hes1-protein--human and Skin-Neoplasms

Class 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.. The 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.. Our 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.. Our 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.

Topics: Carcinoma, Merkel Cell; Cell Line, Tumor; Histone Deacetylase Inhibitors; Humans; Interferon Type I; RNA, Messenger; Skin Neoplasms; Transcription Factor HES-1

Squamous cell carcinoma of the skin (SCC) represents one of the most common cancers in the general population and is associated with a substantial risk of metastasis. Previous work uncovered the functional role of CYFIP1 in epithelial tumors as an invasion inhibitor. It was down-regulated in some cancers and correlated with the metastatic properties of these malignant cells. We investigated its role and expression mechanisms in SCC. We analyzed the expression of CYFIP1 in patient derived SCC, primary keratinocytes and SCC cell lines, and correlated it to the differentiation and NOTCH1 levels. We analyzed the effects of Notch1 manipulation on CYFIP1 expression and confirmed the biding of Notch1 to the CYFIP1 promoter. CYFIP1 expression was down-regulated in SCC and correlated inversely with histological differentiation of tumors. As keratinocyte differentiation depends on Notch1 signaling, we investigated the influence of Notch1 on CYFIP1 expression. CYFIP1 mRNA was highly increased in human Notch1-overexpressing keratinocytes. Further manipulation of the Notch1 pathway in keratinocytes impacted CYFIP1 levels and chromatin immunoprecipitation assay confirmed the direct binding of Notch1 to the CYFIP1 promoter. CYFIP1 may be a link between loss of differentiation and invasive potential in malignant keratinocytes of cutaneous squamous cell carcinoma.

Topics: Adaptor Proteins, Signal Transducing; Carcinoma, Squamous Cell; Cell Differentiation; Cell Line; Cell Movement; Chromatin Immunoprecipitation; Down-Regulation; Humans; Keratinocytes; Promoter Regions, Genetic; Protein Binding; Receptor, Notch1; RNA Interference; RNA, Small Interfering; Signal Transduction; Skin Neoplasms; Tamoxifen; Transcription Factor HES-1

Zfp191 represses differentiation and keeps various cells in the stem/progenitor stage. Here, we report that a Zfp191 homolog protein, ZNF396, is expressed in basal cell carcinoma (BCC) and possibly represses the expression of a Notch system effector molecule, Hes1 (hairy and enhancer of split-1), and prevents BCC cells from undergoing Notch-mediated squamous cell differentiation. ZNF396 immunoreactivity was found in the nucleus of 35 of 38 cutaneous BCC and 4 of 74 squamous cell carcinoma tissue specimens. In non-tumorous epidermal tissues, ZNF396 immunoreactivity was restricted in basal cells. siRNA-mediated silencing of ZNF396 induced the expression of Notch2, Hes1, and involucrin in cultured BCC cells. Finally, we found that siRNA-mediated silencing of ZNF396 gene inhibited the proliferation of TE354.T basal cell carcinoma cells. ZNF396 might repress Notch-Hes1 signaling axis and prevent tumor cells from undergoing squamous differentiation in BCC.

Topics: Basic Helix-Loop-Helix Transcription Factors; Carcinoma, Basal Cell; Carcinoma, Squamous Cell; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Down-Regulation; HEK293 Cells; Homeodomain Proteins; Humans; Protein Precursors; Receptor, Notch2; RNA Interference; RNA, Small Interfering; Skin Neoplasms; Transcription Factor HES-1; Transcription Factors

NOTCH signaling is important for development and tissue homeostasis and is activated in many human cancers. We investigated a role for NOTCH1 signaling in Sézary syndrome (SS), a cutaneous T-cell lymphoma in which CD4+ tumor cells (Sézary cells) are present in the skin, lymph nodes, and peripheral blood. We show consistent expression of activated NOTCH1 by Sézary cells isolated from peripheral blood of SS patients, as well as the SS-derived cell lines SeAx and HuT78. In addition, immunohistochemical stainings of skin biopsies from SS patients showed consistent expression of nuclear NOTCH1 and its downstream target hairy/enhancer of split-1 (HES1) by Sézary cells. We demonstrate that this persistent NOTCH1 activation is not caused by mutations in the coding regions of NOTCH1 and F-box and WD40 domain protein 7 (FBWX7) genes. Inhibition of NOTCH1 signaling by gamma secretase inhibitors decreased cellular viability and induced apoptosis of Sézary cells. These observations argue that NOTCH1 signaling is functionally involved in the pathogenesis of SS, and inhibition of NOTCH1 signaling represents a new therapeutic target for the treatment of SS.

Topics: Amyloid Precursor Protein Secretases; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Benzodiazepinones; Cell Cycle Proteins; Cell Line, Tumor; Enzyme Inhibitors; F-Box Proteins; F-Box-WD Repeat-Containing Protein 7; Female; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Male; Middle Aged; Mutation; Oligopeptides; Protein Structure, Tertiary; PTEN Phosphohydrolase; Receptor, Notch1; Sezary Syndrome; Signal Transduction; Skin Neoplasms; STAT3 Transcription Factor; Transcription Factor HES-1; Ubiquitin-Protein Ligases

While the pro-differentiation and tumour suppressive functions of Notch signalling in keratinocytes are well established, the underlying mechanisms remain poorly understood. We report here that interferon regulatory factor 6 (IRF6), an IRF family member with an essential role in epidermal development, is induced in differentiation through a Notch-dependent mechanism and is a primary Notch target in keratinocytes and keratinocyte-derived SCC cells. Increased IRF6 expression contributes to the impact of Notch activation on growth/differentiation-related genes, while it is not required for induction of 'canonical' Notch targets like p21(WAF1/Cip1), Hes1 and Hey1. Down-modulation of IRF6 counteracts differentiation of primary human keratinocytes in vitro and in vivo, promoting ras-induced tumour formation. The clinical relevance of these findings is illustrated by the strikingly opposite pattern of expression of Notch1 and IRF6 versus epidermal growth factor receptor in a cohort of clinical SCCs, as a function of their grade of differentiation. Thus, IRF6 is a primary Notch target in keratinocytes, which contributes to the role of this pathway in differentiation and tumour suppression.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Cell Cycle Proteins; Cell Differentiation; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; DNA-Binding Proteins; ErbB Receptors; Genes, Tumor Suppressor; Homeodomain Proteins; Humans; Interferon Regulatory Factors; Keratinocytes; Mice; Mice, Inbred NOD; Mice, SCID; Oncogene Protein p21(ras); Promoter Regions, Genetic; Receptor, Notch1; RNA Interference; RNA, Small Interfering; Signal Transduction; Skin Neoplasms; Transcription Factor HES-1