Roseline Godbout

Association of FABP5 Expression With Poor Survival in Triple-Negative Breast Cancer: Implication for Retinoic Acid Therapy

Recent studies using animal models suggest that expression of FABP5 drives the stimulation of cell growth observed in estrogen receptor (ER)-negative breast cancer cells on exposure to retinoic acid (RA). The purpose of this study was to investigate the clinicopathological significance of FABP5 in breast cancer and to evaluate FABP5 as a prognostic marker and a possible novel therapeutic target in breast cancer. Gene expression microarray analysis revealed a significant correlation between elevated FABP5 RNA levels and ER/progesterone receptor (PR)-negative status, high tumor grade, and poor prognosis. Tissue microarray analysis demonstrated similar correlations with cytoplasmic FABP5 protein. Based on multivariate proportional regression analysis, cytoplasmic FABP5 is a significant and independent prognostic marker of overall survival and recurrence-free survival in breast cancer. The effects of FABP5 on tumor growth appear to be mediated primarily through cytoplasmic FABP, because no correlation was found between nuclear FABP5 and ER/PR-negative status, recurrence, and survival. FABP5 knockdown in breast cancer cell lines demonstrates a correlation between FABP5 levels and growth response to RA. We propose a model whereby growth-promoting FABP5 competes with growth-inhibiting CRABP2 for RA, with retention of RA in the cytoplasm by FABP5 preventing the inhibition of tumor growth.

FABP7 expression in glioblastomas: relation to prognosis, invasion and EGFR status

FABP7 expression has been analysed in a series of 123 glioblastomas (68 pure GBM, 55 GBMO, i.e. with oligodendroglial component). FABP7, found in 91/123 samples, showed a pure cytoplasmic expression in 69 cases, and cytoplasmicxa0+xa0nuclear expression in 22 cases. FABP7 expression was associated with pure GBM histology and shorter survival (15.7 months versus 21.5xa0months). Nuclear expression of FABP7 was more specifically related to EGFR amplification and more invasive tumors. These data, although they need to be confirmed by further studies, support the relation between FABP7, astrocytic features, invasion and poor prognosis and suggests that EGFR amplification is associated with nuclear translocation of FABP7.

Differential CRX and OTX2 expression in human retina and retinoblastoma

The histogenesis of retinoblastoma tumors remains controversial, with the cell‐of‐origin variably proposed to be an uncommitted retinal progenitor cell, a bipotent committed cell, or a cell committed to a specific lineage. Here, we examine the expression of two members of the orthodenticle family implicated in photoreceptor and bipolar cell differentiation, cone‐rod homeobox, CRX, and orthodenticle homeobox 2, OTX2, in normal human retina, retinoblastoma cell lines and retinoblastoma tumors. We show that CRX and OTX2 have distinct expression profiles in the developing human retina, with CRX first expressed in proliferating cells and cells committed to the bipolar lineage, and OTX2 first appearing in the photoreceptor lineage. In the mature retina, CRX levels are highest in photoreceptor cells whereas OTX2 is preferentially found in bipolar cells and in the retinal pigmented epithelium. Both CRX and OTX2 are widely expressed in retinoblastoma cell lines and in retinoblastoma tumors, although CRX is more abundant than OTX2 in the differentiated elements of retinoblastoma tumors such as large rosettes, Flexner‐Wintersteiner rosettes and fleurettes. Widespread expression of CRX and OTX2 in retinoblastoma tumors and cell lines suggests a close link between the cell‐of‐origin of retinoblastoma tumors and cells expressing CRX and OTX2.

High levels of aldehyde dehydrogenase transcripts in the undifferentiated chickretina

A cDNA clone corresponding to chicken aldehyde dehydrogenase (ALDH) mRNA was isolated from a library representing the polyadenylated RNAs expressed in the retina of day 3.5 chick embryos. The profile of ALDH RNA expression was examined in different tissues as well as at different stages of development in the chick embryo. A notable feature of this analysis was the high level of ALDH transcripts found in the undifferentiated cells of the retina. A 20-fold decrease in ALDH RNA levels was observed upon retinal differentiation, in contrast to the kidney, liver and gut where tissue maturation was accompanied by an increase in ALDH mRNA levels. The observations reported here suggest an important role for the ALDH enzyme in retinal development. One possibility is that retinal, the aldehyde form of vitamin A, serves as a substrate for ALDH in the developing retina, resulting in the formation of retinoic acid which has been implicated in various differentiation processes.

Aldehyde dehydrogenase 1A3 influences breast cancer progression via differential retinoic acid signaling.

