Rebecca A. Fillmore

MiR-200, a new star miRNA in human cancer

MicroRNAs (miRNAs) are a set of non-coding small RNA molecules in control of gene expression at posttranscriptional/translational level. They not only play crucial roles in normal developmental progress, but also are commonly dysregulated in human diseases, including cancer. MiR-200 is a family of tumor suppressor miRNAs consisting of five members, which are significantly involved in inhibition of epithelial-to-mesenchymal transition (EMT), repression of cancer stem cells (CSCs) self-renewal and differentiation, modulation of cell division and apoptosis, and reversal of chemoresistance. In this article, we summarize the latest findings with regard to the tumor suppressor signatures of miR-200 and the regulatory mechanisms of miR-200 expression. The collected evidence supports that miR-200 is becoming a new star miRNA in study of human cancer.

Breast cancer metastasis suppressor 1 (BRMS1) inhibits osteopontin transcription by abrogating NF-κB activation

BackgroundOsteopontin (OPN), a secreted phosphoglycoprotein, has been strongly associated with tumor progression and aggressive cancers. MDA-MB-435 cells secrete very high levels of OPN. However metastasis-suppressed MDA-MB-435 cells, which were transfected with breast cancer metastasis suppressor 1 (BRMS1), expressed significantly less OPN. BRMS1 is a member of mSin3-HDAC transcription co-repressor complex and has been shown to suppress the metastasis of breast cancer and melanoma cells in animal models. Hence we hypothesized that BRMS1 regulates OPN expression.ResultsThe search for a BRMS1-regulated site on the OPN promoter, using luciferase reporter assays of the promoter deletions, identified a novel NF-κB site (OPN/NF-κB). Electrophoretic mobility shift assays and chromatin immunoprecipitations (ChIP) confirmed this site to be an NF-κB-binding site. We also show a role of HDAC3 in suppression of OPN via OPN/NF-κB.ConclusionOur results show that BRMS1 regulates OPN transcription by abrogating NF-κB activation. Thus, we identify OPN, a tumor-metastasis activator, as a crucial downstream target of BRMS1. Suppression of OPN may be one of the possible underlying mechanisms of BRMS1-dependent suppression of tumor metastasis.

Large isoform of MRJ (DNAJB6) reduces malignant activity of breast cancer

IntroductionMammalian relative of DnaJ (MRJ [DNAJB6]), a novel member of the human DnaJ family, has two isoforms. The smaller isoform, MRJ(S), is studied mainly for its possible role in Huntingtons disease. There are no reports of any biologic activity of the longer isoform, MRJ(L). We investigated whether this molecule plays any role in breast cancer. Our studies were prompted by interesting observations we made regarding the expression of MRJ in breast cancer cell lines and breast cancer tissue microarrays, as described below.MethodsExpression of MRJ(L) from several breast cancer cell lines was evaluated using real-time PCR. Relative levels of the small and large isoforms in breast cancer cell lines were studied using Western blot analysis. A breast cancer progression tissue microarray was probed using anti-MRJ antibody. MRJ(L) was ectopically expressed in two breast cancer cell lines. These cell lines were evaluated for their in vitro correlates of tumor aggressiveness, such as invasion, migration, and anchorage independence. The cell lines were also evaluated for in vivo tumor growth and metastasis. The secreted proteome of the MRJ(L) expressors was analyzed to elucidate the biochemical changes brought about by re-expression of MRJ(L).ResultsWe found that MRJ(L) is expressed at a significantly lower level in aggressive breast cancer cell lines compared with normal breast. Furthermore, in clinical cases of breast cancer expression of MRJ is lost as the grade of infiltrating ductal carcinoma advances. Importantly, MRJ staining is lost in those cases that also had lymph node metastasis. We report that MRJ(L) is a protein with a functional nuclear localization sequence. Expression of MRJ(L) via an exogenous promoter in breast cancer cell line MDA-MB-231 and in MDA-MB-435 (a cell line that metastasizes from the mammary fat pad) decreases their migration and invasion, reduces their motility, and significantly reduces orthotopic tumor growth in nude mice. Moreover, the secreted proteome of the MRJ(L)-expressing cells exhibited reduced levels of tumor progression and metastasis promoting secreted proteins, such as SPP1 (osteopontin), AZGP1 (zinc binding α2-glycoprotein 1), SPARC (osteonectin), NPM1 (nucleophosmin) and VGF (VGF nerve growth factor inducible). On the other hand, levels of the secreted metastasis-suppressor KiSS1 (melanoma metastasis suppressor) were increased in the secreted proteome of the MRJ(L)-expressing cells. We confirmed by quantitative RT-PCR analysis that the secreted profile reflected altered transcription of the respective genes.ConclusionCollectively, our data indicate an important role for a totally uncharacterized isoform of DNAJB6 in breast cancer. We show that MRJ(L) is a nuclear protein that is lost in breast cancer, that regulates several key players in tumor formation and metastasis, and that is functionally able to retard tumor growth.

