Studying non-mammalian models? Not a fool's ERRand!

Through studies in mammalian model systems, the estrogen-receptor-related receptor (ERR) alpha, an orphan nuclear receptor, has been shown to interfere with estrogen signaling and might therefore be an interesting pharmaceutical target in estrogen-related diseases. ERRalpha is also involved in energy storage and consumption, and its modulation might be of relevance in the treatment of obesity and diabetes. Recent data have also been published on the effects of this receptor, as well as other members of the ERR family, in non-mammalian animal model systems. Besides indications concerning their mechanisms of action, this analysis demonstrated a role for ERRalpha in controlling cellular movements, and suggested that ERRs might be implicated in a more subtle range of processes than originally envisioned

Thyroid hormone receptor {beta} (TR{beta}) and liver X receptor (LXR) regulate carbohydrate response element binding protein (ChREBP) expression in a tissue selective manner.

Thyroid hormone- (TR) and Liver X- (LXR)receptors are transcription factors involved in lipogenesis. Both receptors recognize the same consensus DNA response element in vitro. It was previously shown that their signalling pathways interact in the control of cholesterol elimination in the liver. In the present study ChREBP, a major transcription factor controlling the activation of glucose-induced lipogenesis in liver, is characterized as a direct target of thyroid hormones(TH) in liver and white adipose tissue(WAT), the two main lipogenic tissues in mice. Using genetic and molecular approaches ChREBP is shown to be specifically regulated by TRbeta, but not by TRalpha in vivo even in WAT where both TR isoforms are expressed. However this isotype specificity is not found in vitro. This TRbeta specific regulation correlates with the loss of TH-induced lipogenesis in TRbeta-/- mice. Fasting/refeeding experiments show that TRbeta is not required for the activation of ChREBP expression particularly marked in WAT following refeeding. However TH can stimulate ChREBP expression in WAT even under fasting conditions suggesting completely independent pathways. Since ChREBP has been described as an LXR target, the interaction of LXR and TRbeta in ChREBP regulation was assayed both in vitro and in vivo. Each receptor recognizes a different response element on the ChREBP promoter, located only eight base pairs apart.There is a crosstalk between LXR and TRbeta signalling on the ChREBP promoter in liver but not in WAT where LXR does not regulate ChREBP expression. The molecular basis for this crosstalk has been determined in in vitro systems

Down-Regulation of ECRG4, a Candidate Tumor Suppressor Gene, in Human Breast Cancer

INTRODUCTION: ECRG4/C2ORF40 is a potential tumor suppressor gene (TSG) recently identified in esophageal carcinoma. Its expression, gene copy number and prognostic value have never been explored in breast cancer. METHODS: Using DNA microarray and array-based comparative genomic hybridization (aCGH), we examined ECRG4 mRNA expression and copy number alterations in 353 invasive breast cancer samples and normal breast (NB) samples. A meta-analysis was done on a large public retrospective gene expression dataset (n = 1,387) in search of correlations between ECRG4 expression and histo-clinical features including survival. RESULTS: ECRG4 was underexpressed in 94.3% of cancers when compared to NB. aCGH data revealed ECRG4 loss in 18% of tumors, suggesting that DNA loss is not the main mechanism of underexpression. Meta-analysis showed that ECRG4 expression was significantly higher in tumors displaying earlier stage, smaller size, negative axillary lymph node status, lower grade, and normal-like subtype. Higher expression was also associated with disease-free survival (DFS; HR = 0.84 [0.76-0.92], p = 0.0002) and overall survival (OS; HR = 0.72 [0.63-0.83], p = 5.0E-06). In multivariate analysis including the other histo-clinical prognostic features, ECRG4 expression remained the only prognostic factor for DFS and OS. CONCLUSIONS: Our data suggest that ECRG4 is a candidate TSG in breast cancer, the expression of which may help improve the prognostication. If functional analyses confirm this TSG role, restoring ECRG4 expression in the tumor may represent a promising therapeutic approach

Myc Promoter-Binding Protein-1 (MBP-1) Is a Novel Potential Prognostic Marker in Invasive Ductal Breast Carcinoma

