9091 Background: Manipulation of key epigenetic regulators in melanoma proliferation is emerging as a new therapeutic strategy. Bromodomain-containing proteins such as the extraterminal domain (BET) family are components of transcription factor complexes and determinants of epigenetic memory. We investigated the expression of BRD4, a BET family member in melanoma cell lines and tissues, and the effects of its inhibition with the small molecule compounds MS436 and MS417 in in vitro and in vivo models of melanoma. Methods: BRD2 and BRD4 expression were analyzed by immunohistochemistry. We tested the effects of pharmacological or RNAi-mediated inhibition of BRD4 in melanoma cells using crystal violet-based assays for proliferation/colony formation and flow-cytometry for cell cycle analysis. The molecular effects of BRD4 suppression were examined using RNA sequencing, Real-Time quantitative PCR and western blots for p27, p21, MYC, ERK1 and SKP2. In the in vivo xenograft experiments NOD/SCID/IL2γR-/-mice were injected with melanoma cells and treated with MS417. Statistical significance was determined by unpaired t-test (GraphPad). Results: BRD4 was found significantly upregulated in primary and metastatic melanoma tissues compared to melanocytes and nevi (p<0.001). Treatment with BET inhibitors impaired melanoma cell proliferation in vitro and tumor growth and metastatic behavior in vivo, effects that were mostly recapitulated by individual silencing of BRD4. Rapidly after BET displacement, key cell cycle genes (SKP2, ERK1 and c-MYC) were downregulated concomitantly with the accumulation of CDK inhibitors (p21, p27), followed by melanoma cell cycle arrest. BET inhibitor efficacy was not influenced by BRAF or NRAS mutational status. Conclusions: Our results demonstrate for the first time a role for BRD4 in melanoma maintenance and support the role of BET proteins as novel targets in melanoma. Further investigation in the clinical setting is warranted.

Metastatic melanoma remains a mostly incurable disease. Although newly approved targeted therapies are efficacious in a subset of patients, resistance and relapse rapidly ensue. Alternative therapeutic strategies to manipulate epigenetic regulators and disrupt the transcriptional program that maintains tumor cell identity are emerging. Bromodomain and extraterminal domain (BET) family of proteins consists of BRD2, BRD3, BRD4, and testis- specific BRDT, and are epigenome readers known to exert key roles at the interface between chromatin remodeling and transcriptional regulation. We investigated the role of BET proteins in melanoma tumor maintenance and assessed their value as therapeutic targets. Data mining of our previously published gene expression profile of melanoma cell lines and immunostaining of melanoma tissue microarray revealed that BRD4 is significantly upregulated in primary and metastatic melanoma tissues compared to melanocytes and nevi, thus suggesting a potential role for BET family proteins in promoting melanoma tumorigenesis. Treatment with BET inhibitors impaired melanoma cell proliferation and colony formation in vitro. Moreover, tumor growth and metastatic behavior assessed by a xenograft model also revealed impairment of melanoma proliferation in vivo. These effects were mostly recapitulated by individual silencing of BRD4, and not of other BET family members. RNA sequencing of BET inhibitor-treated cells followed by gene ontology analysis showed a striking impact on transcriptional programs controlling cell growth, proliferation, cell-cycle regulation and differentiation. In particular, we found that, rapidly after BET displacement, key cell cycle genes (SKP2, ERK1 and c-MYC) were downregulated concomitantly with the accumulation of CDK inhibitors (p21, p27), followed by melanoma cell cycle arrest. However, single genetic manipulation of these cell cycle genes did not rescue the cytostatic effect of BET inhibition, suggesting that BET inactivation leads to a non-redundant, simultaneous regulation of multiple cell cycle effectors. Interestingly, SKP2 and ERK1 mRNA levels directly correlated with those of BRD4 in a panel of melanoma tissues, suggesting that these two factors may be direct BRD4 targets. Importantly, the effects of the BET inhibitor were not influenced by BRAF or NRAS mutational status, opening the possibility of using these small molecule compounds to treat patients for whom no effective targeted therapy currently exists. Collectively, our results strongly support a critical role for BRD4 in melanoma tumor maintenance, and render it a legitimate and novel target for epigenetic therapy directed against the core transcriptional program of melanoma. Citation Format: Barbara Fontanals-Cirera, Miguel F. Segura, Avital Gaziel-Sovran, Maria V. Guijarro, Doug Hanniford, Pilar Gonzalez-Gomez, Weijia Zhang, Guantao Zhang, Farbod Darvishian, Michael Ohlmeyer, Iman Osman, Ming-Ming Zhou, Eva Hernando. BRD4 is a new therapeutic target in melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A10.

This study sought to improve the utilization of sorghum straw resources and promote the industrial production of new biomass materials. Herein, we fabricated SSP/ZnO/PVA nanocomposite films from sorghum straw powder (SSP), corn starch, polyvinyl alcohol (PVA), and nanostructured ZnO via the casting method. Then, we used response surface methodology to examine the effects of the mass concentrations of SSP, glycerol (Gly), and nanostructured ZnO, as well as the starch–PVA mass ratio on the tensile strength (TS) and water vapor permeability (WVP) of the SSP/ZnO/PVA nanocomposite films. The optimum preparation conditions were as follows: SSP mass concentration of 2.0 g/150 mL, Gly mass concentration of 2.5 g/150 mL, starch–PVA mass ratio of 6:4.5, and nanostructured ZnO mass concentration of 0.7 g/150 mL. The TS and WVP of the prepared films were 47.57% higher and 27.07% lower, respectively, than those of ZnO/PVA composite films without SSP. Scanning electron microscopy and atomic force microscopy showed that the SSP/ZnO/PVA nanocomposite films had smooth surfaces and dense cross-sections, without obvious delamination or phase separation. Fourier transform infrared spectroscopy, X-ray diffraction, and thermogravimetric analyses revealed that SSP was highly compatible with the ZnO/PVA matrix. Thus, SSP addition could improve the crystallinity, thermal stability, and matrix interactions of SSP/ZnO/PVA nanocomposite films.