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...

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MicroRNAs (miRs) are conserved small non-coding RNAs that negatively regulate gene expression. The miR profiles are markedly altered in cancers and some of them have a causal role in tumorigenesis. Here, we report changes in miR expression profile in hepatocellular carcinomas (HCCs) developed in male Fisher rats-fed folic acid, methionine, and choline-deficient (FMD) diet. Comparison of the miR profile by microarray analysis showed altered expression of some miRs in hepatomas compared to the livers from age-matched rats on the normal diet. family of genes was upregulated, miR-122, an abundant liver-specific miR, was downregulated in the tumors. The decrease in hepatic miR-122 was a tumor-specific event because it did not occur in the rats switched to the folate and methyladequate diet after 36 weeks on deficient diet, which did not lead to hepatocarcinogenesis. miR-122 was also silent in a transplanted rat hepatoma. Extrapolation of this study to human primary HCCs revealed that miR-122 expression was significantly (P = 0.013) reduced in 10 out of 20 tumors compared to the pair-matched control tissues. These findings suggest that the downregulation of miR-122 is associated with hepatocarcinogenesis and could be a potential biomarker for liver cancers. RNAs that block translation by imperfect base pairing to the 3′-untranslated regions (3′-UTR) of specific mRNA and by inducing mRNA degradation. Most miRs are expressed as primary transcripts transcribed by pol II, some miRs in clusters are coordinately expressed, and others are generated from introns. Primary miRs (pri-miRs) have 5′ caps and 3′ poly (A) tails, which is processed to mature miR by specific ribonuclease complexes (for review, see Zeng et al. [2005]). miRs play a key role in regulating diverse cellular processes that include development, differentiation, cell growth, apoptosis, viral infection, and metabolism (for review, see Ambros [2004]). KeywordsLike mRNAs, the majority of miRs are expressed predominantly in a tissue-specific manner whereas some are enriched in certain tissue [Lagos-Quintana et al., 2002]. Recently, much attention has been focused on miRs and cancer since miR genes are located at chromosomal regions, characterized by fragile sites and regions of deletion or amplification [Calin et al., 2004]. Some of these miRs deregulated in cancer function as tumor suppressors or oncogenes (for review, see Hwang and Mendell [2006]).Our laboratory has been studying the transcriptional and epigenetic regulation of gene expression in rodent and human primary hepatocellular carcinomas (HCCs) [Majumder et al., 2002;Ghoshal et al., 2004]. To study the altered regulation of gene expression at different stages of hepatocarcinogeneis, we have used a rat model. In this model, Fisher male rats-fed folate and methyl-deficient (FMD) diets develop preneoplastic nodules after 36 weeks and HCCs after 54 weeks Li, 2006]. Recently, we have used this rat model to identify the genes that are regulated tumor-specifically by epigenetic mechanism ]. This mode...

Epithelial-mesenchymal transition (EMT) is a key process in tumor metastatic cascade that is characterized by the loss of cell-cell junctions and cell polarity, resulting in the acquisition of migratory and invasive properties. However, the precise molecular events that initiate this complex EMT process in head and neck cancers are poorly understood. Increasing evidence suggests that tumor microenvironment plays an important role in promoting EMT in tumor cells. We have previously shown that head and neck tumors exhibit significantly higher Bcl-2 expression in tumor-associated endothelial cells and overexpression of Bcl-2 alone in tumor-associated endothelial cells was sufficient to enhance tumor metastasis of oral squamous cell carcinoma in a SCID mouse model. In this study, we show that endothelial cells expressing Bcl-2 (EC-Bcl-2), when co-cultured with head and neck tumor cells (CAL27), significantly enhance EMT-related changes in tumor cells predominantly by the secretion of IL-6. Treatment with recombinant IL-6 or stable IL-6 overexpression in CAL27 cells or immortalized oral epithelial cells (IOE) significantly induced the expression of mesenchymal marker, vimentin, while repressing E-cadherin expression via the JAK/STAT3/Snail signaling pathway. These EMT-related changes were further associated with enhanced tumor and IOE cell scattering and motility. STAT3 knock-down significantly reversed IL-6-mediated tumor and IOE cell motility by inhibiting FAK activation. Furthermore, tumor cells overexpressing IL-6 showed marked increase in lymph node and lung metastasis in a SCID mouse xenograft model. Taken together, these results demonstrate a novel function for IL-6 in mediating EMT in head and neck tumor cells and increasing their metastatic potential.

