Merkel Cell Polyomavirus targets SET/PP2A complex to promote cellular proliferation and migration.

Merkel Cell Carcinoma (MCC) is a rare neuroendocrine skin cancer. In our previous work, we decoded genes specifically deregulated by MCPyV early genes as opposed to other polyomaviruses and established functional importance of NDRG1 in inhibiting cellular proliferation and migration in MCC. In the present work, we found the SET protein, (I2PP2A, intrinsic inhibitor of PP2A) upstream of NDRG1 which was modulated by MCPyV early genes, both in hTERT-HK-MCPyV and MCPyV-positive (+) MCC cell lines. Additionally, MCC dermal tumour nodule tissues showed strong SET expression. Inhibition of the SET-PP2A interaction in hTERT-HK-MCPyV using the small molecule inhibitor, FTY720, increased NDRG1 expression and inhibited cell cycle regulators, cyclinD1 and CDK2. SET inhibition by shRNA and FTY720 also decreased cell proliferation and colony formation in MCPyV(+) MCC cells. Overall, these results pave a path for use of drugs targeting SET protein for the treatment of MCC.

Efficacy Comparison of an Automated Growth-Based System and Plate-Count Method on the Detection of Yeasts and Molds in Personal Care Products.

OBJECTIVE: The present investigation studies the efficacy of an automated growth-based system for a quantitative determination of Candida albicans and Aspergillus brasiliensis in several personal care products. The main purpose of this validation study was to prove that the alternative method's entire performance is not inferior to the conventional pour-plate method for a quantitative determination of yeasts and molds. Thus, a performance equivalence was established in accordance with the United Stated Pharmacopeia (USP-NF) Validation of Alternative Microbiological Methods ˂1223˃. METHODS: C. albicans and A. brasiliensis were pooled to use as inoculum (equivalent to 1.0 × 108 CFU/mL) in the suitability of the method test. PCP's preservatives were chemically neutralized leading to the yeast and mold recovery by means of the alternative microbiological method (AMM) and the pour-plate method. A correlation curve was generated for each PCP by plotting DTs relative to the corresponding log CFU values. RESULTS: Thirty PCPs have been tested for quantification of yeasts and molds using an AMM. An equivalence of results was made through the construction of correlation curves that allowed the establishment of numerically equivalent results between the enumeration data from the reference method (CFU) and the alternative method (Detection times, DTs). Thus, following the guidelines of USP Ch.1223, essential validation parameters were tested, such as equivalence of results (Correlation coeficient, CC >0.95), linearity (R2 >0.9025), accuracy (% recovery >70%), operating range, precision (CV <35%), ruggedness (one-way ANOVA, P > 0.05), specificity, LOD, and LOQ. CONCLUSION: It was shown that all the test results obtained from the alternative method were in statistical agreement with the standard plate-count method (PCM). Thus, this new technology was found to meet all the validation criteria needed to be considered for an alternative method for yeast and mold quantification in the PCPs tested. HIGHLIGHTS: In accordance with the United Stated Pharmacopeia (USP-NF) Validation of Alternative Microbiological Methods ˂1223˃, the implementation of alternative methods can offer benefits in execution and automation while improving accuracy, sensitivity, and precision and reduce the microbiological process time compared to the traditional ones.

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.

Estrogen receptor inhibition mediates radiosensitization of ER-positive breast cancer models.

Endocrine therapy (ET) is an effective first-line therapy for women with estrogen receptor-positive (ER + ) breast cancers. While both ionizing radiation (RT) and ET are used for the treatment of women with ER+ breast cancer, the most effective sequencing of therapy and the effect of ET on tumor radiosensitization remains unclear. Here we sought to understand the effects of inhibiting estrogen receptor (ER) signaling in combination with RT in multiple preclinical ER+ breast cancer models. Clonogenic survival assays were performed using variable pre- and post-treatment conditions to assess radiosensitization with estradiol, estrogen deprivation, tamoxifen, fulvestrant, or AZD9496 in ER+ breast cancer cell lines. Estrogen stimulation was radioprotective (radiation enhancement ratios [rER]: 0.51-0.82). Conversely, when given one hour prior to RT, ER inhibition or estrogen depletion radiosensitized ER+ MCF-7 and T47D cells (tamoxifen rER: 1.50-1.60, fulvestrant rER: 1.76-2.81, AZD9496 rER: 1.33-1.48, estrogen depletion rER: 1.47-1.51). Combination treatment resulted in an increase in double-strand DNA (dsDNA) breaks as a result of inhibition of non-homologous end joining-mediated dsDNA break repair with no effect on homologous recombination. Treatment with tamoxifen or fulvestrant in combination with RT also increased the number of senescent cells but did not affect apoptosis or cell cycle distribution. Using an MCF-7 xenograft model, concurrent treatment with tamoxifen and RT was synergistic and resulted in a significant decrease in tumor volume and a delay in time to tumor doubling without significant toxicity. These findings provide preclinical evidence that concurrent treatment with ET and RT may be an effective radiosensitization strategy.

