed in MCC cells. CD8- and FOXP3-positive cell infiltrations were higher in the invasive front than in the tumor center. Multivariate Cox hazard analysis revealed that high infiltration of cells with low-intensity FOXP3 expression in the invasive front is a favorable prognostic factor (p = 0.025). Thus, targeting TIGIT–CD155 signaling and FOXP3 as well as PD-L1 may be a therapeutic strategy for MCC.

Single-cell dissection of Merkel cell carcinoma heterogeneity unveils transcriptomic plasticity and therapeutic vulnerabilities

Merkel cell carcinoma (MCC), a rare but aggressive skin cancer, remains a challenge in the era of precision medicine. Immune checkpoint inhibitors (ICIs), the only approved therapy for advanced MCC, are impeded by high primary and acquired resistance. Hence, we dissect transcriptomic heterogeneity at single-cell resolution in a panel of patient tumors, revealing phenotypic plasticity in a subset of treatment-naive MCC. The tumor cells in a "mesenchymal-like" state are endowed with an inflamed phenotype that portends a better ICI response. This observation is also validated in the largest whole transcriptomic dataset available from MCC patient tumors. In contrast, ICI-resistant tumors predominantly express neuroepithelial markers in a well-differentiated state with "immune-cold" landscape. Importantly, a subtle shift to "mesenchymal-like" state reverts copanlisib resistance in primary MCC cells, highlighting potential strategies in patient stratification for therapeutics to harness tumor cell plasticity, augment treatment efficacy, and avert resistance.

The effect of GP-2250 on cultured virus-negative Merkel cell carcinoma cells: preliminary results.

Even in the novel immunotherapy era, Merkel cell carcinoma (MCC) remains challenging in its treatment. Apart from Merkel cell polyomavirus (MCPyV) associated MCC, this cancer is linked in about 20% of cases to ultraviolet-induced mutational burden frequently causing aberrations in Notch and PI3K/AKT/mTOR signalling pathways. The recently developed agent GP-2250 is capable to inhibit growth of cells of different cancers, including pancreatic neuroendocrine tumors. The objective of the present study was to investigate the effects of GP-2250 on MCPyV-negative MCC cells. MethodsWe employed three cell lines (MCC13, MCC14.2, MCC26) which were exposed to different GP-2250doses. GP-2250's effects on cell viability, proliferation, and migration were evaluated by means of MTT, BrdU, and scratch assays, respectively. Flow cytometry was performed for the evaluation of apoptosis and necrosis. Western blotting was implemented for the determination of AKT, mTOR, STAT3, and Notch1 protein expression. Cell viability, proliferation, and migration decreased with increasing GP-2250 doses. Flow cytometry revealed a dose response to GP-2250 in all three MCC cell lines. While the viable fraction decreased, the share of necrotic and in a smaller amount the apoptotic cells increased. Regarding Notch1, AKT, mTOR, and STAT3 expression a comparatively time- and dose-dependent decrease of protein expression in the MCC13 and MCC26 cell lines was observed. By contrast, Notch1, AKT, mTOR, and STAT3 expression in MCC14.2 was scarcely altered or even increased by the three dosages of GP-2250 applied. The present study indicates GP-2250 having anti-neoplastic effects in MCPyV-negative tumor cells in regard to viability, proliferation, and migration. Moreover, the substance is capable of downregulating protein expression of aberrant tumorigenic pathways in MCPyV-negative MCC cells.

Methyltransferase-independent function of enhancer of zeste homologue 2 maintains tumorigenicity induced by human oncogenic papillomavirus and polyomavirus