MicroRNAs (miR) are a class of small (f21 nucleotide) noncoding RNAs that, in general, negatively regulate gene expression. Some miRs harboring CGIs undergo methylationmediated silencing, a characteristic of many tumor suppressor genes. To identify such miRs in liver cancer, the miRNA expression profile was analyzed in hepatocellular carcinoma (HCC) cell lines treated with 5-azacytidine (DNA hypomethylating agent) and/or trichostatin A (histone deacetylase inhibitor). The results showed that these epigenetic drugs differentially regulate expression of a few miRs, particularly miR-1-1, in HCC cells. The CGI spanning exon 1 and intron 1 of miR-1-1 was methylated in HCC cell lines and in primary human HCCs but not in matching liver tissues. The miR-1-1 gene was hypomethylated and activated in DNMT1À/À HCT 116 cells but not in DNMT3B null cells, indicating a key role for DNMT1 in its methylation. miR-1 expression was also markedly reduced in primary human hepatocellular carcinomas compared with matching normal liver tissues. Ectopic expression of miR-1 in HCC cells inhibited cell growth and reduced replication potential and clonogenic survival. The expression of FoxP1 and MET harboring three and two miR-1 cognate sites, respectively, in their respective 3 ¶-untranslated regions, was markedly reduced by ectopic miR-1. Up-regulation of several miR-1 targets including FoxP1, MET, and HDAC4 in primary human HCCs and down-regulation of their expression in 5-AzaC-treated HCC cells suggest their role in hepatocarcinogenesis. The inhibition of cell cycle progression and induction of apoptosis after re-expression of miR-1 are some of the mechanisms by which DNA hypomethylating agents suppress hepatocarcinoma cell growth. [Cancer Res 2008;68(13):5049-58]

5-Azacytidine-and 5-aza-deoxycytidine (5-aza-CdR)-mediated reactivation of tumor suppressor genes silenced by promoter methylation has provided an alternate approach in cancer therapy. Despite the importance of epigenetic therapy, the mechanism of action of DNA-hypomethylating agents in vivo has not been completely elucidated. Here we report that among three functional DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B), the maintenance methyltransferase, DNMT1, was rapidly degraded by the proteasomal pathway upon treatment of cells with these drugs.

The methylation status of the CpG island located within the ribosomal RNA (rRNA) promoter in human hepatocellular carcinomas and pair-matched liver tissues was analyzed by bisulfite genomic sequencing. Significant hypomethylation of methyl-CpGs in the rRNA promoter was observed in the tumor samples compared with matching normal tissues, which was consistent with the relatively high level of rRNA synthesis in rapidly proliferating tumors. To study the effect of CpG methylation on RNA polymerase I (pol I)-transcribed rRNA genes, we constructed pHrD-IRES-Luc (human rRNA promoter-luciferase reporter). In this plasmid, Kozak sequence of the pGL3-basic vector was replaced by the internal ribosome entry site (IRES) of encephalomyocarditis viral genome to optimize pol I-driven reporter gene expression. Transfection of this plasmid into HepG2 (human) cells revealed reduced pol I-driven luciferase activity with an increase in methylation density at the promoter. Markedly reduced luciferase activity in Hepa (mouse) cells compared with HepG2 (human) cells showed that pHrD-IRES-Luc is transcribed by pol I. Site-specific methylation of human rRNA promoter demonstrated that methylation of CpG at the complementary strands located in the promoter (؊9, ؊102, ؊347 with respect to the ؉1 site) inhibited luciferase activity, whereas symmetrical methylation of a CpG in the transcribed region (؉152) did not affect the promoter activity. Immunofluorescence studies showed that the methyl-CpG-binding proteins, MBD1, MBD2, MBD3, and MeCP2, are localized both in the nuclei and nucleoli of HepG2 cells. Transient overexpression of MBD2 suppressed luciferase activity specifically from the methylated rRNA promoter, whereas MBD1 and MBD3 inhibited rRNA promoter activity irrespective of the methylation status. Chromatin immunoprecipitation analysis confirmed predominant association of MBD2 with the endogenous methylated rRNA promoter, which suggests a selective role for MBD2 in the methylationmediated inhibition of ribosomal RNA gene expression.

Reactivation of silenced tumor suppressor genes by 5-azacytidine (Vidaza) and its congener 5-aza-2 ¶-deoxycytidine (decitabine) has provided an alternate approach to cancer therapy. We have shown previously that these drugs selectively and rapidly induce degradation of the maintenance DNA methyltransferase (DNMT) 1 by a proteasomal pathway. Because the toxicity of these compounds is largely due to their incorporation into DNA, it is critical to explore novel, nonnucleoside compounds that can effectively reactivate the silenced genes. Here, we report that a quinoline-based compound, designated SGI-1027, inhibits the activity of DNMT1, DNMT3A, and DNMT3B as well M. Sss I with comparable IC 50 (6-13 Mmol/L) by competing with S-adenosylmethionine in the methylation reaction. Treatment of different cancer cell lines with SGI-1027 resulted in selective degradation of DNMT1 with minimal or no effects on DNMT3A and DNMT3B. At a concentration of 2.5 to 5 Mmol/L (similar to that of decitabine), complete degradation of DNMT1 protein was achieved within 24 h without significantly affecting its mRNA level. MG132 blocked SGI-1027-induced depletion of DNMT1, indicating the involvement of proteasomal pathway. Prolonged treatment of RKO cells with SGI-1027 led to demethylation and reexpression of the silenced tumor suppressor genes P16, MLH1, and TIMP3. Further, this compound did not exhibit significant toxicity in a rat hepatoma (H4IIE) cell line. This study provides a novel class of DNA hypomethylating agents that have the potential for use in epigenetic cancer therapy. [Cancer Res 2009;69(10):4277-85]