Aldehyde dehydrogenase (ALDH) 1A enzymes produce retinoic acid (RA), a transcription induction molecule. To investigate if ALDH1A1 or ALDH1A3‐mediated RA signaling has an active role in breast cancer tumorigenesis, we performed gene expression and tumor xenograft studies. Analysis of breast patient tumors revealed that high levels of ALDH1A3 correlated with expression of RA‐inducible genes with retinoic acid response elements (RAREs), poorer patient survival and triple‐negative breast cancers. This suggests a potential link between ALDH1A3 expression and RA signaling especially in aggressive and/or triple‐negative breast cancers. In MDA‐MB‐231, MDA‐MB‐468 and MDA‐MB‐435 cells, ALDH1A3 and RA increased expression of RA‐inducible genes. Interestingly, ALDH1A3 had opposing effects in tumor xenografts, increasing tumor growth and metastasis of MDA‐MB‐231 and MDA‐MB‐435 cells, but decreasing tumor growth of MDA‐MB‐468 cells. Exogenous RA replaced ALDH1A3 in inducing the same opposing tumor growth and metastasis effects, suggesting that ALDH1A3 mediates these effects by promoting RA signaling. Genome expression analysis revealed that ALDH1A3 induced largely divergent gene expression in MDA‐MB‐231 and MDA‐MB‐468 cells which likely resulted in the opposing tumor growth effects. Treatment with DNA methylation inhibitor 5‐aza‐2deoxycytidine restored uniform RA‐inducibility of RARE‐containing HOXA1 and MUC4 in MDA‐MB‐231 and MDA‐MB‐468 cells, suggesting that differences in epigenetic modifications contribute to differential ALDH1A3/RA‐induced gene expression in breast cancer. In summary, ALDH1A3 induces differential RA signaling in breast cancer cells which affects the rate of breast cancer progression.

Involvement of AP-2 in regulation of the R-FABP gene in the developing chick retina.

Little is known regarding the molecular pathways that underlie the retinal maturation process. We are studying the regulation of the retinal fatty-acid-binding protein (R-FABP) gene, highly expressed in retinal precursor cells, to identify DNA regulatory elements and transcriptional factors involved in retinal development. Although the upstream sequence of the R-FABP gene is extremely GC rich, CpG methylation in this region is not implicated in the regulation of this gene because the 5 flanking DNA remains unmethylated with tissue differentiation when there is a dramatic decrease in R-FABP transcript levels. Using a combination of DNase I hypersensitivity experiments, gel shift assays, and DNase I footprinting, we have found three sites of DNA-protein interaction within 205 bp of 5 flanking DNA in the undifferentiated retina and four sites in the differentiated retina. DNA transfection analysis indicates that the first two footprints located within 150 bp of 5 flanking DNA are required for high levels of transcription in primary undifferentiated retinal cultures. The first footprint includes a putative TATA box and Spl binding sites while the second footprint contains a consensus AP-2 DNA binding site. Supershift experiments using antibodies to AP-2 and methylation interference experiments indicate that an AP-2-like transcription factor present in both late-proliferative-stage retina and differentiated retina binds to the upstream region of the R-FABP gene. A combination of data including the expression profile of AP-2 during retinal development and DNA transfection analysis using constructs mutated at critical residues within the AP-2 binding site suggests that AP-2 is a repressor of R-FABP transcription.

The NAD + salvage pathway modulates cancer cell viability via p73

The involvement of the nicotinamide adenine dinucleotide (NAD+) salvage pathway in cancer cell survival is poorly understood. Here we show that the NAD+ salvage pathway modulates cancer cell survival through the rarely mutated tumour suppressor p73. Our data show that pharmacological inhibition or knockdown of nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting enzyme in the NAD+ salvage pathway, enhances autophagy and decreases survival of cancer cells in a p53-independent manner. Such NAMPT inhibition stabilizes p73 independently of p53 through increased acetylation and decreased ubiquitination, resulting in enhanced autophagy and cell death. These effects of NAMPT inhibition can be effectively reversed using nicotinamide mononucleotide (NMN), the enzymatic product of NAMPT. Similarly, knockdown of p73 also decreases NAMPT inhibition-induced autophagy and cell death, whereas overexpression of p73 alone enhances these effects. We show that the breast cancer cell lines (MCF-7, MDA-MB-231 and MDA-MB-468) harbour significantly higher levels of NAMPT and lower levels of p73 than does the normal cell line (MCF-10A), and that NAMPT inhibition is cytotoxic exclusively to the cancer cells. Furthermore, data from 176 breast cancer patients demonstrate that higher levels of NAMPT and lower levels of p73 correlate with poorer patient survival, and that high-grade tumours have significantly higher NAMPT/p73 mRNA ratios. Therefore, the inverse relationship between NAMPT and p73 demonstrable in vitro is also reflected from the clinical data. Taken together, our studies reveal a new NAMPT-p73 nexus that likely has important implications for cancer diagnosis, prognosis and treatment.

