Merkel Cell Carcinoma Cell Lines Research Articles

Roles for miR-375 in Neuroendocrine Differentiation and Tumor Suppression via Notch Pathway Suppression in Merkel Cell Carcinoma

Dysfunction of key miRNA pathways regulating basic cellular processes is a common driver of many cancers. However, the biological roles and/or clinical applications of such pathways in Merkel cell carcinoma (MCC), a rare but lethal cutaneous neuroendocrine (NE) malignancy, have yet to be determined. Previous work has established that miR-375 is highly expressed in MCC tumors, but its biological role in MCC remains unknown. Herein, we show that elevated miR-375 expression is a specific feature of well-differentiated MCC cell lines that express NE markers. In contrast, miR-375 is strikingly down-regulated in highly aggressive, undifferentiated MCC cell lines. Enforced miR-375 expression in these cells induced NE differentiation, and opposed cancer cell viability, migration, invasion, and survival, pointing to tumor-suppressive roles for miR-375. Mechanistically, miR-375-driven phenotypes were caused by the direct post-transcriptional repression of multiple Notch pathway proteins (Notch2 and RBPJ) linked to cancer and regulation of cell fate. Thus, we detail a novel molecular axis linking tumor-suppressive miR-375 and Notch with NE differentiation and cancer cell behavior in MCC. Our findings identify miR-375 as a putative regulator of NE differentiation, provide insight into the cell of origin of MCC, and suggest that miR-375 silencing may promote aggressive cancer cell behavior through Notch disinhibition.

ABCB5-Targeted Chemoresistance Reversal Inhibits Merkel Cell Carcinoma Growth

Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine skin cancer with profound but poorly understood resistance to chemotherapy, which poses a significant barrier to clinical MCC treatment. Here we show that ATP-binding cassette member B5 (ABCB5) confers resistance to standard-of-care MCC chemotherapeutic agents and provide proof-of-principle that ABCB5 blockade can inhibit human MCC tumor growth through sensitization to drug-induced cell cytotoxicity. ABCB5 expression was detected in both established MCC lines and clinical MCC specimens at levels significantly higher than those in normal skin. Carboplatin- and etoposide-resistant MCC cell lines exhibited increased expression of ABCB5, along with enhanced ABCB1 and ABCC3 transcript expression. ABCB5-expressing MCC cells in heterogeneous cancers preferentially survived treatment with carboplatin and etoposide invitro and in human MCC xenograft-bearing mice invivo. Moreover, patients with MCC also exhibited enhanced ABCB5 positivity after carboplatin- and etoposide-based chemotherapy, pointing to clinical significance of this chemoresistance mechanism. Importantly, ABCB5 blockade reversed MCC drug resistance and impaired tumor growth in xenotransplantation models invivo. Our results establish ABCB5 as a chemoresistance mechanism in MCC and suggest utility of this molecular target for improved MCC therapy.

Evaluation of Polo-like kinase 1 as a potential therapeutic target in Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a rare and aggressive malignancy of the skin. Treatment options for MCC include surgery, radiotherapy, and chemotherapy. The purpose of this study was to assess the expression of Polo-like kinase 1 (PLK1) in MCC and the role of the inhibitor, BI2536, as a potential therapeutic option in MCC. PLK1 expression was assessed in tissue samples from 28 patients with MCC and 5 healthy skin samples via immunohistochemistry and furthermore in the 2 MCC cell lines, MCC13 and MCC26, via immunoblotting. The impact of increasing doses of BI2536 alone and in combination with cisplatin or irradiation on cell viability was measured using the CCK-8 assay. Colony forming assays were performed to evaluate long-term effects of combination treatments. Additionally, the induction of apoptotic cell death was measured via flow cytometry. PLK1 is moderately to strongly expressed in 75% of the patients with MCC. The PLK1 inhibitor, BI2536, demonstrated marked inhibition of cell proliferation with IC50 in the low nM range (from 10.07-12.39 nM). Furthermore, BI2536 induces apoptosis in MCC cell lines and acts synergistically in combination with irradiation and cisplatin. Because of the marked upregulation of PLK1 in MCC tumor samples and potent inhibition of cell proliferation using a specific clinically available inhibitor, targeting of PLK1 qualifies as a potential novel therapeutic strategy in MCC. © 2015 Wiley Periodicals, Inc. Head Neck 38: E1918-E1925, 2016.