Background Alpha-enolase is a glycolytic enzyme that catalyses the formation of phosphoenolpyruvate in the cell cytoplasm. \u3b1-Enolase and the predominantly nuclear Myc promoter-binding protein-1 (MBP-1) originate from a single gene through the alternative use of translational starting sites. MBP-1 binds to the P2 c-myc promoter and competes with TATA-box binding protein (TBP) to suppress gene transcription. Although several studies have shown an antiproliferative effect of MBP-1 overexpression on several human cancer cells, to date detailed observations of \u3b1-enolase and MBP-1 relative expression in primary tumors versus normal tissues and their correlation with clinicopathological features have not been undertaken. Methodology and Findings We analyzed \u3b1-enolase and MBP-1 expression in normal breast epithelium and primary invasive ductal breast carcinoma (IDC) from 177 patients by Western blot and immunohistochemical analyses, using highly specific anti-\u3b1-enolase monoclonal antibodies. A significant increase in the expression of cytoplasmic \u3b1-enolase was observed in 98% of the tumors analysed, compared to normal tissues. Nuclear MBP-1 was found in almost all the normal tissues while its expression was retained in only 35% of the tumors. Statistically significant associations were observed among the nuclear expression of MBP-1 and ErbB2 status, Ki-67 expression, node status and tumor grade. Furthermore MBP-1 expression was associated with good survival of patients with IDC. Conclusions MBP-1 functions in repressing c-myc gene expression and the results presented indicate that the loss of nuclear MBP-1 expression in a large number of IDC may be a critical step in the development and progression of breast cancer and a predictor of adverse outcome. Nuclear MBP-1 appears to be a novel and valuable histochemical marker with potential prognostic value in breast cancer

Crosstalk between Integrin αvβ3 and Estrogen Receptor-α Is Involved in Thyroid Hormone-Induced Proliferation in Human Lung Carcinoma Cells

A cell surface receptor for thyroid hormone that activates extracellular regulated kinase (ERK) 1/2 has been identified on integrin αvβ3. We have examined the actions of thyroid hormone initiated at the integrin on human NCI-H522 non-small cell lung carcinoma and NCI-H510A small cell lung cancer cells. At a physiologic total hormone concentration (10−7 M), T4 significantly increased proliferating cell nuclear antigen (PCNA) abundance in these cell lines, as did 3, 5, 3′-triiodo-L-thyronine (T3) at a supraphysiologic concentration. Neutralizing antibody to integrin αvβ3 and an integrin-binding Arg-Gly-Asp (RGD) peptide blocked thyroid hormone-induced PCNA expression. Tetraiodothyroacetic acid (tetrac) lacks thyroid hormone function but inhibits binding of T4 and T3 to the integrin receptor; tetrac eliminated thyroid hormone-induced lung cancer cell proliferation and ERK1/2 activation. In these estrogen receptor-α (ERα)-positive lung cancer cells, thyroid hormone (T4>T3) caused phosphorylation of ERα; the specific ERα antagonist ICI 182,780 blocked T4-induced, but not T3-induced ERK1/2 activation, as well as ERα phosphorylation, proliferating-cell nuclear antigen (PCNA) expression and hormone-dependent thymidine uptake by tumor cells. Thus, in ERα-positive human lung cancer cells, the proliferative action of thyroid hormone initiated at the plasma membrane is at least in part mediated by ERα. In summary, thyroid hormone may be one of several endogenous factors capable of supporting proliferation of lung cancer cells. Activity as an inhibitor of lung cancer cell proliferation induced at the integrin receptor makes tetrac a novel anti-proliferative agent

Cyclin A2 Mutagenesis Analysis: A New Insight into CDK Activation and Cellular Localization Requirements

Cyclin A2 is essential at two critical points in the somatic cell cycle: during S phase, when it activates CDK2, and during the G2 to M transition when it activates CDK1. Based on the crystal structure of Cyclin A2 in association with CDKs, we generated a panel of mutants to characterize the specific amino acids required for partner binding, CDK activation and subcellular localization. We find that CDK1, CDK2, p21, p27 and p107 have overlapping but distinct requirements for association with this protein. Our data highlight the crucial importance of the N-terminal α helix, in conjunction with the α3 helix within the cyclin box, in activating CDK. Several Cyclin A2 mutants selectively bind to either CDK1 or CDK2. We demonstrate that association of Cyclin A2 to proteins such as CDK2 that was previously suggested as crucial is not a prerequisite for its nuclear localization, and we propose that the whole protein structure is involved

The RIP140 Gene Is a Transcriptional Target of E2F1

RIP140 is a transcriptional coregulator involved in energy homeostasis and ovulation which is controlled at the transcriptional level by several nuclear receptors. We demonstrate here that RIP140 is a novel target gene of the E2F1 transcription factor. Bioinformatics analysis, gel shift assay, and chromatin immunoprecipitation demonstrate that the RIP140 promoter contains bona fide E2F response elements. In transiently transfected MCF-7 breast cancer cells, the RIP140 promoter is transactivated by overexpression of E2F1/DP1. Interestingly, RIP140 mRNA is finely regulated during cell cycle progression (5-fold increase at the G1/S and G2/M transitions). The positive regulation by E2F1 requires sequences located in the proximal region of the promoter (−73/+167), involves Sp1 transcription factors, and undergoes a negative feedback control by RIP140. Finally, we show that E2F1 participates in the induction of RIP140 expression during adipocyte differentiation. Altogether, this work identifies the RIP140 gene as a new transcriptional target of E2F1 which may explain some of the effect of E2F1 in both cancer and metabolic diseases