Notch and TGFβ form a positive regulatory loop and regulate EMT in epithelial ovarian cancer cells

Epithelial-mesenchymal transition (EMT) plays a critical role in the progression of epithelial ovarian cancer (EOC). However, the mechanisms that regulate EMT in EOC are not fully understood. Here, we report that activation of Notch1 induces EMT in EOC cells as evidenced by downregulation of E-cadherin and cytokeratins, upregulation of Slug and Snail, as well as morphological changes. Interestingly, activation of Notch1 increases TGFβ/Smad signaling by upregulating the expression of TGFβ and TGFβ type 1 receptor. Time course experiments demonstrate that inhibition of Notch by DAPT (a γ-secretase inhibitor) decreases TGFβ-induced phosphorylation of receptor Smads at late, but not at early, timepoints. These results suggest that Notch activation plays a role in sustaining TGFβ/Smad signaling in EOC cells. Furthermore, inhibition of Notch by DAPT decreases TGFβ induction of Slug and repression of E-cadherin and knockdown of Notch1 decreases TGFβ-induced repression of E-cadherin, indicating that Notch is required, at least in part, for TGFβ-induced EMT in EOC cells. On the other hand, TGFβ treatment increases the expression of Notch ligand Jagged1 and Notch target gene HES1 in EOC cells. Functionally, the combination of Notch1 activation and TGFβ treatment is more potent in promoting motility and migration of EOC cells than either stimulation alone. Taken together, our results indicate that Notch and TGFβ form a reciprocal positive regulatory loop and cooperatively regulate EMT and promote EOC cell motility and migration.

Disabled-1 alternative splicing in human fetal retina and neural tumors.

Background The Reelin-Dab1 signaling pathway plays a critical role in the positioning of migrating neurons, dendrite formation and lamination in the developing central nervous system. We have previously identified two alternatively spliced forms of Dab1 in the developing chick retina: an early form, Dab1-E, expressed in retinal progenitor cells, and a late form, Dab1 or Dab1-L, expressed in amacrine and ganglion cells. Compared to Dab1-L, Dab1-E lacks two exons that encode two Src family kinase (SFK) phosphorylation sites. Principal Findings Both Dab1-L and Dab1-E-like transcripts were identified in human fetal retina. Expression of human Dab1-L in primary chick retinal cultures resulted in Reelin-mediated induction of SFK phosphorylation and formation of neurite-like processes. In contrast, human Dab1-E-expressing cells retained an undifferentiated morphology. The human Dab1 gene is located within a common fragile site, and it has been postulated that it may function as a tumor suppressor. Analysis of Dab1 splice forms in retinoblastoma and neuroblastoma tumor cells revealed relative enrichment of Dab1-L-like (includes exons 7 and 8) and Dab1-E-like (excludes exons 7 and 8) transcripts in retinoblastoma and neuroblastoma, respectively. Treatment of retinoblastoma cell line RB522A with Reelin resulted in increased tyrosine phosphorylation of Dab1. As Nova2 has previously been implicated in the exclusion of exons 9B and 9C in Dab1, we examined the expression of this splicing factor in neuroblastoma and retinoblastoma cell lines. Nova2 was only detected in neuroblastoma cells, suggesting a correlation between Nova2 expression and increased levels of Dab1-E-like splice forms in neuroblastoma. Conclusions These results indicate that alternative splicing of Dab1 is conserved in avian and mammalian species, with Dab1-L driving SFK phosphorylation in both species. Dab1-E- and Dab-L-like isoforms are also expressed in childhood neural tumors, with preferential enrichment of Dab1-L-like and Dab1-E-like isoforms in retinoblastoma and neuroblastoma, respectively.

AP2 Transcription Factor Induces Apoptosis in Retinoblastoma Cells

The underlying cause of human retinoblastoma is complete inactivation of both copies of the RB1 gene. Other chromosome abnormalities, with the most common being extra copies of chromosome arm 6p, are also observed in retinoblastoma. The RB protein has previously been shown to interact with TFAP2 transcription factors. Here, we show that TFAP2A and TFAP2B, which map to chromosome arm 6p, are expressed in the amacrine and horizontal cells of human retina. TFAP2A RNA can readily be detected in retinoblastoma cell lines and tumors; however, the great majority of retinoblastoma cell lines and tumors are completely devoid of TFAP2A protein and TFAP2B RNA/protein. Transfection of TFAP2A and TFAP2B expression constructs into retinoblastoma cells induces apoptosis and inhibits proliferation. Our results suggest that a consequence of loss of RB1 gene function in retinoblastoma cells is inactivation of TFAP2A and TFAP2B function. We propose that inability to differentiate along the amacrine/horizontal cell lineages may underlie retinoblastoma tumor formation.