Retinoblastoma (RB; encoded by RB1) is a tumor suppressor that is frequently disrupted in tumorigenesis and acts in multiple cell types to suppress cell cycle progression. The role of RB in tumor progression, however, is poorly defined. Here, we have identified a critical role for RB in protecting against tumor progression through regulation of targets distinct from cell cycle control. In analyses of human prostate cancer samples, RB loss was infrequently observed in primary disease and was predominantly associated with transition to the incurable, castration-resistant state. Further analyses revealed that loss of the RB1 locus may be a major mechanism of RB disruption and that loss of RB function was associated with poor clinical outcome. Modeling of RB dysfunction in vitro and in vivo revealed that RB controlled nuclear receptor networks critical for tumor progression and that it did so via E2F transcription factor 1-mediated regulation of androgen receptor (AR) expression and output. Through this pathway, RB depletion induced unchecked AR activity that underpinned therapeutic bypass and tumor progression. In agreement with these findings, disruption of the RB/E2F/nuclear receptor axis was frequently observed in the transition to therapy resistance in human disease. Together, these data reveal what we believe to be a new paradigm for RB function in controlling prostate tumor progression and lethal tumor phenotypes. IntroductionRetinoblastoma (RB; encoded by RB1), a tumor suppressor protein, is a critical negative regulator of tumor development. RB prevents tumorigenesis by suppressing cell cycle progression (1). However, the role of RB in tumor progression is poorly understood, and the clinical importance of RB loss during this process has not been well considered. Here, we identified a clinically relevant function for RB in tumor progression, manifest through control of hormone signaling networks.The function of RB in cell cycle control has been well described (1). Conditions favoring cell cycle arrest induce RB hypophosphorylation and activation. Active RB binds to promoters of genes required for S-phase entry (e.g., CCNA2 and MCM7) and, through association with the SWI/SNF complex and corepressor molecules (e.g., Sin3B), elicits transcriptional corepression. Many RB target genes are positively regulated by activator E2F transcription factors, supporting the current model that RB acts by suppressing E2F-mediated transcriptional activation. Indeed, the minimal transcriptional repression and tumor suppression domain of RB contains the E2F binding motif. E2F-independent functions of RB have been identified (2), but the contribution of these functions to tumor suppression is uncertain. Thus, contemporary views of RB suggest that the protein prevents cell cycle deregulation and tumor development through suppression of activator E2Fs.

Plasmacytoid dendritic cells (PDC) produce high levels of type I IFN upon stimulation with viruses, while monocytes and monocyte-derived dendritic cells (MDDC) produce significantly lower levels. To find what determines the high production of type I IFN in PDC, we examined the relative levels of IRF transcription factors, some of which play critical roles in the induction of IFN. Furthermore, to determine whether the differences could result from expression of distinct IFNA subtypes, the profile of IFNA genes expressed was examined. PDC responded equally well to stimulation with HSV-1 and Sendai virus (SV) by producing high levels of type I IFN, whereas the MDDC and monocyte response to SV were lower, and neither responded well to HSV-1. All three populations constitutively expressed most of the IRF genes. However, real-time RT-PCR demonstrated increased levels of IRF-7 transcripts in PDC compared with monocytes. As determined by intracellular flow cytometry, the PDC constitutively expressed significantly higher levels of IRF-7 protein than the other populations while IRF-3 levels were similar among populations. Analysis of the profile of IFNA genes expressed in virus-stimulated PDC, monocytes and MDDC demonstrated that each population expressed IFNA1 as the major subtype but that the range of the subtypes expressed in PDC was broader, with some donor and stimulus-dependent variability. We conclude that PDC but not MDDC are uniquely preprogrammed to respond rapidly and effectively to a range of viral pathogens with high levels of IFN-alpha production due to the high levels of constitutively expressed IRF-7.

The role of mammary epithelial cell (MEC) NF-kB in tumor progression in vivo is unknown, as murine NF-kB components and kinases either are required for murine survival or interfere with normal mammary gland development. As NF-kB inhibitors block both tumor-associated macrophages (TAM) and MEC NF-kB, the importance of MEC NF-kB to tumor progression in vivo remained to be determined. Herein, an MEC-targeted inducible transgenic inhibitor of NF-kB (IkBaSR) was developed in ErbB2 mammary oncomice. Inducible suppression of NF-kB in the adult mammary epithelium delayed the onset and number of new tumors. Within similar sized breast tumors, TAM and tumor neoangiogenesis was reduced. Coculture experiments demonstrated MEC NF-kB enhanced TAM recruitment. Genome-wide expression and proteomic analysis showed that IkBaSR inhibited tumor stem cell pathways. IkBaSR inhibited breast tumor stem cell markers in transgenic tumors, reduced stem cell expansion in vitro, and repressed expression of Nanog and Sox2 in vivo and in vitro. MEC NF-kB contributes to mammary tumorigenesis. As we show that NF-kB contributes to expansion of breast tumor stem cells and heterotypic signals that enhance TAM and vasculogenesis, these processes may contribute to NF-kB-dependent mammary tumorigenesis.

