Enhanced expression of an insulin growth factor-like binding protein (mac25) in senescent human mammary epithelial cells and induced expression with retinoic acid.

mac25, the subject of this report, was selected by the differential display of mRNA method in a search for genes overexpressed in senescent human mammary epithelial cells. mac25 had previously been cloned as a discrete gene, preferentially expressed in normal, leptomeningial cells compared with meningioma tumors. mac25 is another member of the insulin growth factor-binding protein (IGFBP) family. Insulin-like growth factors are potent mitogens for mammary epithelial cells, and the IGFBPs have been shown to modulate this mitogenic activity. We report here that mac25, unlike most IGFBPs, is down-regulated at the transcription level in mammary carcinoma cell lines, suggesting a tumor-suppressor role. The gene was mapped to chromosome 4q12. We found that mac25 accumulates in senescent cells and is up-regulated in normal, growing mammary epithelial cells by all-trans-retinoic acid or the synthetic retinoid fenretinide. These findings suggest that mac25 may be a downstream effector of retinoid chemoprevention in breast epithelial cells and that its tumor-suppressive role may involve a senescence pathway.

Expression of HOXB2, a retinoic acid signaling target in pancreatic cancer and pancreatic intraepithelial neoplasia

Purpose: Despite significant progress in understanding the molecular pathology of pancreatic cancer and its precursor lesion: pancreatic intraepithelial neoplasia (PanIN), there remain no molecules with proven clinical utility as prognostic or therapeutic markers. Here, we used oligonucleotide microarrays to interrogate mRNA expression of pancreatic cancer tissue and normal pancreas to identify novel molecular pathways dysregulated in the development and progression of pancreatic cancer. Experimental Design: RNA was hybridized to Affymetrix Genechip HG-U133 oligonucleotide microarrays. A relational database integrating data from publicly available resources was created to identify candidate genes potentially relevant to pancreatic cancer. The protein expression of one candidate, homeobox B2 (HOXB2), in PanIN and pancreatic cancer was assessed using immunohistochemistry. Results: We identified aberrant expression of several components of the retinoic acid (RA) signaling pathway (RARa, MUC4, Id-1, MMP9, uPAR, HB-EGF, HOXB6, and HOXB2), many of which are known to be aberrantly expressed in pancreatic cancer and Pan IN. HOXB2, a downstream target of RA, was up-regulated 6.7-fold in pancreatic cancer compared with normal pancreas. Immunohistochemistry revealed ectopic expression of HOXB2 in 15% of early Pan IN lesions and 48 of 128 (38%) pancreatic cancer specimens. Expression of HOXB2 was associated with nonresectable tumors and was an independent predictor of poor survival in resected tumors. Conclusions: We identified aberrant expression of RA signaling components in pancreatic cancer, including HOXB2, which was expressed in a proportion of PanIN lesions. Ectopic expression of HOXB2 was associated with a poor prognosis for all patients with pancreatic cancer and was an independent predictor of survival in patients who underwent resection.

Expression of the retinoic acid catabolic enzyme CYP26B1 in the human brain to maintain signaling homeostasis

Funding was provided by the Wellcome Trust grant WT081633MA-NCE and Biological Sciences Research Council Grant BB/K001043/1. Prof Fragoso is the recipient of a Post Doctoral Science without Borders grant from the Brazilian National Council for Scientific and Technological Development (CNPq, 237450/2012-7).

Preclinical studies of the combined effect of anti-MYCN PNA and Retinoic Acid as potential approach to treat Neuroblastoma

Neuroblastoma (NB) is the deadliest cancer in early childhood. Around 25% of patients pre- sent MYCN-amplification (MNA) which is linked to poor prognosis, metastasis, and therapy- resistance. While retinoic acid (RA) is beneficial only for some NB patients, the cause of its resistance is still unknown. Thus, there remains a need for new therapies to treat NB. I show that MYCN-specific inhibition by the antigene oligonucleotide BGA002 in combination with 13-cis RA (BGA002-RA) overcome resistance in MNA-NB cell lines, leading to potent MYCN mRNA expression and protein decrease. Moreover, BGA002-RA reactivated neuron differentiation or led to apoptosis in MNA-NB cell lines, and inhibited invasiveness capacity. Since NB and PI3K/mTOR pathway are strictly related MYCN down-regulation by BGA002 led to mTOR pathway inhibition in MNA-NB, that was strengthened by BGA002-RA. I further analyzed if MYCN silencing may induce autophagy reactivation, and indeed BGA002-RA caused a massive increase in lysosomes and macrovacuoles in MNA-NB cells. In addition, while MYCN is known to induce angiogenesis, BGA002-RA in vivo treatment elim- inated the tumor vascularization in a MNA-NB mice model, and significantly increased the survival. Overall, these results indicate that MYCN modulation mediates the therapeutic efficacy of RA and the development of RA resistance in MNA-NB. Furthermore, by targeting MYCN, we show a cancer-specific way of mTOR pathway inhibition only in MNA-NB, avoiding side effects of targeting mTOR in normal cells. These findings warrant clinical testing of BGA002-RA as a potential strategy to overcome RA resistance in MNA-NB.

