RESUMO
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.
Assuntos
Carcinoma de Célula de Merkel , Movimento Celular , Proliferação de Células , Poliomavírus das Células de Merkel , Proteína Fosfatase 2 , Humanos , Poliomavírus das Células de Merkel/fisiologia , Poliomavírus das Células de Merkel/genética , Proteína Fosfatase 2/metabolismo , Proteína Fosfatase 2/genética , Carcinoma de Célula de Merkel/virologia , Carcinoma de Célula de Merkel/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Cloridrato de Fingolimode/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Linhagem Celular Tumoral , Chaperonas de Histonas/metabolismo , Chaperonas de Histonas/genética , Infecções por Polyomavirus/virologia , Neoplasias Cutâneas/virologia , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 2 Dependente de Ciclina/genéticaRESUMO
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.
Assuntos
Carcinoma de Célula de Merkel , Proteínas Oncogênicas Virais , Infecções por Papillomavirus , Polyomavirus , Neoplasias Cutâneas , Humanos , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Papillomavirus Humano , Infecções por Papillomavirus/complicações , Metiltransferases , Carcinoma de Célula de Merkel/metabolismo , Proteínas Oncogênicas Virais/genética , Neoplasias Cutâneas/metabolismoRESUMO
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.
RESUMO
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.
Assuntos
Autofagia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinogênese/metabolismo , Vesículas Extracelulares/metabolismo , Receptor ErbB-2/metabolismo , Animais , Proteínas Relacionadas à Autofagia/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Endossomos/metabolismo , Feminino , Complexo de Golgi/metabolismo , Humanos , Neoplasias Mamárias Animais/patologia , Camundongos , Complexo de Endopeptidases do Proteassoma/metabolismo , Transporte ProteicoRESUMO
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.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Movimento Celular/genética , Proliferação de Células/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Poliomavírus das Células de Merkel/genética , Poliomavírus das Células de Merkel/fisiologia , Antígenos Virais de Tumores/genética , Antígenos Virais de Tumores/metabolismo , Carcinogênese/genética , Carcinoma de Célula de Merkel/virologia , Linhagem Celular , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Regulação Viral da Expressão Gênica , Humanos , Queratinócitos/virologia , Infecções por Polyomavirus/virologia , Pele/patologia , Neoplasias Cutâneas/genética , Transcriptoma , Infecções Tumorais por Vírus/virologiaRESUMO
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.
Assuntos
Antígenos Virais de Tumores/genética , Carcinoma de Célula de Merkel/etiologia , Carcinoma de Célula de Merkel/metabolismo , Poliomavírus das Células de Merkel/genética , Fenótipo , Infecções por Polyomavirus/complicações , Fatores de Transcrição SOXB1/genética , Antígenos Virais de Tumores/imunologia , Antígenos Virais de Tumores/metabolismo , Carcinoma de Célula de Merkel/patologia , Ciclo Celular/genética , Linhagem Celular Tumoral , Linhagem da Célula/genética , Transformação Celular Viral , Técnicas de Silenciamento de Genes , Humanos , Queratinócitos , Células de Merkel/metabolismo , Poliomavírus das Células de Merkel/imunologia , Neuritos/metabolismo , Neurônios/metabolismo , Infecções por Polyomavirus/imunologia , Infecções por Polyomavirus/virologia , Fatores de Transcrição SOXB1/metabolismo , Infecções Tumorais por Vírus/complicações , Infecções Tumorais por Vírus/imunologia , Infecções Tumorais por Vírus/virologiaRESUMO
Merkel cell polyomavirus (MCV) plays a causal role in â¼80% 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.
RESUMO
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.