Abstract 3875: Merkel cell carcinomas in Australia have distinct mutation profiles reflecting viral etiology and UV-related DNA damage

Abstract Background/Aims: Merkel cell carcinoma (MCC) is rare but aggressive cutaneous high-grade neuroendocrine cancer. Viral infection and sun-exposure are known risk factors for development of MCC, the latter being important in Australia, which has the highest reported incidence of MCC worldwide. There is growing evidence to suggest that MCC associated with the Merkel cell polyoma virus (MCPyV) is clinically, biologically and genetically distinct to those tumors without viral infection. We aimed to assess mutations arising in MCC in the Australian population by targeted massively-parallel sequencing (MPS) to gain new biological insight and identify novel therapeutic opportunities in this disease. Methods: MCPyV was detected in tumours by PCR amplification of large-T antigen coding DNA. The tumor cohort was comprised of seven MCPyV-positive and 16 MCPyV-negative tumors reflecting the reported prevalence of these subtypes in the Australian population. We also sequenced two viral negative MCC cell lines. Hybridization-based DNA capture was used for enrichment of 625 cancer genes for high-depth MPS of tumor and the matching germline DNA, where available. Single nucleotide variants and small insertions and deletions were detected using GATK and muTect variant calling tools and annotated using ENSEMBL variant effect predictor. Somatic variants were enriched in the data by filtering out any variants found in matching germline samples or population-based polymorphism databases Results: High mutation burden was found exclusively in the MCPyV-negative cases with the hallmark signature of UV induced DNA damage. Somatic mutations were identified in known cancer genes specific to the MCPyV-negative tumors. Deleterious RB1 and TP53 mutations were found in 17 and 16 of the MCPyV-negative cases, respectively. Canonical mutations were identified in PIK3CA, HRAS as well as truncating NF1 mutations, indicating that activation of the PI3K and RAS-MAPK pathways are important for pathogenesis of MCPyV-negative tumors. Treatment of two cell lines harboring either PIK3CA or HRAS mutations, respectively, showed in vitro sensitivity to the dual PI3K/mTOR inhibitor PF-04691502. Conclusions: The identification of a UV-induced DNA damage signature and mutations in PI3K and MAPK pathways specific to viral negative tumors confirms the distinct routes to pathogenesis in this disease subtype. This study provides new insight into the biology of viral negative MCC and potential opportunities for the deployment of targeted therapies in this patient group. Citation Format: Stephen Q. Wong, Kelly Waldeck, Ismael A. Vergara, Jason Li, Richard Lupat, Timothy Semple, Carleen Cullinane, Gisela Mir Arnau, Meredith Johnston, Annette Hogg, Anthony T. Papenfuss, Stephen Fox, Grant McArthur, Anthony Gill, Rodney J. Hicks, Richard W. Tothill. Merkel cell carcinomas in Australia have distinct mutation profiles reflecting viral etiology and UV-related DNA damage. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3875. doi:10.1158/1538-7445.AM2015-3875

Assessment of Cancer Cell Line Representativeness Using Microarrays for Merkel Cell Carcinoma

When using cell lines to study cancer, phenotypic similarity to the original tumor is paramount. Yet, little has been done to characterize how closely Merkel cell carcinoma (MCC) cell lines model native tumors. To determine their similarity to MCC tumor samples, we characterized MCC cell lines via gene expression microarrays. Using whole transcriptome gene expression signatures and a computational bioinformatic approach, we identified significant differences between variant cell lines (UISO, MCC13, and MCC26) and fresh frozen MCC tumors. Conversely, the classic WaGa and Mkl-1 cell lines more closely represented the global transcriptome of MCC tumors. When compared to publicly available cancer lines, WaGa and Mkl-1 cells were similar to other neuroendocrine tumors, but the variant cell lines were not. WaGa and Mkl-1 cells grown as xenografts in mice had histological and immunophenotypical features consistent with MCC, while UISO xenograft tumors were atypical for MCC. Spectral karyotyping and short tandem repeat analysis of the UISO cells matched the original cell line’s description, ruling out contamination. Our results validate the use of transcriptome analysis to assess the cancer cell line representativeness and indicate that UISO, MCC13, and MCC26 cell lines are not representative of MCC tumors, whereas WaGa and Mkl-1 more closely model MCC.