Merkel cell polyomavirus (MCV) and high-risk human papillomavirus (HPV) are human tumor viruses that cause Merkel cell carcinoma (MCC) and oropharyngeal squamous cell carcinoma (OSCC), respectively. HPV E7 and MCV large T (LT) oncoproteins target the retinoblastoma tumor suppressor protein (pRb) through the conserved LxCxE motif. We identified enhancer of zeste homolog 2 (EZH2) as a common host oncoprotein activated by both viral oncoproteins through the pRb binding motif. EZH2 is a catalytic subunit of the polycomb 2 (PRC2) complex that trimethylates histone H3 at lysine 27 (H3K27me3). In MCC tissues EZH2 was highly expressed, irrespective of MCV status. Loss-of-function studies revealed that viral HPV E6/E7 and T antigen expression are required for Ezh2 mRNA expression and that EZH2 is essential for HPV(+)OSCC and MCV(+)MCC cell growth. Furthermore, EZH2 protein degraders reduced cell viability efficiently and rapidly in HPV(+)OSCC and MCV(+)MCC cells, whereas EZH2 histone methyltransferase inhibitors did not affect cell proliferation or viability within the same treatment period. These results suggest that a methyltransferase-independent function of EZH2 contributes to tumorigenesis downstream of two viral oncoproteins, and that direct targeting of EZH2 protein expression could be a promising strategy for the inhibition of tumor growth in HPV(+)OSCC and MCV(+)MCC patients.

Effect of selinexor on lipogenesis in virus-positive Merkel cell carcinoma cell lines.

Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine cutaneous carcinoma aetiologically linked to the Merkel cell polyomavirus (MCPyV). Immune checkpoint inhibitors are currently the first-line therapy for metastatic MCC; however, the treatment is effective in only about half of patients, highlighting the need for alternative therapies. Selinexor (KPT-330) is a selective inhibitor of nuclear exportin 1 (XPO1) and has been shown to inhibit MCC cell growth in vitro, but the pathogenesis has not been established. Decades of research have established that cancer cells significantly upregulate lipogenesis to meet an increased demand for fatty acids and cholesterol. Treatments that inhibit lipogenic pathways may halt cancer cell proliferation. To determine the effect of increasing doses of selinexor on fatty acid and cholesterol synthesis in MCPyV-positive MCC (MCCP) cell lines and aid in elucidating the mechanism by which selinexor prevents and reduces MCC growth. MKL-1 and MS-1 cell lines were treated with increasing doses of selinexor for 72 h. Protein expression quantification was determined using chemiluminescent Western immunoblotting and densitometric analysis. Fatty acids and cholesterol were quantified using free fatty acid assay and cholesterol ester detection kits. Selinexor causes statistically significant reductions of the lipogenic transcription factors sterol regulatory element-binding proteins 1 and 2, and lipogenic enzymes acetyl-CoA carboxylase, fatty acid synthase, squalene synthase and 3β-hydroxysterol Δ-24-reductase in a dose-dependent manner in two MCCP cell lines. Although inhibiting the fatty acid synthesis pathway results in meaningful decreases in fatty acids, the cellular cholesterol levels did not demonstrate such reductions. For patients with metastatic MCC refractory to immune checkpoint inhibitors, selinexor may provide clinical benefit through the inhibition of the lipogenesis pathway; however, further research and clinical trials are needed to evaluate these findings.

4-[(5-Methyl-1H-pyrazol-3-yl)amino]-2H-phenyl-1-phthalazinone Inhibits MCPyV T Antigen Expression in Merkel Cell Carcinoma Independent of Aurora Kinase A.

Merkel cell carcinoma (MCC) is frequently caused by the Merkel cell polyomavirus (MCPyV), and MCPyV-positive tumor cells depend on expression of the virus-encoded T antigens (TA). Here, we identify 4-[(5-methyl-1H-pyrazol-3-yl)amino]-2H-phenyl-1-phthalazinone (PHT)-a reported inhibitor of Aurora kinase A-as a compound inhibiting growth of MCC cells by repressing noncoding control region (NCCR)-controlled TA transcription. Surprisingly, we find that TA repression is not caused by inhibition of Aurora kinase A. However, we demonstrate that β-catenin-a transcription factor repressed by active glycogen synthase kinase 3 (GSK3)-is activated by PHT, suggesting that PHT bears a hitherto unreported inhibitory activity against GSK3, a kinase known to function in promoting TA transcription. Indeed, applying an in vitro kinase assay, we demonstrate that PHT directly targets GSK3. Finally, we demonstrate that PHT exhibits in vivo antitumor activity in an MCC xenograft mouse model, suggesting a potential use in future therapeutic settings for MCC.