The molecular mechanism by which virus induces expression of the early inflammatory genes has not yet been completely elucidated. Previous studies indicated that the virus-mediated transcription of type I interferon (IFN) genes required activation of two members of IFN regulatory factor (IRF) family, IRF-3 and IRF-7, where the expression of IRF-7 was found to be indispensable for the induction of IFNA genes. To determine the factors that regulate expression of IRF-7 gene, as well as its inducibility by type I IFNs, we have isolated and characterized the promoter and first intron of the human IRF-7 gene. This region shows a presence of two potential interferon-sensitive response elements (ISRE/ IRF-E). However, only the ISRE present in the first intron was functional and conferred interferon inducibility in a transient transfection assay. Using a pull-down assay with an oligodeoxynucleotide corresponding to this ISRE immobilized to magnetic beads, we have demonstrated that this ISRE binds ISGF3 complex and IRF-1 from the extract of IFN-treated cells but not from the untreated cells. We have further shown that the previously observed lack of expression of IRF-7 in 2fTGH fibrosarcoma cell line, correlated with hypermethylation of the CpG island in the human IRF-7 promoter. The repression of the promoter activity was relieved by treatment with DNA methyltransferase inhibitor 5-azadeoxycytidine. In vitro methylation of IRF-7 promoter silenced IRF-7 directed expression of luciferase gene in HeLa cells that express endogenous IRF-7 gene. Whether silencing of IRF-7 by methylation is instrumental for the process of tumorigenesis remains to be determined.Expression of eukaryotic genes is regulated at multiple levels including the accessibility of promoter DNA for binding of basic transcriptional machinery or the specific transcription factors and chromatin structure around the potential promoters. The molecular mechanism by which virus activates expression of the early inflammatory genes has not yet been completely elucidated. It was shown, however, that activation of the NFB family of transcription factors in infected cells plays a critical role in the transcriptional activation of many cytokine and chemokine genes, since large number of these genes contains NFB-binding sites in their promoters. Recently the importance of another family of transcription factors as the mediators of virus induced signaling has emerged. These factors designated interferon (IFN)

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Cyclin D1 belongs to a family of proteins that regulate progression through the G 1 -S phase of the cell cycle by binding to cyclin-dependent kinase (cdk)-4 to phosphorylate the retinoblastoma protein and release E2F transcription factors for progression through cell cycle. Several cancers, including breast, colon, and prostate, overexpress the cyclin D1 gene. However, the correlation of cyclin D1 overexpression with E2F target gene regulation or of cdk-dependent cyclin D1 activity with tumor development has not been identified. This suggests that the role of cyclin D1 in oncogenesis may be independent of its function as a cell cycle regulator. One such function is the role of cyclin D1 in cell adhesion and motility. Filamin A (FLNa), a member of the actin-binding filamin protein family, regulates signaling events involved in cell motility and invasion. FLNa has also been associated with a variety of cancers including lung cancer, prostate cancer, melanoma, human bladder cancer, and neuroblastoma. We hypothesized that elevated cyclin D1 facilitates motility in the invasive MDA-MB-231 breast cancer cell line. We show that MDA-MB-231 motility is affected by disturbing cyclin D1 levels or cyclin D1-cdk4/6 kinase activity. Using mass spectrometry, we find that cyclin D1 and FLNa coimmunoprecipitate and that lower levels of cyclin D1 are associated with decreased phosphorylation of FLNa at Ser2152 and Ser1459. We also identify many proteins related to cytoskeletal function, biomolecular synthesis, organelle biogenesis, and calcium regulation whose levels of expression change concomitant with decreased cell motility induced by decreased cyclin D1 and cyclin D1-cdk4/6 activities.

IRF-7 plays an essential role in virus-activated transcription of IFNA genes. To analyze functional domains of IRF-7 we have constructed an amino-terminal deletion mutant of IRF-7 (237-514) which exerted a dominant negative (DN) effect on virus-induced expression of the endogenous Type I IFN genes. Focusing on the molecular mechanism underlying the dominant negative effect of IRF-7 DN, we found that virus-activated transcription of endogenous IFNA genes requires full-length IRF-7 and that Serine 483 and 484 play an essential role. While IRF-7 DN had no effect on virus-stimulated nuclear translocation of IRF-3 and IRF-7, the binding of IRF-7 DN to IRF-3 and IRF-7 was detected by GST pull-down assay as well as by immunoprecipitation in infected cells, indicating that IRF-7 DN targets both IRF-7 and IRF-3. The region by which IRF-7 interacts with IRF-3 was mapped between amino acid 418 and 473. Overexpression of IRF-7 DN in virus-infected 2FTGH cells resulted in an inhibition of IFN synthesis and in a significant reduction of binding of both IRF-3 and IRF-7 to the IFNA1 promoter. Interestingly, the IRF-7 DN-mediated suppression of IFNA gene expression can be negated by overexpression of IRF-3. Altogether these results suggest that the IRF-3/IRF-7 complexes are biologically active and are involved in virus-activated transcription of endogenous IFNA genes.

The Dachshund (dac) gene, initially cloned as a dominant inhibitor of the Drosophila hyperactive EGFR mutant ellipse, encodes a key component of the cell fate determination pathway involved in Drosophila eye development. Analysis of more than 2,200 breast cancer samples showed improved survival by some 40 months in patients whose tumors expressed DACH1. Herein, DACH1 and estrogen receptor-α (ERα) expressions were inversely correlated in human breast cancer. DACH1 bound and inhibited ERα function. Nuclear DACH1 expression inhibited estradiol (E2)-induced DNA synthesis and cellular proliferation. DACH1 bound ERα in immunoprecipitation-Western blotting, associated with ERα in chromatin immunoprecipitation, and inhibited ERα transcriptional activity, requiring a conserved DS domain. Proteomic analysis identified proline, glutamic acid, and leucine rich protein 1 (PELP1) as a DACH1-binding protein. The DACH1 COOH terminus was required for binding to PELP1. DACH1 inhibited induction of ERα signaling. E2 recruited ERα and disengaged corepressors from DACH1 at an endogenous ER response element, allowing PELP1 to serve as an ERα coactivator. DACH1 expression, which is lost in poor prognosis human breast cancer, functions as an endogenous inhibitor of ERα function.