45 Polo-like-kinase-1 as a potential therapeutic target in merkel cell carcinoma

Background Merkel cell carcinoma (MCC) is a rare and aggressive skin malignancy. Late stage MCC treatment options include radio- and chemotherapy. The aim of this study was to assess the expression of polo-like-kinase-1 (PLK1) in MCC and whether the PLK1 inhibitor BI2536 is a potential therapeutic option in MCC. Material and methods To evaluate PLK1 expression in patient specimens a tissue microarray was constructed for immunohistochemical staining. The two MCC cell lines MCC13 and MCC26 were exposed to increasing doses of BI2536 in combination with cisplatin or irradiation. Suppression of PLK1 was measured by Western blot analysis. Clonogenic assays were performed after irradiation to evaluate long-term results. Apoptotic and necrotic fractions were measured via flow cytometry. Results BI2536 is capable of inhibiting cell proliferation with an IC50 of 12nM. Combination with cisplatin is synergistic and a partially synergistic effect in combination with irradiation was detected. Clonogenic survival is inhibited synergistically. BI2536 induces apoptosis in MCC cell lines. Conclusion PLK1 seems to be a promising target in the treatment of MCC. Treatment with BI2536, a potent inhibitor of PLK1, resulted in suppression of MCC growth in vitro. Our data warrant further investigations for the potential use of BI2536 in MCC.

31 MCPyV and the immune system: Target and modulator

Merkel cell carcinoma (MCC) is a aggressive skin cancer associated with the Merkel cell polyomavirus (MCPyV). Presence of tumor-infiltrating lymphocytes and immune competence are strongly correlated with the course of the disease. MCPyV encoded proteins should be highly effective targets for cytotoxic immune responses as they are both foreign to the host and specific for the infection. Srutinizing for T-cell responses against MCPyV proteins revealed responses against HLA-A1, -A2, -A3, -A11 and -B7 restricted epitopes derived from both Large and Small T antigen in MCC patients. Moreover, we demonstrate the processing and presentation of the oncoprotein derived peptides, as well as the functional activity of the T antigen-reactive T cells. Despite the expression of immunogenic viral proteins and the presence of reactive cytotoxic T cell, MCCs are able to evade the immune system. Infected transformed cells in general express activating NKG2D ligands such as MICA/B activating cellular immune responses. However, in none of the MCPyV positive MCC cell lines MICA/B were expressed on transcriptional or translational level. Immunohistochemistry of 54 MCC tumors further revealed that in the majority MICA was not detectable. Since MICA/B expression is frequently silenced by histone deacetylation the effect of the HDAC inhibitor SAHA was tested revealing an induction of MICA/B resulting in an boosted lysis by cytotoxic lymphocytes. Accordingly, ChIP analysis demonstrated enhanced Histone H3 acetylation of the MICA/B promoters. Thus, MCPyV derived epitopes elicit cytotoxic T-cell responses which can be rendered functional effective by induction of NKG2D Ligand expression.

