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14-3-3σ, a gene upregulated by p53 in response to DNA damage, exists as part of a positive-feedback loop which activates p53 and is a human cancer epithelial marker downregulated in various cancer types. 14-3-3σ levels are critical for maintaining p53 activity in response to DNA damage and regulating signal mediators such as Akt. Here, we identify Mammalian Constitutive Photomorphogenic 1 (COP1) as a novel E3 ubiquitin ligase for targeting 14-3-3σ through proteasome degradation. We show for the first time that COP9 signalosome subunit 6 (CSN6) associates with COP1 and is involved in 14-3-3σ ubiquitin-mediated degradation. Mechanistic studies show that CSN6 expression leads to stabilization of COP1 through reducing COP1 self-ubiquitination and decelerating COP1’s turnover rate. We also show that CSN6-mediated 14-3-3σ ubiquitination is compromised when COP1 is knocked down. Thus, CSN6 mediates 14-3-3σ ubiquitination through enhancing COP1 stability. Subsequently, we show that CSN6 causes 14-3-3σ downregulation, thereby activating Akt and promoting cell survival. Also, CSN6 overexpression leads to increased cell growth, transformation and promotes tumorigenicity. Significantly, 14-3-3σ expression can correct the abnormalities mediated by CSN6 expression. These data suggest that the CSN6-COP1 axis is involved in 14-3-3σ degradation, and that deregulation of this axis will promote cell growth and tumorigenicity.

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Background: Triple negative breast cancers (TNBCs) account for about 15% of breast cancer diagnosis in the USA, and occur at a higher percentage among African-American women (about 34%). Although they constitute a small percentage, the lack of targeted therapy of these hormone receptor-negative cancers has led to poor prognosis and overall survival. The main cause of TNBC-associated death is tumor metastasis from the primary site to distant sites. Characterization of drivers of EMT, one of the major drivers of metastasis could potentially identify therapeutic targets to prevent and potentially block metastasis. Interactions between tumor cells and inflammatory cells, including tumor-infiltrating neutrophils (TILs) play an important role in initiating and driving the process of metastasis. Our group previously reported that Neutrophil Elastase (NE), a secreted elastin from neutrophils as a mitogen that promotes tumor growth and activation of EGFR and TLR4 signaling pathways. We have also shown that molecular and pharmacological inhibition of NE leads to 90% reduction in metastatic foci in the lung. In this study, we interrogated the role of NE in EMT and its ability to affect key cellular functions involved in tumor metastasis, using TNBC cell lines as our model system. Methods: Using a genetic knockdown model of NE in murine and several human breast cancer cell lines we performed cellular proliferation, migration and invasion assays to evaluate its role in essential EMT-associated functions. We also interrogated the specific roles of the epithelial junction protein E-cadherin and mesenchymal transcription factor Snail in the ability of NE to drive a mesenchymal phenotype in the breast cancer cells. Results: Silencing of NE in all cells examined to date led to a significant increase in E-cadherin, a junction protein associated with an epithelial phenotype. We also observed a significant decrease at the mRNA level of Snail, a major transcription factor associated with epithelial-mesenchymal transition and Axl, a receptor tyrosine kinase that is highly associated with mesenchymal cells. Performing functional assays, we showed that NE silencing had a significant effect on cellular invasion through Matrigel. Studies are on going to validate the effects on cellular invasion and EMT mediated by exogenous NE uptake by the breast cancer cells. Conclusions: In summary, we identified NE as a potential driver of EMT and a mesenchymal phenotype in breast cancer cell lines. NE promotes cellular invasion and causes upregulation of factors associated with EMT. Studies are on going to characterize the major pathways downstream of NE that mediate the effects and to determine the function of exogenous NE that tumor cells are exposed to in their microenvironment. This study will lead to identification of mechanisms of NE-mediated effects on EMT and potential therapeutic targets of tumor metastasis. Citation Format: Kavitha Balaji, Chun-Hui Su, Said Akli, Kelly K. Hunt, Khandan Keyomarsi. Neutrophil Elastase plays a key role in epithelial-mesenchymal transition and metastasis in triple-negative breast cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1422. doi:10.1158/1538-7445.AM2015-1422