Kip1 in the resistant OHT R cells caused enhanced cell death when exposed to tamoxifen. This is the first study demonstrating a relationship between miR-221/222 expression and HER2/neu overexpression in primary breast tumors that are generally resistant to tamoxifen therapy. This finding also provides the rationale for the application of altered expression of specific miRNAs as a predictive tamoxifen-resistant breast cancer marker.Breast cancer is the most common malignancy in women, accounting for 31% of all female cancers. An estimated 178,480 new cases of invasive breast cancer was diagnosed in the United States in 2007, and 40,460 women will die of this cancer. Over two-thirds of breast cancers exhibit high concentrations of estrogen receptor, which contribute to tumor growth and progression. Blocking the steroid hormone pathway with tamoxifen and/or oophorectomy has been shown to be effective in this patient population. The Early Breast Cancer Trialists' Collaborative Group overview demonstrated a significant improvement in 15-year survival with the addition of adjuvant tamoxifen for 5 years following surgery (1). Furthermore, tamoxifen can also reduce the incidence of contralateral breast cancer and has been approved as a prophylactic agent to prevent breast cancer. Despite this accomplishment in the management of women with potentially endocrine-responsive breast cancers, a significant proportion of these women will experience disease progression due to either an intrinsic or acquired resistance to tamoxifen.Nongenomic activation of epidermal growth factor receptor/ HER2 signaling by tamoxifen is an important factor contributing to tamoxifen resistance. This leads to activation of both the p42/44 mitogen-activated protein kinase (MAPK) and Akt signaling pathways, which favor cell proliferation and survival. These changes could be blocked by the selective epidermal growth factor receptor tyrosine kinase inhibitor gefitnib, suggesting that epidermal growth factor receptor/HER2 signaling is directly involved in tamoxifen resistance (2). The preclinical data are corroborated by clinical observations that tumors expressing HER2 exhibit poor outcome when treated with tamoxifen (3). None of the molecular mechanisms proposed for tamoxifen resistance (for review, see Ref. 4) have led to the development of a gene expression profile that can consistently identify resistant tumors and benefit these patients from upfront use of alternative drugs such as aromatase inhibitors.Recent studies have highlighted the key regulatory roles of microRNAs (miRNAs) 3 in all fundamental cellular processes in animals and plants. Altered expression of miRNAs in primary human cancers has been used for tumor diagnosis, classification, staging, and prognosis (5). These small noncoding RNAs regulate expression of their target proteins primarily by inhibiting translation of the target mRNA and in some cases by inducing rapid decay of the message (6). A study with 76 neo-* This work was supported, in whole or in part, by National Institut...

Key Points• EBV infection leads to PRMT5 overexpression and global epigenetic changes that are essential to drive B-lymphocyte transformation.• Highly selective PRMT5 inhibitors represent a novel, first-in-class drug that restores critical regulatory checkpoints in lymphoma cells.Epigenetic events that are essential drivers of lymphocyte transformation remain incompletely characterized. We used models of Epstein-Barr virus (EBV)-induced B-cell transformation to document the relevance of protein arginine methyltransferase 5 (PRMT5) to regulation of epigenetic-repressive marks during lymphomagenesis. EBV 1 lymphomas and transformed cell lines exhibited abundant expression of PRMT5, a type II PRMT enzyme that promotes transcriptional silencing of target genes by methylating arginine residues on histone tails. PRMT5 expression was limited to EBV-transformed cells, not resting or activated B lymphocytes, validating it as an ideal therapeutic target. We developed a first-in-class, small-molecule PRMT5 inhibitor that blocked EBV-driven B-lymphocyte transformation and survival while leaving normal B cells unaffected. Inhibition of PRMT5 led to lost recruitment of a PRMT5/p65/HDAC3-repressive complex on the miR96 promoter, restored miR96 expression, and PRMT5 downregulation. RNA-sequencing and chromatin immunoprecipitation experiments identified several tumor suppressor genes, including the protein tyrosine phosphatase gene PTPROt, which became silenced during EBV-driven B-cell transformation. Enhanced PTPROt expression following PRMT5 inhibition led to dephosphorylation of kinases that regulate B-cell receptor signaling. We conclude that PRMT5 is critical to EBV-driven B-cell transformation and maintenance of the malignant phenotype, and that PRMT5 inhibition shows promise as a novel therapeutic approach for B-cell lymphomas. (Blood. 2015;125(16):2530-2543