Downregulation of MHC-I expression is prevalent but reversible in Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is an aggressive, polyomavirus-associated skin cancer. Robust cellular immune responses are associated with excellent outcomes in patients with MCC, but these responses are typically absent. We determined the prevalence and reversibility of major histocompatibility complex class I (MHC-I) downregulation in MCC, a potentially reversible immune-evasion mechanism. Cell-surface MHC-I expression was assessed on five MCC cell lines using flow cytometry as well as immunohistochemistry on tissue microarrays representing 114 patients. Three additional patients were included who had received intralesional IFN treatment and had evaluable specimens before and after treatment. mRNA expression analysis of antigen presentation pathway genes from 35 MCC tumors was used to examine the mechanisms of downregulation. Of note, 84% of MCCs (total n = 114) showed reduced MHC-I expression as compared with surrounding tissues, and 51% had poor or undetectable MHC-I expression. Expression of MHC-I was lower in polyomavirus-positive MCCs than in polyomavirus-negative MCCs (P < 0.01). The MHC-I downregulation mechanism was multifactorial and did not depend solely on HLA gene expression. Treatment of MCC cell lines with ionizing radiation, etoposide, or IFN resulted in MHC-I upregulation, with IFNs strongly upregulating MHC-I expression in vitro, and in 3 of 3 patients treated with intralesional IFNs. MCC tumors may be amenable to immunotherapy, but downregulation of MHC-I is frequently present in these tumors, particularly those that are positive for polyomavirus. This downregulation is reversible with any of several clinically available treatments that may thus promote the effectiveness of immune-stimulating therapies for MCC.

TERT promoter mutations and gene amplification: promoting TERT expression in Merkel cell carcinoma.

Telomerase activation through the induction of its catalytic component TERT is essential in carcinogenesis. The regulatory mechanism and clinical significance underlying cancer-specific TERT expression have been extensively investigated in various human malignancies, but little is known about these in Merkel cell carcinoma (MCC), an aggressive neuroendocrine skin tumor. Here we addressed these issues by determining TERT promoter mutations, gene amplification, mRNA expression and association with clinical variables in MCC. TERT mRNA was expressed in 6/6 MCC cell lines and 41 of 43 tumors derived from 35 MCC patients. Telomerase activity was detectable in all 6 cell lines and 11 tumors analyzed. TERT promoter mutations were identified in 1/6 cell lines and 4/35 (11.4%) MCC cases. The mutation exhibited UV signature and occurred in sun-exposed areas. Increased TERT gene copy numbers were observed in 1/6 cell lines and 11/14 (79%) tumors, and highly correlated with its mRNA expression (r = 0.7419, P = 0.0024). Shorter overall survival was significantly associated with higher TERT mRNA levels in MCC patients (P = 0.032). Collectively, TERT expression and telomerase activity is widespread in MCC, and may be attributable to TERT promoter mutations and gene amplification. Higher TERT expression predicts poor patient outcomes.

Merkel Cell Carcinoma Dependence on Bcl-2 Family Members for Survival

Merkel cell carcinoma (MCC), a rare but aggressive cutaneous neoplasm with high metastatic potential, has a poor prognosis at late stages of disease with no proven chemotherapeutic regimens. Using an enriched culture medium, we established and characterized 11 MCC cell lines for Bcl-2 family profiling and functional studies. Immunoblot analysis revealed collectively high protein levels of pro-survival Bcl-2 members in cell lines and a panel of MCC tumors. Down-regulation of individual Bcl-2 proteins by RNAi promoted death in a subset of MCC cell lines, whereas simultaneous inhibition of multiple family members using the small molecule antagonist ABT-263 led to dramatic induction of cell death in 10 of 11 lines. ABT-263 induced Bax-dependent apoptosis with rapid cleavage of caspase-3 and PARP, regardless of Bcl-2 family profile or presence of Merkel cell polyomavirus. Furthermore, ABT-263 treatment led to rapid and sustained growth suppression of MCC xenografts from a representative cell line, accompanied by a striking increase in apoptosis. Our results establish that concurrent inhibition of multiple pro-survival Bcl-2 proteins leads to effective induction of apoptosis, and strongly support the concept that targeting MCC addiction to these molecules may be useful therapeutically by reversing an intrinsic resistance to cell death.