Autophagy Blockade Limits HER2+ Breast Cancer Tumorigenesis by Perturbing HER2 Trafficking and Promoting Release Via Small Extracellular Vesicles.

Autophagy modulation is an emerging strategy for cancer therapy. By deleting an essential autophagy gene or disrupting its autophagy function, we determined a mechanism of HER2+ breast cancer tumorigenesis by directly regulating the oncogenic driver. Disruption of FIP200-mediated autophagy reduced HER2 expression on the tumor cell surface and abolished mammary tumorigenesis in MMTV-Neu mice. Decreased HER2 surface expression was due to trafficking from the Golgi to the endocytic pathways instead of the plasma membrane. Autophagy inhibition led to HER2 accumulation in early and late endosomes associated with intraluminal vesicles and released from tumor cells in small extracellular vesicles (sEVs). Increased HER2 release from sEVs correlated with reduced tumor cell surface levels. Blocking sEVs secretion rescued HER2 levels in tumor cells. Our results demonstrate a role for autophagy to promote tumorigenesis in HER2+ breast cancer. This suggests that blocking autophagy could supplement current anti-HER2 agents for treating the disease.

Merkel Cell Polyomavirus Downregulates N-myc Downstream-Regulated Gene 1, Leading to Cellular Proliferation and Migration.

Merkel cell polyomavirus (MCPyV) is the first human polyomavirus etiologically associated with Merkel cell carcinoma (MCC), a rare and aggressive form of skin cancer. Similar to other polyomaviruses, MCPyV encodes early T antigen genes, viral oncogenes required for MCC tumor growth. To identify the unique oncogenic properties of MCPyV, we analyzed the gene expression profiles in human spontaneously immortalized keratinocytes (NIKs) expressing the early genes from six distinct human polyomaviruses (PyVs), including MCPyV. A comparison of the gene expression profiles revealed 28 genes specifically deregulated by MCPyV. In particular, the MCPyV early gene downregulated the expression of the tumor suppressor gene N-myc downstream-regulated gene 1 (NDRG1) in MCPyV gene-expressing NIKs and hTERT-MCPyV gene-expressing human keratinocytes (HK) compared to their expression in the controls. In MCPyV-positive MCC cells, the expression of NDRG1 was downregulated by the MCPyV early gene, as T antigen knockdown rescued the level of NDRG1. In addition, NDRG1 overexpression in hTERT-MCPyV gene-expressing HK or MCC cells resulted in a decrease in the number of cells in S phase and cell proliferation inhibition. Moreover, a decrease in wound healing capacity in hTERT-MCPyV gene-expressing HK was observed. Further analysis revealed that NDRG1 exerts its biological effect in Merkel cell lines by regulating the expression of the cyclin-dependent kinase 2 (CDK2) and cyclin D1 proteins. Overall, NDRG1 plays an important role in MCPyV-induced cellular proliferation.IMPORTANCE Merkel cell carcinoma was first described in 1972 as a neuroendocrine tumor of skin, most cases of which were reported in 2008 to be caused by a PyV named Merkel cell polyomavirus (MCPyV), the first PyV linked to human cancer. Thereafter, numerous studies have been conducted to understand the etiology of this virus-induced carcinogenesis. However, it is still a new field, and much work is needed to understand the molecular pathogenesis of MCC. In the current work, we sought to identify the host genes specifically deregulated by MCPyV, as opposed to other PyVs, in order to better understand the relevance of the genes analyzed on the biological impact and progression of the disease. These findings open newer avenues for targeted drug therapies, thereby providing hope for the management of patients suffering from this highly aggressive cancer.

Conversion of Sox2-dependent Merkel cell carcinoma to a differentiated neuron-like phenotype by T antigen inhibition.