The HDAC inhibitor domatinostat induces type I interferon α in Merkel cell carcinoma by HES1 repression

BackgroundClass I selective histone deacetylase inhibitors (HDACi) have been previously demonstrated to not only increase major histocompatibility complex class I surface expression in Merkel cell carcinoma (MCC) cells by restoring the antigen processing and presentation machinery, but also exert anti-tumoral effect by inducing apoptosis. Both phenomena could be due to induction of type I interferons (IFN), as has been described for HDACi. However, the mechanism of IFN induction under HDACi is not fully understood because the expression of IFNs is regulated by both activating and inhibitory signaling pathways. Our own preliminary observations suggest that this may be caused by suppression of HES1.MethodsThe effect of the class I selective HDACi domatinostat and IFNα on cell viability and the apoptosis of MCPyV-positive (WaGa, MKL-1) and -negative (UM-MCC 34) MCC cell lines, as well as, primary fibroblasts were assessed by colorimetric methods or measuring mitochondrial membrane potential and intracellular caspase-3/7, respectively. Next, the impact of domatinostat on IFNA and HES1 mRNA expression was measured by RT-qPCR; intracellular IFNα production was detected by flow cytometry. To confirm that the expression of IFNα induced by HDACi was due to the suppression of HES1, it was silenced by RNA interference and then mRNA expression of IFNA and IFN-stimulated genes was assessed.ResultsOur studies show that the previously reported reduction in viability of MCC cell lines after inhibition of HDAC by domatinostat is accompanied by an increase in IFNα expression, both of mRNA and at the protein level. We confirmed that treatment of MCC cells with external IFNα inhibited their proliferation and induced apoptosis. Re-analysis of existing single-cell RNA sequencing data indicated that induction of IFNα by domatinostat occurs through repression of HES1, a transcriptional inhibitor of IFNA; this was confirmed by RT-qPCR. Finally, siRNA-mediated silencing of HES1 in the MCC cell line WaGa not only increased mRNA expression of IFNA and IFN-stimulated genes but also decreased cell viability.ConclusionOur results demonstrate that the direct anti-tumor effect of HDACi domatinostat on MCC cells is at least in part mediated via decreased HES1 expression allowing the induction of IFNα, which in turn causes apoptosis.

Distinct Regulation of EZH2 and its Repressive H3K27me3 Mark in Polyomavirus-Positive and -Negative Merkel Cell Carcinoma

Merkel cell carcinoma (MCC) is an aggressive skin cancer for which Merkel cell polyomavirus integration and expression of viral oncogenes small T and Large T have been identified as major oncogenic determinants. Recently, a component of the PRC2 complex, the histone methyltransferase enhancer of zeste homolog 2 (EZH2) that induces H3K27 trimethylation as a repressive mark has been proposed as a potential therapeutic target in MCC. Because divergent results have been reported for the levels of EZH2 and trimethylation of lysine 27 on histone 3, we analyzed these factors in a large MCC cohort to identify the molecular determinants of EZH2 activity in MCC and to establish MCC cell lines' sensitivity to EZH2 inhibitors. Immunohistochemical expression of EZH2 was observed in 92% of MCC tumors (156 of 170), with higher expression levels in virus-positive than virus-negative tumors (P= 0.026). For the latter, we showed overexpression of EZHIP, a negative regulator of the PRC2 complex. Invitro, ectopic expression of the large T antigen in fibroblasts led to the induction of EZH2 expression, whereas the knockdown of T antigens in MCC cell lines resulted in decreased EZH2 expression. EZH2 inhibition led to selective cytotoxicity on virus-positive MCC cell lines. This study highlights the distinct mechanisms of EZH2 induction between virus-negative and -positive MCC.