The HSP70 Modulator MAL3-101 Inhibits Merkel Cell Carcinoma

Merkel Cell Carcinoma (MCC) is a rare and highly aggressive neuroendocrine skin cancer for which no effective treatment is available. MCC represents a human cancer with the best experimental evidence for a causal role of a polyoma virus. Large T antigens (LTA) encoded by polyoma viruses are oncoproteins, which are thought to require support of cellular heat shock protein 70 (HSP70) to exert their transforming activity. Here we evaluated the capability of MAL3-101, a synthetic HSP70 inhibitor, to limit proliferation and survival of various MCC cell lines. Remarkably, MAL3-101 treatment resulted in considerable apoptosis in 5 out of 7 MCC cell lines. While this effect was not associated with the viral status of the MCC cells, quantitative mRNA expression analysis of the known HSP70 isoforms revealed a significant correlation between MAL3-101 sensitivity and HSC70 expression, the most prominent isoform in all cell lines. Moreover, MAL3-101 also exhibited in vivo antitumor activity in an MCC xenograft model suggesting that this substance or related compounds are potential therapeutics for the treatment of MCC in the future.

Characterization of the Merkel Cell Carcinoma miRNome

MicroRNAs have been implicated in various skin cancers, including melanoma, squamous cell carcinoma, and basal cell carcinoma; however, the expression of microRNAs and their role in Merkel cell carcinoma (MCC) have yet to be explored in depth. To identify microRNAs specific to MCC (MCC-miRs), next-generation sequencing (NGS) of small RNA libraries was performed on different tissue samples including MCCs, other cutaneous tumors, and normal skin. Comparison of the profiles identified several microRNAs upregulated and downregulated in MCC. For validation, their expression was measured via qRT-PCR in a larger group of MCC and in a comparison group of non-MCC cutaneous tumors and normal skin. Eight microRNAs were upregulated in MCC: miR-502-3p, miR-9, miR-7, miR-340, miR-182, miR-190b, miR-873, and miR-183. Three microRNAs were downregulated: miR-3170, miR-125b, and miR-374c. Many of these MCC-miRs, the miR-183/182/96a cistron in particular, have connections to tumorigenic pathways implicated in MCC pathogenesis. In situ hybridization confirmed that the highly expressed MCC-miR, miR-182, is localized within tumor cells. Furthermore, NGS and qRT-PCR reveal that several of these MCC-miRs are highly expressed in the patient-derived MCC cell line, MS-1. These data indicate that we have identified a set of MCC-miRs with important implications for MCC research.

Chronic mTOR activation promotes cell survival in Merkel cell carcinoma

Merkel cell carcinoma (MCC) is an aggressive skin cancer with rising incidence. In this study, we demonstrate that mTOR activation and suppressed autophagy is common in MCCs. mTOR inhibition in two primary human MCC cell lines induces autophagy and cell death that is independent of caspase activation but can be attenuated by autophagy inhibition. This is the first study to evaluate mTOR and autophagy in MCC. Our data suggests a potential role of autophagic cell death upon mTOR inhibition and thus uncovers a previously underappreciated role of mTOR signaling and cell survival, and merits further studies for potential therapeutic targets.

Response of Merkel Cell Polyomavirus-Positive Merkel Cell Carcinoma Xenografts to a Survivin Inhibitor

Merkel cell carcinoma (MCC) is a neuroendocrine skin cancer associated with high mortality. Merkel cell polyomavirus (MCV), discovered in 2008, is associated with ~80% of MCC. The MCV large tumor (LT) oncoprotein upregulates the cellular oncoprotein survivin through its conserved retinoblastoma protein-binding motif. We confirm here that YM155, a survivin suppressor, is cytotoxic to MCV-positive MCC cells in vitro at nanomolar levels. Mouse survival was significantly improved for NOD-Scid-Gamma mice treated with YM155 in a dose and duration dependent manner for 3 of 4 MCV-positive MCC xenografts. One MCV-positive MCC xenograft (MS-1) failed to significantly respond to YM155, which corresponds with in vitro dose-response activity. Combination treatment of YM155 with other chemotherapeutics resulted in additive but not synergistic cell killing of MCC cell lines in vitro. These results suggest that survivin targeting is a promising therapeutic approach for most but not all MCV-positive MCCs.

