Dahu Chen

Dynamic assembly of chromatin complexes during cellular senescence: implications for the growth arrest of human melanocytic nevi

The retinoblastoma (RB)/p16INK4a pathway regulates senescence of human melanocytes in culture and oncogene‐induced senescence of melanocytic nevi in vivo. This senescence response is likely due to chromatin modifications because RB complexes from senescent melanocytes contain increased levels of histone deacetylase (HDAC) activity and tethered HDAC1. Here we show that HDAC1 is prominently detected in p16INK4a‐positive, senescent intradermal melanocytic nevi but not in proliferating, recurrent nevus cells that localize to the epidermal/dermal junction. To assess the role of HDAC1 in the senescence of melanocytes and nevi, we used tetracycline‐based inducible expression systems in cultured melanocytic cells. We found that HDAC1 drives a sequential and cooperative activity of chromatin remodeling effectors, including transient recruitment of Brahma (Brm1) into RB/HDAC1 mega‐complexes, formation of heterochromatin protein 1β (HP1β)/SUV39H1 foci, methylation of H3‐K9, stable association of RB with chromatin and significant global heterochromatinization. These chromatin changes coincide with expression of typical markers of senescence, including the senescent‐associated β‐galactosidase marker. Notably, formation of RB/HP1β foci and early tethering of RB to chromatin depends on intact Brm1 ATPase activity. As cells reached senescence, ejection of Brm1 from chromatin coincided with its dissociation from HP1β/RB and relocalization to protein complexes of lower molecular weight. These results provide new insights into the role of the RB pathway in regulating cellular senescence and implicate HDAC1 as a likely mediator of early chromatin remodeling events.

SKI pathways inducing progression of human melanoma.

The proteins SKI and SnoN are implicated in processes as diverse as differentiation, transformation and tumor progression. Until recently, SKI was solely viewed as a nuclear protein with a principal function of inhibiting TGF-β signaling through its association with the Smad proteins. However, new studies suggest that SKI plays additional roles not only inside but also outside the nucleus. In normal melanocytes and primary non-invasive melanomas, SKI localizes predominantly in the nucleus, whereas in primary invasive melanomas SKI displays both nuclear and cytoplasmic localization. Intriguingly, metastatic melanoma tumors display nuclear and cytoplasmic or predominantly cytoplasmic SKI distribution. Cytoplasmic SKI is functional, as it associates with Smad3 and prevents its nuclear localization mediated by TGF-β. SKI can also function as a transcriptional activator, targeting the β -catenin pathway and activating MITF and NrCAM, two proteins involved in survival, migration and invasion. Intriguingly, SKI appears to live a dual life, one as a tumor suppressor and another as a transforming protein. Loss of one copy of mouse ski increases susceptibility to tumorigenesis in mice, whereas its overexpression is associated with cancer progression of human melanoma, esophageal, breast and colon. The molecular reasons for such dramatic change in SKI function appear to result from new acquired activities. In this review, we discuss the mechanisms by which SKI regulates crucial pathways involved in the progression of human malignant melanoma.

Increased expression of enzymes of triglyceride synthesis is essential for the development of hepatic steatosis

Molecular mechanisms underpinning nonalcoholic fatty liver disease (NAFLD) are not well understood. The earliest step of NAFLD is hepatic steatosis, which is one of the main characteristics of aging liver. Here, we present a molecular scenario of age-related liver steatosis. We show that C/EBPα-S193D knockin mice have age-associated epigenetic changes and develop hepatic steatosis at 2 months of age. The underlying mechanism of the hepatic steatosis in old wild-type (WT) mice and in young S193D mice includes increased amounts of tripartite p300-C/EBPα/β complexes that activate promoters of five genes that drive triglyceride synthesis. Knockdown of p300 in old WT mice inhibits hepatic steatosis. Indeed, transgenic mice expressing dominant-negative p300 have fewer C/EBPα/β-p300 complexes and do not develop age-dependent hepatic steatosis. Notably, the p300-C/EBPα/β pathway is activated in the livers of patients with NAFLD. Thus, our results show that p300 and C/EBP proteins are essential participants in hepatic steatosis.

SKI knockdown inhibits human melanoma tumor growth in vivo.

The SKI protein represses the TGF‐β tumor suppressor pathway by associating with the Smad transcription factors. SKI is upregulated in human malignant melanoma tumors in a disease‐progression manner and its overexpression promotes proliferation and migration of melanoma cells in vitro. The mechanisms by which SKI antagonizes TGF‐β signaling in vivo have not been fully elucidated. Here we show that human melanoma cells in which endogenous SKI expression was knocked down by RNAi produced minimal orthotopic tumor xenograft nodules that displayed low mitotic rate and prominent apoptosis. These minute tumors exhibited critical signatures of active TGF‐β signaling including high levels of nuclear Smad3 and p21Waf1, which are not found in the parental melanomas. To understand how SKI promotes tumor growth we used gain‐ and loss‐of‐function approaches and found that simultaneously to blocking the TGF‐β‐growth inhibitory pathway, SKI promotes the switch of Smad3 from tumor suppression to oncogenesis by favoring phosphorylations of the Smad3 linker region in melanoma cells but not in normal human melanocytes. In this context, SKI is required for preventing TGF‐β‐mediated downregulation of the oncogenic protein c‐MYC, and for inducing the plasminogen activator inhibitor‐1, a mediator of tumor growth and angiogenesis. Together, the results indicate that SKI exploits multiple regulatory levels of the TGF‐β pathway and its deficiency restores TGF‐β tumor suppressor and apoptotic activities in spite of the likely presence of oncogenic mutations in melanoma tumors.