B-cell lymphoma-extra large is a promising drug target in Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is an aggressive skin tumour with neuroendocrine differentiation. Immunotherapies are effective in the treatment of patients with advanced-stage MCC, but for patients whose tumours cannot be controlled by the immune system, alternative approaches are urgently needed. To identify overexpressed oncogenes as potential drug targets for MCC. NanoString platform, digital droplet polymerase chain reaction (ddPCR) and fluorescence in situ hybridization (FISH) assays were used to determine copy number variations (CNVs); BCL2L1 and PARP1 mRNA expression levels were determined by quantitative real-time polymerase chain reaction (qRT-PCR), B-cell lymphoma extra-large (Bcl-xL) and poly (ADP-ribose) polymerase 1 (PARP1) protein by immuno-blot. Specific Bcl-xL inhibitors and a PARP1 inhibitor were used alone or in combination to test their antitumour effect. Screening for CNVs in 13 classic Merkel cell polyomavirus (MCPyV)-positive and MCPyV-negative MCC cell lines revealed BCL2L1 gains and amplifications, confirmed by ddPCR in 10 cell lines. By ddPCR and FISH, we demonstrated that BCL2L1 gains are present in tumour tissue. BCL2L1 copy number gains were associated with increased Bcl-xL mRNA and protein expression. However, high Bcl-xL expression was not restricted to MCC cells harbouring a BCL2L1 gain/amplification, suggesting additional epigenetic means of regulation. The functional relevance of Bcl-xL in MCC cells was demonstrated by the fact that specific Bcl-xL inhibitors (A1331852 and WEHI-539) led to the induction of apoptosis. Owing to the strong expression and activation of PARP1 in MCC cell lines, we next tested the combination of Bcl-xL inhibitors with the PARP1 inhibitor olaparib, which showed synergistic antitumour effects. Bcl-xL, which is highly expressed in MCC, appears to be an attractive therapeutic target for the treatment of this tumour, especially as the effect of specific Bcl-xL inhibitors is synergistically enhanced by simultaneous PARP inhibition.

Now you see me: lights on Merkel Cell Carcinoma

Merkel Cell Carcinoma (MCC) is a rare primary cutaneous cancer with aggressive behaviour and poor prognosis. Although MCC cells express somatostatin receptors (SSTR), SSTR-targeted PET/CT is not routinely performed in clinical practice. In contrast, the use of [18F]FDG PET/CT is more widespread and its prognostic role is well established. We present the case of an MCC patient suspected recurrence who underwent restaging with both [18F]FDG and [68 Ga]Ga-DOTA-TOC PET/CT. [18F]FDG PET/CT showed pathological uptake only in mediastinal lymph nodes, but SSTR imaging also revealed multiple liver and skeletal metastases, leading to significant disease upstaging and relevant changes in the therapeutic management.

Combined Merkel Cell Carcinoma with Nodal Presentation: Report of a Case Diagnosed with Excisional but Not Incisional Biopsy and Literature Review.

Merkel cell carcinoma (MCC) is a rare primary neuroendocrine carcinoma (NEC) of the skin. As compared to pure MCCs, combined MCCs are aggressive and exhibit a higher probability of metastasis. A correct diagnosis might be missed, especially when the biopsy sample is too small or too superficial. We report a 79-year-old Taiwanese male who presented with lymphadenopathy suspicious for lymphoma. A nodal biopsy showed metastatic NEC. A skin tumor in the lower back was identified, and an incisional biopsy showed only squamous cell carcinoma (SCC). A subsequent excisional biopsy was performed based on the advice of the senior pathologist because of the presence of metastatic nodal NEC. Finally, a diagnosis of combined MCC and SCC was confirmed. Our literature review identified 13 cases of combined MCC with nodal metastasis as initial presentations, all with an aggressive clinical course. Both the MCC and non-MCC components could be present in the metastatic nodes. Metastases of pure MCC cells were observed in three combined MCCs in sun-protected areas, probably pointing to a distinct pathogenesis. Excision or punch biopsy to include the deep dermal NEC component is recommended as timely diagnosis is mandatory for appropriate management of patients with this rare skin cancer.

The Antineoplastic Effect of Dimethyl Fumarate on Virus-Negative Merkel Cell Carcinoma Cell Lines: Preliminary Results.