The effect of resveratrol in combination with irradiation and chemotherapy

Merkel cell carcinoma (MCC) is a rare, but highly malignant tumor of the skin. In case of systemic disease, possible therapeutic options include irradiation or chemotherapy. The aim of this study was to evaluate whether the flavonoid resveratrol enhances the effect of radiotherapy or chemotherapy in MCC cell lines. The two MCC cell lines MCC13 and MCC26 were treated with increasing doses of resveratrol. Combination experiments were conducted with cisplatin and etoposide. Colony forming assays were performed after sequential irradiation with 1, 2, 3, 4, 6, and 8 Gy and apoptosis was assessed with flow cytometry. Expression of cancer drug targets was analyzed by real-time PCR array. Resveratrol is cytotoxic in MCC cell lines. Cell growth is inhibited by induction of apoptosis. The combination with cisplatin and etoposide resulted in a partially synergistic inhibition of cell proliferation. Resveratrol and irradiation led to a synergistic reduction in colony formation compared to irradiation alone. Evaluation of gene expression did not show significant difference between the cell lines. Due to its radiosensitizing effect, resveratrol seems to be a promising agent in combination with radiation therapy. The amount of chemosensitizing depends on the cell lines tested.

Expression of β-catenin and cyclin D1 in Merkel cell carcinomas of the head and neck

Merkel cell carcinomas (MCC) are very aggressive tumors of the sun-exposed skin with a high potential to metastasize. Little is known about the genesis of MCC and very few prognostic markers have been detected so far. The Wnt pathway protein β-catenin and the cell cycle protein cyclin D1 are two promotors of tumor growth and are expressed in a variety of malignant neoplasms such as lymphomas, thyroid, breast cancer, and many others. Tissue samples of 27 patients with MCC were immunohistochemically stained for β-catenin and cyclin D1 and correlated with overall survival of patients. In addition, western blot analysis was carried out in the two MCC cell lines MCC-13 and MCC-26. β-catenin showed a cytoplasmatic expression of 10-30 % in 11 samples and an expression lower than 10 % in eight samples. Nuclear staining was visible in two samples. None of the 27 samples expressed cyclin D1. Neither cyclin D1 nor β-catenin was expressed in a statistically significant manner, concluding that the development of MCCs is independent of β-catenin and cyclin D1 expression and these proteins are not suitable as prognostic markers. We could describe the expression pattern of cyclin D1 for the first time.

Merkel Cell Polyomavirus–Positive Merkel Cell Carcinoma Cells Do Not Require Expression of the Viral Small T Antigen

Increasing evidence suggests that Merkel cell carcinoma (MCC) is caused by the Merkel cell polyomavirus (MCV). The viral sequence encodes for two potential oncoproteins, i.e., the small T antigen (sT) and the large T antigen (LT). Indeed, sT has recently been shown to bear transforming activity. Here, we confirm this observation by demonstrating focus formation upon expression of MCV sT in NIH3T3 fibroblasts. On the other hand, however, we provide evidence that established MCC cells do not require sT for growth and survival. Silencing of sT protein expression by two different sT-specific short hairpin RNAs (shRNAs) leads to variable degrees of growth retardation in MCV-positive MCC cell lines. However, these effects are not sT specific, as proliferation of MCV-negative cell lines is similarly affected by these sT shRNAs. Furthermore, ectopic expression of shRNA-insensitive sT does not revert the growth inhibition implicated by sT silencing. Finally, the unambiguous and specific growth inhibition induced by means of an shRNA targeting both T antigens, can be completely rescued by ectopic expression of LT alone, thus demonstrating a dispensable role of sT. Altogether, our results indicate that MCV LT is more relevant in maintaining the proliferation and survival of established MCC cell lines.

Merkel cell carcinoma versus metastatic small cell primary bronchogenic carcinoma