Nmi (N‐Myc interactor) inhibits Wnt/β‐catenin signaling and retards tumor growth

We found that the expression levels of N‐Myc interactor (Nmi) were low in aggressive breast cancer cell lines when compared with less aggressive cell lines. However, the lower levels in the aggressive lines were inducible by interferon‐γ (IFN‐γ). Because Nmi has been reported to be a transcription cofactor that augments IFN‐γ induced transcription activity, we decided to test whether Nmi regulates expression of Dkk1, which is also inducible by IFN‐γ. We established stable clones constitutively expressing Nmi in MDA‐MB‐231 (breast) and MDA‐MB‐435 (melanoma) cell lines. Dkk1 was significantly up‐regulated in the Nmi expressing clones concurrent with reduced levels of the critical transcription cofactor of Wnt pathway, β‐catenin. Treatment of the Nmi expressors with blocking antibody to Dkk1 restored β‐catenin protein levels. c‐Myc is a known downstream target of activated β‐catenin signaling. Treatment of Nmi expressors with the proteosome inhibitor MG132, resulted in elevated β‐catenin levels with concomitant elevation of c‐Myc levels. Our functional studies showed that constitutive expression of Nmi reduced the ability of tumor cells for the invasion, anchorage independent growth and tumor growth in vivo. Collectively, the data suggest that overexpression of Nmi inhibits the Wnt/β‐catenin signaling via up‐regulation of Dkk1 and retards tumor growth.

NUPR1 interacts with p53, transcriptionally regulates p21 and rescues breast epithelial cells from doxorubicin-induced genotoxic stress.

Nuclear protein 1 (NUPR1/com1/p8) has been shown to interact with transcriptional regulators such as p300, PTIP, estrogen receptor-beta, and SMAD. NUPR1 also has been implicated in the regulation of cell cycle and apoptosis. An increase in NUPR1 expression has been seen with serum starvation and in response to compounds such as cycloheximide, ceramide, and staurosporine. There are several overtly conflicting reports about the exact role of NUPR1 in tumor biology. This work investigates the nature of the relationship between NUPR1 and the cdk-inhibitor p21 (Waf1/Cip1) expression. We show that the expression of resident and doxorubicin-induced p21 paralleled that of endogenous NUPR1 levels. NUPR1 formed a complex with p53 and p300 and bound the p21 promoter and transcriptionally upregulated p21 expression. Moreover, NUPR1 allowed cells to progress through cell cycle in presence of doxorubicin. Since NUPR1 upregulated p21, concomitant with phosphorylation of Rb and upregulation of the anti-apoptotic protein, Bcl-x(L) we propose that NUPR1 expression imparts a cell growth and survival advantage. Importantly, we also report that NUPR1 conferred resistance to two chemotherapeutic drugs, Taxol and doxorubicin.