Attenuation of colon inflammation through activators of the retinoid X receptor (RXR)/peroxisome proliferator-activated receptor gamma (PPARgamma) heterodimer. A basis for new therapeutic strategies.

The peroxisome proliferator-activated receptor gamma (PPARgamma) is highly expressed in the colon mucosa and its activation has been reported to protect against colitis. We studied the involvement of PPARgamma and its heterodimeric partner, the retinoid X receptor (RXR) in intestinal inflammatory responses. PPARgamma(1/)- and RXRalpha(1/)- mice both displayed a significantly enhanced susceptibility to 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis compared with their wild-type littermates. A role for the RXR/PPARgamma heterodimer in the protection against colon inflammation was explored by the use of selective RXR and PPARgamma agonists. TNBS-induced colitis was significantly reduced by the administration of both PPARgamma and RXR agonists. This beneficial effect was reflected by increased survival rates, an improvement of macroscopic and histologic scores, a decrease in tumor necrosis factor alpha and interleukin 1beta mRNA levels, a diminished myeloperoxidase concentration, and reduction of nuclear factor kappaB DNA binding activity, c-Jun NH(2)-terminal kinase, and p38 activities in the colon. When coadministered, a significant synergistic effect of PPARgamma and RXR ligands was observed. In combination, these data demonstrate that activation of the RXR/PPARgamma heterodimer protects against colon inflammation and suggest that combination therapy with both RXR and PPARgamma ligands might hold promise in the clinic due to their synergistic effects.

Similarities and dissimilarities of branching and septation during lung development.

The lungs of small premature babies are at a developmental stage of finalizing their airway tree by a process called branching morphogenesis, and of creating terminal gas exchange units by a mechanism called septation. If the branching process is disturbed, the lung has a propensity to be hypoplastic. If septation is impaired, the terminal gas exchange units, the alveoli, tend to be enlarged and reduced in number, an entity known as bronchopulmonary dysplasia. Here, we review current knowledge of key molecules influencing branching and septation. In particular, we discuss the molecular similarities and dissimilarities between the two processes of airspace enlargement. Understanding of the molecular mechanisms regulating branching and septation may provide perinatologists with targets for improving lung growth and maturation.

Cutting edge: IL-7 regulates the peripheral pool of adult ROR gamma+ lymphoid tissue inducer cells.

During fetal life, CD4(+)CD3(-) lymphoid tissue inducer (LTi) cells are required for lymph node and Peyer's patch development in mice. In adult animals, CD4(+)CD3(-) cells are found in low numbers in lymphoid organs. Whether adult CD4(+)CD3(-) cells are LTi cells and are generated and maintained through cytokine signals has not been directly addressed. In this study we show that adult CD4(+)CD3(-) cells adoptively transferred into neonatal CXCR5(-/-) mice induced the formation of intestinal lymphoid tissues, demonstrating for the first time their bona fide LTi function. Increasing IL-7 availability in wild-type mice either by IL-7 transgene expression or treatment with IL-7/anti-IL-7 complexes increased adult LTi cell numbers through de novo generation from bone marrow cells and increased the survival and proliferation of LTi cells. Our observations demonstrate that adult CD4(+)lineage(-) cells are LTi cells and that the availability of IL-7 determines the size of the adult LTi cell pool.

Influence of a fat overload on lipogenic regulators in metabolic syndrome patients

Several epidemiological studies have related an increase of lipids in the postprandial state to an individual risk for the development of CVD, possibly due to the increased plasma levels of TAG and fatty acids (FA) through enzymes of FA metabolism. The interaction between nutrition and the human genome determines gene expression and metabolic response. The aim of the present study was to evaluate the influence of a fat overload on the gene mRNA levels of lipogenic regulators in peripheral blood mononuclear cells (PBMC) from patients with the metabolic syndrome. The study included twenty-one patients with criteria for the metabolic syndrome who underwent a fat overload. Measurements were made before and after the fat overload of anthropometric and biochemical variables and also the gene mRNA levels of lipogenic factors. The main results were that the fat overload led to an increased mRNA levels of sterol regulatory element binding protein-1 (SREBP1), retinoid X receptor α (RXRα) and liver X receptor α (LXRα) in PBMC, and this increase was associated with the FA synthase (FASN) mRNA levels. We also found that TAG levels correlated with FASN mRNA levels. In addition, there was a positive correlation of SREBP1 with RXRα and of LXRα with the plasma lipoperoxide concentration. The fat overload led to an increase in regulators of lipogenesis in PBMC from patients with the metabolic syndrome.