Merkel cell carcinoma (MCC) is a rare, difficult-to-treat skin cancer once immunotherapy has failed. MCC is associated either with the clonal integration of the Merkel cell polyomavirus (MCPyV) or mutagenic UV-radiation. Fumaric acid esters, including dimethyl fumarate (DMF), have been shown to inhibit cell growth in cutaneous melanoma and lymphoma. We aimed to explore the effects of DMF on MCPyV-negative MCC cell lines. Three MCC cell lines (MCC13, MCC14.2, and MCC26) were treated with different doses of DMF. The cytotoxic effects and cell proliferation were assessed by the MTT cytotoxicity assay and BrdU proliferation assay at different time points. A significant reduction in cell viability and proliferation were demonstrated for all the cell lines used, with DMF proving to be effective.

Regulation of Transcriptional Activity of Merkel Cell Polyomavirus Large T-Antigen by PKA-Mediated Phosphorylation.

Merkel cell polyomavirus (MCPyV) is the major cause of Merkel cell carcinoma (MCC), an aggressive skin cancer. MCPyV large T-antigen (LTag) and small T-antigen (sTag) are the main oncoproteins involved in MCPyV-induced MCC. A hallmark of MCPyV-positive MCC cells is the expression of a C-terminal truncated LTag. Protein kinase A (PKA) plays a fundamental role in a variety of biological processes, including transcription by phosphorylating and thereby regulating the activity of transcription factors. As MCPyV LTag has been shown to be phosphorylated and acts as a transcription factor for the viral early and late promoter, we investigated whether LTag can be phosphorylayted by PKA, and whether this affects the transcript activity of LTag. Using a phosphorylation prediction algorithm, serine 191, 203, and 265 were identified as putative phosphorylation sites for PKA. Mass spectrometry of in vitro PKA-phosphorylated peptides confirmed phosphorylation of S203 and S265, but not S191. Full-length LTag inhibited early and late promoter activity of MCPyV, whereas the truncated MKL2 LTag variant stimulated both promoters. Single non-phosphorylable, as well as phosphomimicking mutations did not alter the inhibitory effect of full-length LTag. However, the non-phosphorylable mutations abrogated transactivation of the MCPyV promoters by MKL2 LTag, whereas phosphomimicking substitutions restored the ability of MKL2 LTag to activate the promoters. Triple LTag and MKL2 LTag mutants had the same effect as the single mutants. Activation of the PKA signaling pathway did not enhance MCPyV promoter activity, nor did it affect LTag expression levels in MCPyV-positive Merkel cell carcinoma (MCC) cells. Our results show that phosphorylation of truncated LTag stimulates viral promoter activity, which may contribute to higher levels of the viral oncoproteins LTag and sTag. Interfering with PKA-induced LTag phosphorylation/activity may be a therapeutic strategy to treat MCPyV-positive MCC patients.

Merkel cell polyomavirus small T antigen is a viral transcription activator that is essential for viral genome maintenance.

Merkel cell polyomavirus (MCV) is a small DNA tumor virus that persists in human skin and causes Merkel cell carcinoma (MCC) in immunocompromised individuals. The multi-functional protein MCV small T (sT) activates viral DNA replication by stabilizing large T (LT) and promotes cell transformation through the LT stabilization domain (LTSD). Using MCVΔsT, a mutant MCV clone that ablates sT, we investigated the role of sT in MCV genome maintenance. sT was dispensable for initiation of viral DNA replication, but essential for maintenance of the MCV genome and activation of viral early and late gene expression for progression of the viral lifecycle. Furthermore, in phenotype rescue studies, exogenous sT activated viral DNA replication and mRNA expression in MCVΔsT through the LTSD. While exogenous LT expression, which mimics LT stabilization, increased viral DNA replication, it did not activate viral mRNA expression. After cataloging transcriptional regulator proteins by proximity-based MCV sT-host protein interaction analysis, we validated LTSD-dependent sT interaction with four transcriptional regulators: Cux1, c-Jun, BRD9, and CBP. Functional studies revealed Cux1 and c-Jun as negative regulators, and CBP and BRD9 as positive regulators of MCV transcription. CBP inhibitor A-485 suppressed sT-induced viral gene activation in replicating MCVΔsT and inhibited early gene expression in MCV-integrated MCC cells. These results suggest that sT promotes viral lifecycle progression by activating mRNA expression and capsid protein production through interaction with the transcriptional regulators. This activity is essential for MCV genome maintenance, suggesting a critical role of sT in MCV persistence and MCC carcinogenesis.