Smooth muscle γ-actin promoter regulation by RhoA and serum response factor signaling

Abstract Smooth muscle γ-actin (SMGA) is both an early marker of smooth muscle cell differentiation, which demonstrates an expression pattern restricted to smooth muscle and the post meiotic spermatocyte. Serum response factor (SRF) DNA-binding is an important regulator of muscle differentiation, including SMGA expression during smooth muscle cell differentiation. RhoA, a low molecular weight GTPase protein, can regulate cardiac, skeletal, and smooth muscle differentiation through SRF-dependent mechanisms. This studys purpose was to examine RhoA expression during smooth muscle cell development, and determine if the SMGA promoter activity is sensitive to RhoA-mediated signaling through SRF. Additionally, the study identified the promoter regulation modifying SMGA expression by RhoA signaling. Western blot analysis of embryonic chick gizzard whole protein extracts during 5 to 14 days of development demonstrated a large induction of RhoA (10-fold) and β1 integrin expression at day 8, which corresponds to the time SMGA expression and differentiation are occurring. Transient transfections in CV-1 fibroblast cells demonstrated that co-overexpression of SRF and RhoA could induce a 40-fold induction of −176 bp SMGA promoter activity. Mutational analysis demonstrated that serum response element (SRE)-1, but not SRE2, was necessary for RhoA/SRF activation of the SMGA promoter. Deletion analysis revealed that although SRE1 was necessary for SMGA promoter activation by RhoA and SRF, it was not sufficient, implicating a possible obligatory role of additional promoter sequences in the response. Overexpression of a mutated SRF protein that was unable to bind DNA demonstrated that the 40-fold RhoA/SRF activation was largely dependent on SRF binding to the SMGA promoter. Thus, as the SMGA promoter appears to be a target of RhoA-mediated transcriptional regulation, the uncovering of these signaling mechanisms effecting SMGA promoter activity should provide a regulatory paradigm that can then be examined during the regulation of other smooth muscle genes.

Structural and Functional Analysis of Domains Mediating Interaction Between the Bagpipe Homologue, Nkx3.1 and Serum Response Factor

Nkx3.1 is a member of the NK2 class of homeodomain proteins and is expressed in development, being an early marker of the sclerotome and prostate gland. It has been shown to be a critical factor for prostate differentiation and function. Previous studies suggested that Nkx3.1 interacts with Serum Response Factor (SRF) to transactivate the Smooth Muscle γ-Actin (SMGA) promoter. In studies presented here, we examined the molecular mechanisms underlying the functional synergy of these factors upon SMGA transcription. We demonstrate that full length Nkx3.1 physically interacts with SRF in the absence of DNA and that these factors are able to co-associate in cellular context using a mammalian two-hybrid system. The segment of SRF responsible for Nkx3.1 interaction was mapped to a ~30 amino acid region (AAs 142–171) at the N-terminal segment of the MADS box. Two separate regions of Nkx3.1 were found to mediate interactions with SRF. Interestingly, recognized domains of NK2 proteins, namely the TN, homeodomain DNA binding segment, and the NK2-SD do not participate in SRF interactions. One of the Nkx3.1 SRF binding domains was mapped to the N-terminal of the protein consistent with recent studies of these proteins using NMR spectroscopy by Gelmann and colleagues (1). A second SRF binding region was mapped to amino acids C-terminal to the homeodomain. Structural predictions indicate that both of the SRF interacting segments are largely hydrophobic in character and β-strand in structure. With co-transfection transcriptional analyses we found that interaction between SRF and Nkx3.1 as well as DNA binding by both factors was required for the observed transcriptional synergy. Thus our studies have identified novel protein-protein interacting domains within Nkx3.1 and SRF that operate in concert with their respective DNA binding domains to mediate functional transcriptional synergy of these factors to regulate SMGA gene activation.

New concepts concerning prostate cancer screening

Prostate Cancer (CaP) is rapidly becoming a worldwide health issue. While CaP mortality has decreased in recent years, coincident with the widespread use of Prostate-Specific Antigen (PSA) screening, it remains the most common solid tumor in men and is the second leading cause of cancer death in the United States. The frequency of CaP is growing not only in western cultures, but also its incidence is dramatically increasing in eastern nations. Recently, examination of data from long-term trials and follow up has cast a shadow on the effectiveness of employing PSA as a primary screening tool for CaP. In this review, we not only summarize opinions from this examination and synthesize recommendations from several groups that suggest strategies for utilizing PSA as a tool, but also call for research into biomarkers for CaP diagnosis and disease progression. We also describe our recent work that identified a smooth muscle contractile protein in prostate epithelia, namely smooth muscle gamma actin, and indicate the potential for this molecule as a new unique footprint and as a CaP marker.