Background: MicroRNAs-221/222 and 181b are up-regulated in tamoxifen-resistant breast cancer. Results: Anti-miRs-222/181b regressed tamoxifen-resistant xenografts. Down-regulation of TIMP3, a common target of these miRs, facilitated growth factor signaling by regulating metalloproteases. Conclusion: Survival of tamoxifen-resistant breast cancer is dependent on miR-mediated suppression of TIMP3. Significance: Anti-miRs-222/221 and -181b can be used to render tamoxifen-resistant tumors responsive to the drug.

In an effort to understand the epigenetic regulation of ribosomal RNA gene (rDNA) expression we have previously demonstrated the role of DNA methyltransferases and methyl CpG binding proteins in rRNA synthesis. Here, we studied the role of protein arginine methyltransferase PRMT5 and the two methylated histones H3R8Me2 and H4R3Me2, in rDNA expression in Epstein Barr virus- transformed primary B-cells (LCLs) and in HeLa cells responding to serum-regulated growth. Chromatin immunoprecipitation assay showed that histones H3 and H4 associated with rRNA promoters were differentially methylated at arginine residues 8 and 3, respectively, depending on its transcriptional activity. Association of PRMT5 and methylated H3 with the unmethylated promoters in resting B-cells was significantly reduced in rapidly growing LCLs. Unlike PRMT5 and H3R8Me2, histone H4 associated with both methylated and unmethylated rRNA promoters in resting B-cells was methylated at the R3 residue. However, a dramatic decrease in R3 methylation of H4 recruited to the unmethylated rRNA promoters was observed in LCLs while it remained unaltered in the fraction bound to the methylated promoters. Differential interaction of PRMT5 and methylation of H3 and H4 associated with the rRNA promoters was also observed when serum starved HeLa cells were allowed to grow in serum replenished media. Ectopic expression of PRMT5 suppressed activity of both unmethylated and methylated rRNA promoter in transient transfection assay whereas siRNA mediated knockdown of PRMT5 increased rRNA synthesis in HeLa cells. These data suggest a key role of PRMT5 and the two methylated histones in regulating rRNA promoter activity.

Metallothioneins (MT) are potent scavengers of free radicals that are silenced in primary hepatocellular carcinomas (HCC) of human and rodent origin. To examine whether loss of MT promotes hepatocarcinogenesis, male Mt-1 and Mt-2 double knockout (MTKO) and wild-type (WT) mice were exposed to diethylnitrosamine (DEN) and induction of HCC was monitored at 23 and 33 weeks. The size and number of liver tumors, the ratio between liver and body weight, and liver damage were markedly elevated in the MTKO mice at both time points compared with the WT mice. At 23 weeks, MTKO mice developed HCC whereas WT mice developed only preneoplastic nodules suggesting that loss of MT accelerates hepatocarcinogenesis. MTKO tumors also exhibited higher superoxide anion levels. Although NF-kB activity increased in the liver nuclear extracts of both genotypes after DEN exposure, the complex formed in MTKO mice was predominantly p50/65 heterodimer (transcriptional activator) as opposed to p50 homodimer (transcriptional repressor) in WT mice. Phosphorylation of p65 at Ser276 causing its activation was also significantly augmented in DEN-exposed MTKO livers. NFkB targets that include early growth response genes and proinflammatory cytokines were significantly upregulated in MTKO mice. Concurrently, there was a remarkable increase ($100-fold) in Pai-1 expression; significant increase in c-Jun, c-Fos, c-Myc, Ets2, and ATF3 expressions; and growth factor signaling that probably contributed to the increased tumor growth in MTKO mice. Taken together, these results demonstrate that MTs protect mice from hepatocarcinogen-induced liver damage and carcinogenesis, underscoring their potential therapeutic application against hepatocellular cancer.