Viral cancers show oncogene addiction to viral oncoproteins, which are required for survival and proliferation of the dedifferentiated cancer cell. Human Merkel cell carcinomas (MCCs) that harbor a clonally integrated Merkel cell polyomavirus (MCV) genome have low mutation burden and require viral T antigen expression for tumor growth. Here, we showed that MCV+ MCC cells cocultured with keratinocytes undergo neuron-like differentiation with neurite outgrowth, secretory vesicle accumulation, and the generation of sodium-dependent action potentials, hallmarks of a neuronal cell lineage. Cocultured keratinocytes are essential for induction of the neuronal phenotype. Keratinocyte-conditioned medium was insufficient to induce this phenotype. Single-cell RNA sequencing revealed that T antigen knockdown inhibited cell cycle gene expression and reduced expression of key Merkel cell lineage/MCC marker genes, including HES6, SOX2, ATOH1, and KRT20 Of these, T antigen knockdown directly inhibited Sox2 and Atoh1 expression. MCV large T up-regulated Sox2 through its retinoblastoma protein-inhibition domain, which in turn activated Atoh1 expression. The knockdown of Sox2 in MCV+ MCCs mimicked T antigen knockdown by inducing MCC cell growth arrest and neuron-like differentiation. These results show Sox2-dependent conversion of an undifferentiated, aggressive cancer cell to a differentiated neuron-like phenotype and suggest that the ontology of MCC arises from a neuronal cell precursor.

Characterization of a Merkel Cell Polyomavirus-Positive Merkel Cell Carcinoma Cell Line CVG-1.

Merkel cell polyomavirus (MCV) plays a causal role in ∼80% of Merkel cell carcinomas (MCC). MCV is clonally integrated into the MCC tumor genome, which results in persistent expression of large T (LT) and small T (sT) antigen oncoproteins encoded by the early locus. In MCV-positive MCC tumors, LT is truncated by premature stop codons or deletions that lead to loss of the C-terminal origin binding (OBD) and helicase domains important for replication. The N-terminal Rb binding domain remains intact. MCV-positive cell lines derived from MCC explants have been valuable tools to study the molecular mechanism of MCV-induced Merkel cell carcinogenesis. Although all cell lines have integrated MCV and express truncated LT antigens, the molecular sizes of the LT proteins differ between cell lines. The copy number of integrated viral genome also varies across cell lines, leading to significantly different levels of viral protein expression. Nevertheless, these cell lines share phenotypic similarities in cell morphology, growth characteristics, and neuroendocrine marker expression. Several low-passage MCV-positive MCC cell lines have been established since the identification of MCV. We describe a new MCV-positive MCV cell line, CVG-1, with features distinct from previously reported cell lines. CVG-1 tumor cells grow in more discohesive clusters in loose round cell suspension, and individual cells show dramatic size heterogeneity. It is the first cell line to encode an MCV sT polymorphism resulting in a unique leucine (L) to proline (P) substitution mutation at amino acid 144. CVG-1 possesses a LT truncation pattern near identical to that of MKL-1 cells differing by the last two C-terminal amino acids and also shows an LT protein expression level similar to MKL-1. Viral T antigen knockdown reveals that, like other MCV-positive MCC cell lines, CVG-1 requires T antigen expression for cell proliferation.

A situational analysis of breast cancer early detection services in Trinidad and Tobago.

PURPOSE: A situational analysis of breast cancer (BC) early detection services was carried out to investigate whether Trinidad and Tobago (T&T) has the framework for successful organized national screening. METHODS: An online survey was designed to assess the availability, accessibility, quality control and assurance (QC&A), and monitoring and evaluation (M&E) mechanisms for public and private BC early detection. A focus group with local radiologists (n = 3) was held to identify unaddressed challenges and make recommendations for improvement. RESULTS: Major public hospitals offer free detection services with wait times of 1-6 months for an appointment. Private institutions offer mammograms for TTD$240 (USD$37) at minimum with same day service. Both sectors report a lack of trained staff. Using 1.2 mammograms per 10,000 women ≥40 years as sufficient, the public sector's rate of 0.19 mammograms per 10,000 women ≥40 years for screening and diagnosis is inadequate. Program M&E mechanisms, QC&A guidelines for machinery use, delays in receipt of pathology reports, and unreliable drug access are further unaddressed challenges. CONCLUSION: T&T must first strengthen its human and physical resources, implement M&E and QC&A measures, strengthen cancer care, and address other impediments to BC early detection before investing in nationally organized BC screening.