451 Blockade of tumor cell-intrinsic PD-1 inhibits Merkel cell carcinoma growth

Merkel Cell Carcinoma (MCC) is a rare, but highly aggressive cancer of the skin. In MCC patients, inhibitors targeting the programmed cell death-1 (PD-1) immune checkpoint pathway have yielded remarkable antitumor activity by stimulating cancer-specific T-cell immunity. Here, we identify a previously unknown role for PD-1 as a growth-promoting receptor intrinsic to MCC cells. In four established human MCC cell lines, MKL-1, MKL-2, MS-1, and WaGa, RT-PCR amplification and sequencing revealed expression of the full PD-1 (PDCD1) mRNA coding sequence.

Genomic integration and expression of Felis catus papillomavirus type 2 oncogenes in feline Merkel cell carcinoma.

The involvement of Felis catus papillomavirus type 2 (FcaPV2) in feline Merkel cell carcinoma (MCC) has been previously hypothesized. In this study, the expression and localization of FcaPV2 oncogene mRNA, the integration of FcaPV2 genes, and p53 mutations in feline MCC were examined by RNAscope in situ hybridization (ISH), whole genome sequencing (WGS), and Sanger DNA sequencing, respectively. Furthermore, the morphological and molecular characteristics of FcaPV2-positive (FMX-MCC01) and FcaPV2-negative (AS-MCC01) MCC cell lines were compared in vitro and in vivo using immunofluorescence, ISH, xenotransplantation into mice, and immunohistochemistry. ISH for FcaPV2 E6/E7 detected viral RNA in 18/21 FcaPV2-positive MCC and not in 1/1 FcaPV2-negative MCC. WGS of 2 FcaPV2-positive cases revealed the integration of FcaPV2 genes in both cases. In cultured cells and xenograft tissues of FMX-MCC01, most cells were positive for E6/E7 by ISH and p16CDKN2A, a few cells were positive for the retinoblastoma protein (pRb), and all cells were negative for p53. In cultured cells and xenograft tissues of AS-MCC01, all cells were negative for p16CDKN2A, most cells were positive for pRb, and some cells were positive for p53. Missense mutations in p53 were identified in 8/10 FcaPV2-positive and 1/1 FcaPV2-negative MCC. These results suggest that the expression of integrated FcaPV2 oncogenes might be associated with reduced expression of the tumor suppressor proteins pRb and p53 and might contribute to the development of feline MCC. On the other hand, p53 mutations may be involved in both FcaPV2-positive and FcaPV2-negative MCC tumorigenesis.

Repurposing disulfiram for the treatment of Merkel cell carcinoma

Background and aims: Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer. Although two immune checkpoint inhibitors are approved for the treatment of advanced stage MCC, less than half of patients achieve durable benefit. Therefore, we sought to identify new and effective treatments for MCC. Methods: To identify novel treatments for MCC, we performed high-throughput screening of ∼4,000 small molecules on MCC cell lines. Additionally, we conducted mechanistic studies using molecular profiling, cell viability assays, and antioxidant assays.

The paranuclear dot in Merkel cell carcinoma, how does it form and what does it do?

Background and aims: Staining for Cytokeratin 20 (CK20) and other intermediate filaments in a paranuclear dot pattern is a histological feature used to diagnose the neuroendocrine skin cancer Merkel cell carcinoma (MCC). The functional significance of these paranuclear protein aggregates is unknown. Therefore, we sought to investigate the structure and function of the paranuclear dot in MCC. Methods: In native tumors and MCC cell lines we used: immunostaning, microscopy, chemical inhibition, siRNA knockdown, and cell death modulators to study the paranuclear dot.

