Hematopoietic- and neurologic-expressed sequence 1 (Hn1) depletion in B16.F10 melanoma cells promotes a differentiated phenotype that includes increased melanogenesis and cell cycle arrest

Departments of Pharmacology & Therapeutics, University of Florida, College of Medicine, Gainesville, FL 32610-0267, USA.
Differentiation (Impact Factor: 3.44). 06/2009; 78(1):35-44. DOI: 10.1016/j.diff.2009.04.001
Source: PubMed


The Hematopoietic- and neurologic-expressed sequence 1 (Hn1) gene encodes a small protein that is highly conserved among species. Hn1 expression is upregulated in regenerating neural tissues, including the axotomized adult rodent facial motor nerve and dedifferentiating retinal pigment epithelial cells of the Japanese newt. It is also expressed in numerous tissues during embryonic development as well as in regions of the adult brain that exhibit high plasticity. Hn1 has also been reported as a marker for human ovarian carcinoma and it is expressed in high-grade human gliomas. This study was directed toward understanding the function of Hn1 in a murine melanoma cell line. Hn1 mRNA and protein were identified in B16.F10 cells and in tumors formed from these cells. Inhibition of Hn1 protein expression with siRNA increased melanogenesis. Hn1-depleted cells expressed higher levels of the melanogenic proteins tyrosinase and Trp2 and an increased interaction between actin and Rab27a. The in vitro cell growth rate of Hn1-depleted cells was significantly reduced due to G1/S cell cycle arrest. This was consistent with a reduction in the phosphorylation of retinoblastoma protein as well as lower levels of p27 and increased expression of p21. Decreased expression of c-Met, the receptor for hepatocyte growth factor, was also detected in the Hn1-depleted cells, however HGF-dependent stimulation of phosphorylated-ERK was unaffected. Hn1 depletion also led to increased basal levels of phosphorylated p38 MAPK, while basal ERK phosphorylation was reduced. Moreover, Hn1-depleted cells had reduced expression of transcription factors MITF and USF-1, and increased expression of TFE3. These data, coupled with reports on Hn1 expression in regeneration and development, suggest that Hn1 functions as a suppressor of differentiation in cells undergoing repair or proliferation.

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    • ", siRNA-mediated depletion of HN1 in both melanoma and prostate cells results in delayed G1/S-phase transition and increases the p21, cyclin D1, and cyclin B1 levels, suggesting that HN1 significantly contributes to the cell cycle regulation [Laughlin et al., 2009; Varisli et al., 2011]. Moreover, gene expression analysis and in vivo studies demonstrated that HN1 mRNA also increases during wound healing and retina regeneration [Zujovic et al., 2005; Goto et al., 2006], and the cells ectopically overexpressing HN1 heal wounds faster than controls [Varisli et al., 2012]. "
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    ABSTRACT: Previously, it has been reported that HN1 is involved in cytoplasmic retention and degradation of androgen receptor in an AKT dependent manner. As HN1 is a hormone inducible gene, and has been shown that it is upregulated in various cancers, we studied the importance of HN1 function in β-catenin signaling in prostate cancer cell line, PC-3 and mammary cancer cell line MDA-MB231. Here, we demonstrated that HN1 physically associates with GSK3β/β-catenin destruction complex and abundantly localizes to cytoplasm, especially when the GSK3β is phosphorylated on S9 residue. Further, ectopic HN1 expression results an increase in the β-catenin degradation leading to loss of E-cadherin interaction, concurrently contributing to actin re-organization, colony formation and migration in cancer cell lines. Thus, we report that HN1 is an essential factor for β-catenin turnover and signaling, augments cell growth and migration in prostate cancer cells. J. Cell. Biochem. © 2014 Wiley Periodicals, Inc.
    Full-text · Article · Jan 2015 · Journal of Cellular Biochemistry
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    • "In general, HN1 is an interaction partner of the inactive GSK3b/b-catenin/APC complex, which promotes ubiquitin-dependent proteasomal degradation of b-catenin, and contributes to migration in prostate cancer [15]. Conversely, knocking down HN1 suppresses cell proliferation by inducing cell cycle arresting [12]. By qRT-PCR, Western blot, and luciferase reporter assays, we found that HN1 was a direct target of miR-132. "
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    ABSTRACT: Accumulating evidence indicates that miRNAs play critical roles in tumorigenesis and cancer progression. This study aims to investigate the role and the underlying mechanism of miR-132 in breast cancer. Here, we report that miR-132 is significantly down-regulated in breast cancer tissues and cancer cell lines. Additional study identifies HN1 as a novel direct target of miR-132. MiR-132 down-regulates HN1 expression by binding to the 3′ UTR of HN1 transcript, thereby, suppressing multiple oncogenic traits such as cancer cell proliferation, invasion, migration and metastasis in vivo and in vitro. Overexpression of HN1 restores miR-132-suppressed malignancy. Importantly, higher HN1 expression is significantly associated with worse overall survival of breast cancer patients. Taken together, our data demonstrate a critical role of miR-132 in prohibiting cell proliferation, invasion, migration and metastasis in breast cancer through direct suppression of HN1, supporting the potential utility of miR-132 as a novel therapeutic strategy against breast cancer.
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    • "Astrocytic phosphoprotein PEA-15 is a protein kinase C substrate and is highly abundant in the brain of mammalian systems [54]. Interestingly, HN1 protein has been reported as a suppressor of differentiation in cells that are undergoing proliferation [55] and may have played an important role in the generation of a homogeneous cell population in OmB. Petzold et al. [56] identified neurofilament heavy polypeptide as one brain-specific marker for neurodegeneration. "
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    ABSTRACT: Fish cell cultures are becoming more widely used models for investigating molecular mechanisms of physiological response to environmental challenge. In this study, we derived two immortalized Mozambique tilapia (Oreochromis mossambicus) cell lines from brain (OmB) and lip epithelium (OmL), and compared them to a previously immortalized bulbus arteriosus (TmB) cell line. The OmB and OmL cell lines were generated without or with Rho-associated kinase (ROCK) inhibitor/3T3 feeder layer supplementation. Although both approaches were successful, ROCK inhibitor/feeder layer supplementation was found to offer the advantages of selecting for epithelial-like cell type and decreasing time to immortalization. After immortalization (≥ passage 5), we characterized the proteomes of the newly derived cell lines (OmB and OmL) using LCMS and identified several unique cell markers for each line. Subsequently, osmotolerance for each of the three cell lines following acute exposure to elevated sodium chloride was evaluated. The acute maximum osmotolerance of these tilapia cell lines (>700 mOsm/kg) was markedly higher than that of any other known vertebrate cell line, but was significantly higher in the epithelial-like OmL cell line. To validate the physiological relevance of these tilapia cell lines, we quantified the effects of acute hyperosmotic challenge (450 mOsm/kg and 700 mOsm/kg) on the transcriptional regulation of two enzymes involved in biosynthesis of the compatible organic osmolyte, myo-inositol. Both enzymes were found to be robustly upregulated in all three tilapia cell lines. Therefore, the newly established tilapia cells lines represent valuable tools for studying molecular mechanisms involved in the osmotic stress response of euryhaline fish.
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