Ablation of miR-10b Suppresses Oncogene-Induced Mammary Tumorigenesis and Metastasis and Reactivates Tumor-Suppressive Pathways

The invasive and metastatic properties of many human tumors have been associated with upregulation of the miRNA miR-10b, but its functional contributions in this setting have not been fully unraveled. Here, we report the generation of miR-10b-deficient mice, in which miR-10b is shown to be largely dispensable for normal development but critical to tumorigenesis. Loss of miR-10b delays oncogene-induced mammary tumorigenesis and suppresses epithelial-mesenchymal transition, intravasation, and metastasis in a mouse model of metastatic breast cancer. Among the target genes of miR-10b, the tumor suppressor genes Tbx5 and Pten and the metastasis suppressor gene Hoxd10 are significantly upregulated by miR-10b deletion. Mechanistically, miR-10b promotes breast cancer cell proliferation, migration, and invasion through inhibition of the expression of the transcription factor TBX5, leading to repression of the tumor suppressor genes DYRK1A and PTEN In clinical specimens of breast cancer, the expression of TBX5, HOXD10, and DYRK1A correlates with relapse-free survival and overall survival outcomes in patients. Our results establish miR-10b as an oncomiR that drives metastasis, termed a metastamiR, and define the set of critical tumor suppressor mechanisms it overcomes to drive breast cancer progression. Cancer Res; 76(21); 6424-35. ©2016 AACR.

SKI is critical for repressing the growth inhibitory function of TGF-β in human melanoma

Dear Sir, We read with interest the review of Javelaud et al. (2008) entitled ‘Transforming growth factor-b in cutaneous melanoma’. The purpose of this letter is to point out some issues for readers that were apparently misinterpreted or overlooked in this review. We showed several years ago that the oncogenic protein SKI is expressed in melanoma tumors in a disease progression manner (Reed et al., 2001). The biological significance of such over-expression was established by demonstrating that down-regulation of SKI levels by antisense strategies (RNAi was not readily available at that time) inhibited clonogenic growth, up-regulated p21 and impaired CDK2 kinase activity in two independent melanoma cell lines. Our data were confirmed by others in non-melanocytic human tumors including esophageal squamous cell carcinoma (Fukuchi et al., 2004) and pancreatic cancer (Heider et al., 2007). The review by Javelaud et al. questions our data in a way that can confuse and mislead readers. The authors state that ‘... it has been established that a number of TGF-b target genes are up-regulated in melanoma cells exposed to TGF-b, in particular those involved in invasion and metastasis’. They erroneously conclude that such findings are an indication that SKI does not repress TGF-b signaling in melanoma (the reference provided was a published abstract not available in PubMed). Such a claim is very misleading because TGF-b has dual functions as both tumor suppressor and tumor promoter. Indeed, we agree that TGF-b is up-regulated during melanoma progression (Reed et al., 1994). However, our work and that of others has established that SKI curtails specifically the TGF-b tumor suppressor function, which leads to cell cycle arrest and ⁄ or apoptosis. Javelaud et al. appear to ignore these findings. In fact, we were the first to suggest that by curtailing the tumor suppressor activity of TGF-b, SKI allows this cytokine to stimulate autocrine and paracrine mechanisms leading to stroma remodeling, invasion, angiogenesis and metastasis (Medrano, 2003). Also, we and many others have demonstrated that SKI acts downstream of the TGF-b receptor and does not act by interfering with TbRI phosphorylation of Smad2 ⁄ 3. It is also important to remember that (a) SKI does not disrupt Smad3–Smad4 heteromer formation; (b) recruitment of SKI to the Smad3 ⁄ 4 complex through binding to either Smad3 or Smad4 is both necessary and sufficient for repression (Ueki and Hayman, 2003). The Javelaud review also failed to discuss the fact that SKI localizes to the cytoplasm in some metastatic melanoma tumors, thereby entrapping activated Smads and preventing their nuclear translocation (Reed et al., 2001). The authors must be aware of these findings because one of them co-authored a paper demonstrating that the SKI-related protein SnoN can also localize to the cytoplasm of cells (Krakowski et al., 2005). Javelaud et al. also claim that TGF-b induces rapid degradation of the SKI protein in melanoma cell lines. This statement is also misleading. At low TGF-b concentrations, SKI is resistant to proteasomal degradation, a result also observed by others in cultured fibroblasts (Liu et al., 2008). TGF-b is also unable to degrade SnoN in esophageal cancer cells (Edmiston et al., 2005), consistent with endogenous or transfected SKI being resistant to TGF-b degradation in melanoma tumor cells. The Javelaud et al. review does not discuss how the SKI protein stability is affected by its own protein levels, or how TGF-b dosage affects the kinetics of SKI down-regulation and recovery. TGF-b is secreted by melanoma tumor and stroma cells; therefore, it is abundant in the melanoma microenvironment. So, if the SKI protein were so sensitive to degradation by TGF-b in vivo, it would have been practically impossible to detect SKI by immunohistochemical methods, is well documented in paraffin sections of melanomas, esophageal, pancreatic and gastric cancers. SKI interacts with other important partners, including the retinoblastoma protein RB, HDAC1, mSin3, MeCP2, the kinase HIPK-2, and the lim-only protein FHL2 (Reed

The Multiple Roles of the Oncogenic Protein SKI in Human Malignant Melanoma

Cellular localization, association with different protein partners, and posttranslational modifications can dramatically change protein function. SKI and the highly homologous protein snoN are potent repressors of transforming growth factor-β signaling through their association with the Smad proteins. In fact, SKI can act as molecular switch converting the Smad proteins from an activating to a repressing entity on chromatin. SKI also plays additional roles in melanomas: in association with the LIM protein FHL2 activates β-catenin signaling, a pathway associated with cancer progression. This chapter reviews the transcriptional co-repressor and co-activator activities of SKI and discusses their biological significance for melanoma tumor progression.