Cancer-associated fibroblasts exert a pro-angiogenic activity in Merkel cell carcinoma

Background and aims: The tumor microenvironment (TME) is a complex niche enveloping a tumor formed by extracellular matrix, blood vessels, immune cells, and fibroblasts constantly interacting with cancer cells. Although TME is increasingly recognized as a major player in cancer initiation and progression in many tumor types, its involvement in Merkel cell carcinoma (MCC) pathogenesis is currently unknown. Here, we provide the first molecular and functional characterization of cancer- associated fibroblasts (CAFs), the major TME component, in MCC patient-derived xenografts. Methods: CAFs were established from eight patients undergoing surgical excision of MCC. To define the functional role of CAFs on MCC progression and metastasis, CAFs were co-injected along with an established MCC cell line, MKL-1, in a spontaneous MCC metastasis mouse model we recently published. Explanted tumors were deeply analyzed by immunohistochemistry and bulk RNA transcriptomic analysis. Furthermore, in vitro functional assays were performed to dissect the mechanisms involved in the proangiogenic role of CAFs. Results: Subcutaneous co-injection of patient-derived CAFs and the MCC cell line MKL-1 into SCID mice significantly promotes tumor growth, metastasis, and angiogenesis. Transcriptome analysis of the explanted tumors further highlighted the role of CAFs in MCC angiogenesis. In addition, in vitro tube formation assay provided evidence that the growth-promoting activity of MCC-derived CAFs is mediated by the APA/AngII-III/AT1R axis, with the expression of aminopeptidase A (APA) in CAFs being the upstream triggering event Conclusion: Altogether, our findings highlight the proangiogenic role of MCC-CAFs and point to APA as a potential marker. Background and aims: The tumor microenvironment (TME) is a complex niche enveloping a tumor formed by extracellular matrix, blood vessels, immune cells, and fibroblasts constantly interacting with cancer cells. Although TME is increasingly recognized as a major player in cancer initiation and progression in many tumor types, its involvement in Merkel cell carcinoma (MCC) pathogenesis is currently unknown. Here, we provide the first molecular and functional characterization of cancer- associated fibroblasts (CAFs), the major TME component, in MCC patient-derived xenografts. Methods: CAFs were established from eight patients undergoing surgical excision of MCC. To define the functional role of CAFs on MCC progression and metastasis, CAFs were co-injected along with an established MCC cell line, MKL-1, in a spontaneous MCC metastasis mouse model we recently published. Explanted tumors were deeply analyzed by immunohistochemistry and bulk RNA transcriptomic analysis. Furthermore, in vitro functional assays were performed to dissect the mechanisms involved in the proangiogenic role of CAFs. Results: Subcutaneous co-injection of patient-derived CAFs and the MCC cell line MKL-1 into SCID mice significantly promotes tumor growth, metastasis, and angiogenesis. Transcriptome analysis of the explanted tumors further highlighted the role of CAFs in MCC angiogenesis. In addition, in vitro tube formation assay provided evidence that the growth-promoting activity of MCC-derived CAFs is mediated by the APA/AngII-III/AT1R axis, with the expression of aminopeptidase A (APA) in CAFs being the upstream triggering event Conclusion: Altogether, our findings highlight the proangiogenic role of MCC-CAFs and point to APA as a potential marker.

Therapeutic Potential of 5'-Methylschweinfurthin G in Merkel Cell Polyomavirus-Positive Merkel Cell Carcinoma.

Merkel cell carcinoma (MCC) is a rare but aggressive form of skin cancer predominantly caused by the human Merkel cell polyomavirus (MCPyV). Treatment for MCC includes excision and radiotherapy of local disease, and chemotherapy or immunotherapy for metastatic disease. The schweinfurthin family of natural compounds previously displayed potent and selective growth inhibitory activity against the NCI-60 panel of human-derived cancer cell lines. Here, we investigated the impact of schweinfurthin on human MCC cell lines. Treatment with the schweinfurthin analog, 5′-methylschweinfurth G (MeSG also known as TTI-3114), impaired metabolic activity through induction of an apoptotic pathway. MeSG also selectively inhibited PI3K/AKT and MAPK/ERK pathways in the MCPyV-positive MCC cell line, MS-1. Interestingly, expression of the MCPyV small T (sT) oncogene selectively sensitizes mouse embryonic fibroblasts to MeSG. These results suggest that the schweinfurthin family of compounds display promising potential as a novel therapeutic option for virus-induced MCCs.