Introduction: Merkel cell carcinoma (MCC) of the skin is a rare, aggressive, malignant neuroendocrine neoplasm. The tumor classically demonstrates positive immunohistochemistry (IHC) staining for chromogranin A(ChrA), cytokeratin 20 (CK20), neuron specific enolase (NSE) and/or achaete-acute complex-like 1 (MASH1). The newly identified Merkel cell polyomavirus (MCPyV) has been found to be associated with most MCC cases. The primary histologic differential diagnoses of cutaneous MCC is small cell primary bronchogenic carcinoma (SCLC); moreover, both are of neuroendocrine origin. SCLC accounts for approximately 10-15% of all primary lung cancer cases; this histologic subtype is a distinct entity with biological and oncological features distinct from non-small cell lung cancer (NSCLC). In contradistinction to MCC, SCLC is classically IHC positive for cytokeratin 7 (CK7) and transcription factor (TTF-1). Similar to SCLC, MCC cell lines may be classified into two different biochemical subgroups designated as Classic and Variant. In our review and case report, we aim to emphasize the importance of a multidisciplinary approach to the approach to this difficult differential diagnosis. We also aim to comment about features of the cells of origin of MCC and SCLC; to summarize the microscopic features of both tumors; and to review their respective epidemiologic, clinical, prognostic and treatment features. We want to emphasize the initial workup study of the differential diagnosis patient, including evaluating clinical lymph nodes, a clinical history of any respiratory abnormality, and chest radiogram. If a diagnosis of primary cutaneous MCC is confirmed, classic treatment includes excision of the primary tumor with wide margins, excision of a sentinel lymph node, and computed tomography, positron emission tomography and/or Fluorine-18fluorodeoxyglucose positron emission tomography scan studies.

A Proteomic Study of Human Merkel Cell Carcinoma.

Merkel Cell Carcinoma (MCC) is an aggressive neuroendocrine cancer of the skin. The incidence has been quadrupled with a 5-year mortality rate of 46%, presently there is no cure for metastatic disease. Despite the contribution of Merkel cell polyomavirus, the molecular events of MCC carcinogenesis are poorly defined. To better understand MCC carcinogensis, we have performed the first quantitative proteomic comparison of formalin-fixed, paraffin-embedded (FFPE) MCC tissues using another neuroendocrine tumor (carcinoid tumor of the lung) as controls. Bioinformatic analysis of the proteomic data has revealed that MCCs carry distinct protein expression patterns. Further analysis of significantly over-expressed proteins suggested the involvement of MAPK, PI3K/Akt/mTOR, wnt, and apoptosis signaling pathways. Our previous study and that from others have shown mTOR activation in MCCs. Therefore, we have focused on two downstream molecules of the mTOR pathway, lactate dehydrogenase B (LDHB) and heterogeneous ribonucleoprotein F (hnRNPF). We confirm over-expression of LDHB and hnRNPF in two primary human MCC cell lines, 16 fresh tumors, and in the majority of 80 tissue microarray samples. Moreover, mTOR inhibition suppresses LDHB and hnRNPF expression in MCC cells. The results of the current study provide insight into MCC carcinogenesis and provide rationale for mTOR inhibition in pre-clinical studies.

Characterization of an early passage Merkel cell polyomavirus-positive Merkel cell carcinoma cell line, MS-1, and its growth in NOD scid gamma mice

Merkel cell carcinoma (MCC) is an aggressive skin cancer with a high mortality rate. The majority of MCC (70-80%) harbor clonally integrated Merkel cell polyomavirus (MCV) in the tumor genome and express viral T antigen oncoproteins. The characterization of an early passage MCV-positive MCC cell line MS-1 is described, and its cellular, immunohistochemical, and virological features to MCV-negative (UISO, MCC13, and MCC26) and MCV-positive cell lines (MKL-1 and MKL-2) were compared. The MS-1 cellular genome harbors integrated MCV, which preserves an identical viral sequence from its parental tumor. Neither VP2 gene transcripts nor VP1 protein are detectable in MS-1 or other MCV-positive MCC cell lines tested. Mapping of viral and cellular integration sites in MS-1 and MCC tumor samples demonstrates no consistent viral or cellular gene integration locus. All MCV-positive cell lines show cytokeratin 20 positivity and grow in suspension. When injected subcutaneously into NOD scid gamma (NSG) mice, MS-1 forms a discrete macroscopic tumor. Immunophenotypic analysis of the MS-1 cell line and xenografts in mice show identical profiles to the parental tumor biopsy. Hence, MS-1 is an early passage cell line that provides a useful in vitro model to characterize MCV-positive MCC.