UBE2N is a K63-specific ubiquitin conjugase linked to various immune disorders and cancer. Here, we demonstrate that UBE2N and its partners UBE2V1 and UBE2V2 are highly expressed in malignant melanoma. Silencing of UBE2N and its partners significantly decreased melanoma cell proliferation and subcutaneous tumor growth. This was accompanied by increased expression of E-cadherin, p16, and MC1R and decreased expression of melanoma malignancy markers including SOX10, Nestin, and ABCB5. Mass spectrometry-based phosphoproteomic analysis revealed that UBE2N loss resulted in distinct alterations to the signaling landscape: MEK/ERK signaling was impaired, FRA1 and SOX10 gene regulators were downregulated, and p53 and p16 tumor suppressors were upregulated. Similar to inhibition of UBE2N and MEK, silencing FRA1 decreased SOX10 expression and cell proliferation. Conversely, exogenous expression of active FRA1 increased pMEK and SOX10 expression, and restored anchorage-independent cell growth of cells with UBE2N loss. Systemic delivery of NSC697923, a small-molecule inhibitor of UBE2N, significantly decreased melanoma xenograft growth. These data indicate that UBE2N is a novel regulator of the MEK/FRA1/SOX10 signaling cascade and is indispensable for malignant melanoma growth. Our findings establish the basis for targeting UBE2N as a potential treatment strategy for melanoma. These findings identify ubiquitin conjugase UBE2N and its variant partners as novel regulators of MAPK signaling and potential therapeutic targets in melanoma. .
The objective of this study was to find the optimum dose of flaxseed that would decrease prostaglandins and alter estrogen pathway endpoints implicated in ovarian cancer. Fifty 1.5-year-old chickens per group were fed different percentages of flaxseed (5%, 10%, 15%) for four months then sacrificed to collect blood and tissues. Levels of flaxseed lignan metabolites, Enterolactone (EL) and Enterodiol (ED), were measured in the serum, liver and ovaries by LC-MS/MS while omega-3 and 6 fatty acid levels were measured by GC. Effect of varying flaxseed doses was assessed by measuring levels of prostaglandin E2, estrogen metabolites (16-hydroxyestrone (16-OHE1), 2-hydroxyestrone (2-OHE1)) and analyzing the expression of E2 metabolizing enzymes (CYP3A4, CYP1B1, CYP1A1) and ERα in the ovaries. The ratio of omega-3 fatty acids/ omega-6 fatty acids increased with increase in flaxseed supplementation of diet corresponding to a dose dependent decrease in COX-2 protein and prostaglandin E2 levels. EL and ED increased in the liver, ovary and serum with increasing concentrations of flaxseed. Flaxseed decreased the expression of ERα in the ovary. The ratio of 2-hydroxyestrone to 16-hydroxyestrone in the serum increased significantly in the 15% flaxseed diet with a corresponding increase in CYP1A1 in the liver and a decrease in CYP3A4 in the ovary, respectively. CYP1B1 mRNA also decreased with flaxseed diet in the ovary. The 15% flaxseed supplemented diet significantly decreased inflammatory prostaglandin E2, ERα, CYP3A4, CYP1B1 and 16-OHE1 while it increased CYP1A1 and 2-OHE1, thus reducing the inflammatory and pro-carcinogenic microenvironment of the ovary.
Flaxseed has been studied for decades for its health benefits that include anti-cancer, cardioprotective, anti-diabetic, anti-inflammatory properties. The biologically active components that mediate these effects are the omega-3 fatty acids and the lignan, secoisolaricirescinol diglucoside. We have previously shown that whole flaxseed supplemented diet decreases the severity and incidence of ovarian cancer while a 15% dose of flaxseed is most protective against inflammation and estrogen-induced chemical and genotoxicity. The objective of this study was to dissect the independent effects of the two flaxseed components on estrogen signaling and metabolism. Two and half year old hens were fed either a control diet, 15% whole flaxseed diet, defatted flax meal diet or 5% flax oil diet for 3 months after which the animals were sacrificed and blood and tissues were harvested. Whole flaxseed diet caused a decrease in expression of ERα. ERα target gene expression was assessed using RT2 profiler PCR array. Some targets involved in the IGF/insulin signaling pathway (IRS1, IGFBP4, IGFBP5) were downregulated by flaxseed and its components. Flaxseed diet also downregulated AKT expression. A number of targets related to NF-κB signaling were altered by flaxseed diet including a series of targets implicated in cancer. Whole flaxseed diet also affected E2 metabolism by increasing CYP1A1 expression with a corresponding increase in the onco-protective E2 metabolite, 2-methoxyestradiol. The weak anti-estrogens, enterolactone, enterodiol and 2-methoxyestradiol, might be working synergistically to generate a protective effect on the ovaries from hens on whole flaxseed diet by altering the estrogen signaling and metabolism.
The study reported here demonstrates that a flaxseed supplemented diet causes ovarian tumors in the laying hen to undergo apoptosis, resulting in a reduction of tumor burden, reducing the frequency and severity of ovarian cancer. We have previously shown in normal ovaries that flaxseed and its components downregulate ERalpha and alter the expression of enzymes that metabolize estrogen. In this study, we analyzed the effects of the two main components of whole flaxseed, ligan and omega 3 fatty acids, on estrogen metabolism and the estrogen receptor in ovarian tumors. ER alpha expression was upregulated in the ovarian tumors and was not affected by diet. Liver CYP1A1 expression was significantly increased by the whole flaxseed diet with a corresponding increase in 2-methoxyestradiol plasma levels. We also observed increased p38 and ERK 1/2 MAPK activation in the ovary as well as an increase in apoptosis in the tumor epithelium. SMAD 7, a factor involved in the 2-methoxyestradiol-mediated apoptosis pathway was also upregulated in tumors from the whole flaxseed diet group. 2-methoxyestradiol-induced anti-tumor effects were further validated by in human ovarian cancer cells. This study details the effect of flaxseed diet on estrogen metabolism and demonstrates the anti-ovarian cancer effects of 2-methoxyestradiol.
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