Functional interactions of peroxisome proliferator-activated receptor, retinoid-X receptor, and Sp1 in the transcriptional regulation of the acyl-coenzyme-A oxidase promoter.

Peroxisome proliferator-activated receptor (PPARs) are members of the nuclear receptor superfamily. For transcriptional activation of their target genes, PPARs heterodimerize with the retinoid-X receptor (RXR). The convergence of the PPAR and RXR signaling pathways has been shown to have an important function in lipid metabolism. The promoter of the gene encoding the acyl-coenzyme-A oxidase (ACO), the rate-limiting enzyme in peroxisomal beta-oxidation of fatty acids, is a target site of PPAR action. In this study, we examined the role and the contribution of both cis-and trans-acting factors in the transcriptional regulation of this gene using transient transfections in insect cells. We identified several functional cis-acting elements present in the promoter of the ACO gene and established that PPAR-dependent as well as PPAR-independent mechanisms can activate the ACO promoter in these cells. We show that the PPAR/RXR heterodimer exerts its effect through two response elements within the ACO promoter, in synergy with the transcription factor Sp1 via five Sp1-binding sites. Furthermore, this functional interaction also occurs when Sp1 is co-expressed with PPAR or RXR alone, indicating that activation can occur independently of PPAR/RXR heterodimers.

Human pregnane X receptor is expressed in breast carcinomas, potential heterodimers formation between hPXR and RXR-alpha.

BACKGROUND The human pregnane X receptor (hPXR) is an orphan nuclear receptor that induces transcription of response elements present in steroid-inducible cytochrome P-450 gene promoters. This activation requires the participation of retinoid X receptors (RXRs), needed partners of hPXR to form heterodimers. We have investigated the expression of hPXR and RXRs in normal, premalignant, and malignant breast tissues, in order to determine whether their expression profile in localized infiltrative breast cancer is associated with an increased risk of recurrent disease. METHODS Breast samples from 99 patients including benign breast diseases, in situ and infiltrative carcinomas were processed for immunohistochemistry and Western-blot analysis. RESULTS Cancer cells from patients that developed recurrent disease showed a high cytoplasmic location of both hPXR isoforms. Only the infiltrative carcinomas that relapsed before 48 months showed nuclear location of hPXR isoform 2. This location was associated with the nuclear immunoexpression of RXR-alpha. CONCLUSION Breast cancer cells can express both variants 1 and 2 of hPXR. Infiltrative carcinomas that recurred showed a nuclear location of both hPXR and RXR-alpha; therefore, the overexpression and the subcellular location changes of hPXR could be considered as a potential new prognostic indicator.

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Retinoid X Receptors (RXR) were initially identified as nuclear receptors binding with stereo-selectivity the vitamin A derivative 9-cis retinoic acid, although the relevance of this molecule as endogenous activator of RXRs is still elusive. Importantly, within the nuclear receptor superfamily, RXRs occupy a peculiar place, as they are obligatory partners for a number of other nuclear receptors, thus integrating the corresponding signaling pathways. In this chapter, we describe the structural features allowing RXR to form homo- and heterodimers, and the functional consequences of this unique ability. Furthermore, we discuss the importance of studying RXR activity at a genome-wide level in order to comprehensively address the biological implications of their action that is fundamental to understand to what extent RXRs could be exploited as new therapeutic targets.

Signaling cross-talk between peroxisome proliferator-activated receptor/retinoid X receptor and estrogen receptor through estrogen response elements.

Peroxisome proliferator-activated receptors (PPARs) and retinoid X receptors (RXRs) are nuclear hormone receptors that are activated by fatty acids and 9-cis-retinoic acid, respectively. PPARs and RXRs form heterodimers that activate transcription by binding to PPAR response elements (PPREs) in the promoter of target genes. The PPREs described thus far consist of a direct tandem repeat of the AGGTCA core element with one intervening nucleotide. We show here that the vitellogenin A2 estrogen response element (ERE) can also function as a PPRE and is bound by a PPAR/RXR heterodimer. Although this heterodimer can bind to several other ERE-related palindromic response elements containing AGGTCA half-sites, only the ERE is able to confer transactivation of test reporter plasmids, when the ERE is placed either close to or at a distance from the transcription initiation site. Examination of natural ERE-containing promoters, including the pS2, very-low-density apolipoprotein II and vitellogenin A2 genes, revealed considerable differences in the binding of PPAR/RXR heterodimers to these EREs. In their natural promoter context, these EREs did not allow transcriptional activation by PPARs/RXRs. Analysis of this lack of stimulation of the vitellogenin A2 promoter demonstrated that PPARs/RXRs bind to the ERE but cannot transactivate due to a nonpermissive promoter structure. As a consequence, PPARs/RXRs inhibit transactivation by the estrogen receptor through competition for ERE binding. This is the first example of signaling cross-talk between PPAR/RXR and estrogen receptor.