Galectin-3 Facilitates Cell Motility in Gastric Cancer by Up-Regulating Protease-Activated Receptor-1(PAR-1) and Matrix Metalloproteinase-1(MMP-1)

BACKGROUND: Galectin-3 is known to regulate cancer metastasis. However, the underlying mechanism has not been defined. Through the DNA microarray studies after galectin-3 silencing, we demonstrated here that galectin-3 plays a key role in up-regulating the expressions of protease-activated receptor-1 (PAR-1) and matrix metalloproteinase-1 (MMP-1) PAR-1 thereby promoting gastric cancer metastasis. METHODOLOGY/PRINCIPAL FINDINGS: We examined the expression levels of Galectin-3, PAR-1, and MMP-1 in gastric cancer patient tissues and also the effects of silencing these proteins with specific siRNAs and of over-expressing them using specific lenti-viral constructs. We also employed zebrafish embryo model for analysis of in vivo gastric cancer cell invasion. These studies demonstrated that: a) galectin-3 silencing decreases the expression of PAR-1. b) galectin-3 over-expression increases cell migration and invasion and this increase can be reversed by PAR-1 silencing, indicating that galectin-3 increases cell migration and invasion via PAR-1 up-regulation. c) galectin-3 directly interacts with AP-1 transcriptional factor, and this complex binds to PAR-1 promoter and drives PAR-1 transcription. d) galectin-3 also amplifies phospho-paxillin, a PAR-1 downstream target, by increasing MMP-1 expression. MMP-1 silencing blocks phospho-paxillin amplification and cell invasion caused by galectin-3 over-expression. e) Silencing of either galectin-3, PAR-1 or MMP-1 significantly reduced cell migration into the vessels in zebrafish embryo model. f) Galectin-3, PAR-1, and MMP-1 are highly expressed and co-localized in malignant tissues from gastric cancer patients. CONCLUSIONS/SIGNIFICANCE: Galectin-3 plays the key role of activating cell surface receptor through production of protease and boosts gastric cancer metastasis. Galectin-3 has the potential to serve as a useful pharmacological target for prevention of gastric cancer metastasis

Mitochondrial Apoptosis and FAK Signaling Disruption by a Novel Histone Deacetylase Inhibitor, HTPB, in Antitumor and Antimetastatic Mouse Models

BACKGROUND: Compound targeting histone deacetylase (HDAC) represents a new era in molecular cancer therapeutics. However, effective HDAC inhibitors for the treatment of solid tumors remain to be developed. METHODOLOGY/PRINCIPAL FINDINGS: Here, we propose a novel HDAC inhibitor, N-Hydroxy-4-(4-phenylbutyryl-amino) benzamide (HTPB), as a potential chemotherapeutic drug for solid tumors. The HDAC inhibition of HTPB was confirmed using HDAC activity assay. The antiproliferative and anti-migratory mechanisms of HTPB were investigated by cell proliferation, flow cytometry, DNA ladder, caspase activity, Rho activity, F-actin polymerization, and gelatin-zymography for matrix metalloproteinases (MMPs). Mice with tumor xenograft and experimental metastasis model were used to evaluate effects on tumor growth and metastasis. Our results indicated that HTPB was a pan-HDAC inhibitor in suppressing cell viability specifically of lung cancer cells but not of the normal lung cells. Upon HTPB treatment, cell cycle arrest was induced and subsequently led to mitochondria-mediated apoptosis. HTPB disrupted F-actin dynamics via downregulating RhoA activity. Moreover, HTPB inhibited activity of MMP2 and MMP9, reduced integrin-β1/focal adhesion complex formation and decreased pericellular poly-fibronectin assemblies. Finally, intraperitoneal injection or oral administration of HTPB efficiently inhibited A549 xenograft tumor growth in vivo without side effects. HTPB delayed lung metastasis of 4T1 mouse breast cancer cells. Acetylation of histone and non-histone proteins, induction of apoptotic-related proteins and de-phosphorylation of focal adhesion kinase were confirmed in treated mice. CONCLUSIONS/SIGNIFICANCE: These results suggested that intrinsic apoptotic pathway may involve in anti-tumor growth effects of HTPB in lung cancer cells. HTPB significantly suppresses tumor metastasis partly through inhibition of integrin-β1/FAK/MMP/RhoA/F-actin pathways. We have provided convincing preclinical evidence that HTPB is a potent HDAC targeted inhibitor and is thus a promising candidate for lung cancer chemotherapy