The development of targeted therapies for antiestrogenresistant breast cancer requires a detailed understanding of its molecular characteristics. To further elucidate the molecular events underlying acquired resistance to the antiestrogens tamoxifen and fulvestrant, we established drug-resistant sublines from a single colony of hormonedependent breast cancer MCF7 cells. These model systems allowed us to examine the cellular and molecular changes induced by antiestrogens in the context of a uniform clonal background. Global changes in both basal and estrogeninduced gene expression profiles were determined in hormonesensitive and hormonal-resistant sublines using Affymetrix Human Genome U133 Plus 2.0 Arrays. Changes in DNA methylation were assessed by differential methylation hybridization, a high-throughput promoter CpG island microarray analysis. By comparative studies, we found distinct gene expression and promoter DNA methylation profiles associated with acquired resistance to fulvestrant versus tamoxifen. Fulvestrant resistance was characterized by pronounced upregulation of multiple growth-stimulatory pathways, resulting in estrogen receptor A (ERA)-independent, autocrineregulated proliferation. Conversely, acquired resistance to tamoxifen correlated with maintenance of the ERA-positive phenotype, although receptor-mediated gene regulation was altered. Activation of growth-promoting genes, due to promoter hypomethylation, was more frequently observed in antiestrogen-resistant cells compared with gene inactivation by promoter hypermethylation, revealing an unexpected insight into the molecular changes associated with endocrine resistance. In summary, this study provides an in-depth understanding of the molecular changes specific to acquired resistance to clinically important antiestrogens. Such knowledge of resistance-associated mechanisms could allow for identification of therapy targets and strategies for resensitization to these well-established antihormonal agents. (Cancer Res 2006; 66(24): 11954-66)

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The microRNA miR-21 is overexpressed in many human cancers, wherein accumulating evidence indicates that it functions as an oncogene. Here, we report that the cytokine IFN rapidly induces miR-21 expression in human and mouse cells. Signal transducer and activator of transcription 3 (STAT3) was implicated in this pathway based on the lack of IFN effect on miR-21 expression in prostate cancer cells with a deletion in the STAT3 gene. STAT3 ablation abrogated IFN induction of miR-21, confirming the important role of STAT3 in regulating miR-21. Chromatin immunoprecipitation analysis showed that STAT3 directly bound the miR-21 promoter in response to IFN. Experiments in mouse embryo fibroblasts with a genetic deletion of the p65 NF-κB subunit showed that IFN-induced miR-21 expression was also dependent on NF-κB. STAT3 silencing blocked both IFN-induced p65 binding to the miR-21 promoter and p65 nuclear translocation. Thus, IFN-induced miR-21 expression is coregulated by STAT3 and NF-κB at the level of the miR-21 promoter. Several cell death regulators were identified as downstream targets of miR-21, including PTEN and Akt. Functional experiments in prostate cancer cells directly showed that miR-21 plays a critical role in suppressing IFN-induced apoptosis. Our results identify miR-21 as a novel IFN target gene that functions as a key feedback regulator of IFN-induced apoptosis.

Chemokines and inflammatory cytokines are key regulators of immunity and inflammation during viral infections. Hepatitis C virus (HCV) is a hepatotropic RNA virus frequently associated with chronic liver inflammation and hepatocellular carcinoma. Intrahepatic levels of chemokines and cytokines are elevated in chronic HCV infections, but the underlying mechanisms remain unclear. We find that Toll like receptor-3 (TLR3) senses HCV infection in cultured hepatoma cells, leading to NF-κB activation and the production of numerous chemokines and inflammatory cytokines, such as RANTES, MIP-1α, MIP-1β, IP-10 and IL-6. The chemokine/cytokine induction occurred late in HCV infection and was abrogated when HCV was UV-inactivated prior to infection, indicating a dependence on cellular recognition of HCV replication products. Gel shift and chromatin immunoprecipitation assays revealed that NF-κB plays a pivotal role in HCV-induced chemokine/cytokine transcription. Mutations specifically disrupting the dsRNA binding activity of TLR3 ablated the chemokine/cytokine response to HCV infection, indicating that HCV dsRNA was the pathogen associated molecular pattern triggering TLR3 signaling. In vitro synthesized HCV dsRNAs with a minimal length of ~80-100 bp activated TLR3-dependent chemokine expression, regardless of the genome position from which they derive. In contrast, HCV ssRNAs, including those derived from the structured 3′NTR highly potent for RIG-I activation, failed to do so. Moreover, robust production of chemokines and inflammatory cytokines was also observed in primary human hepatocytes following stimulation with extracellular poly-I:C, a TLR3 ligand. Conclusion Our data suggest that TLR3-mediated chemokine and inflammatory cytokine responses may play an important role in host immune responses to HCV and the pathogenesis of HCV-associated liver diseases.

Alterations in histones, chromatin-related proteins, and DNA methylation contribute to transcriptional silencing in cancer, but the sequence of these molecular events is not well understood. Here we demonstrate that on disruption of estrogen receptor (ER) ␣ signaling by small interfering RNA, polycomb repressors and histone deacetylases are recruited to initiate stable repression of the progesterone receptor (PR) gene, a known ER␣ target, in breast cancer cells. The event is accompanied by acquired DNA methylation of the PR promoter, leaving a stable mark that can be inherited by cancer cell progeny. Reestablishing ER␣ signaling alone was not sufficient to reactivate the PR gene; reactivation of the PR gene also requires DNA demethylation. Methylation microarray analysis further showed that progressive DNA methylation occurs in multiple ER␣ targets in breast cancer genomes. The results imply, for the first time, the significance of epigenetic regulation on ER␣ target genes, providing new direction for research in this classical signaling pathway.