Franck Grall

PDEF, a Novel Prostate Epithelium-specific Ets Transcription Factor, Interacts with the Androgen Receptor and Activates Prostate-specific Antigen Gene Expression

Prostate cancer, the most frequent solid cancer in older men, is a leading cause of cancer deaths. Although proliferation and differentiation of normal prostate epithelia and the initial growth of prostate cancer cells are androgen-dependent, prostate cancers ultimately become androgen-independent and refractory to hormone therapy. The prostate-specific antigen (PSA) gene has been widely used as a diagnostic indicator for androgen-dependent and -independent prostate cancer. Androgen-induced and prostate epithelium-specific PSA expression is regulated by a proximal promoter and an upstream enhancer via several androgen receptor binding sites. However, little progress has been made in identifying androgen-independent regulatory elements involved in PSA gene regulation. We report the isolation of a novel, prostate epithelium-specific Ets transcription factor, PDEF (prostate-derived Etsfactor), that among the Ets family uniquely prefers binding to a GGAT rather than a GGAA core. PDEF acts as an androgen-independent transcriptional activator of the PSA promoter. PDEF also directly interacts with the DNA binding domain of androgen receptor and enhances androgen-mediated activation of the PSA promoter. Our results, as well as the critical roles of other Ets factors in cellular differentiation and tumorigenesis, strongly suggest that PDEF is an important regulator of prostate gland and/or prostate cancer development.

Serum proteomics and biomarkers in hepatocellular carcinoma and chronic liver disease.

Purpose: Proteomic profiling using surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF MS) enables the identification of biomarkers for cancer. We evaluated the sensitivity and specificity of SELDI-TOF MS for detection of established hepatocellular cancer (HCC) and compared it against α-fetoprotein (AFP), Lens culinaris agglutinin–reactive AFP (AFP-L3), and prothrombin induced by vitamin K absence-II (PIVKA-II). Experimental Design: Forty-one patients with HCC and 51 patients with hepatitis C cirrhosis were enrolled. Serum was analyzed by SELDI-TOF MS using three Ciphergen protein array types. Results: An 11-peak algorithm for HCC detection was identified. Using the AFP cutoff of 20 ng/mL, the sensitivity was 73% and the specificity was 71%. Using the AFP-L3 cutoff of 10% yielded a sensitivity of 63% and a specificity of 94%. Using the PIVKA-II cutoff of 125 milliabsorbance units (mAU), the sensitivity was 84% and the specificity was 69%. Overall, the sensitivity and specificity of SELDI-TOF MS for HCC were 79% and 86%, respectively. In multivariate analysis, the 11-peak SELDI profile was predictive of HCC independent of AFP, PIVKA, and AFP-L3. Among eight patients with the largest tumor size of <2 cm, SELDI-TOF MS correctly identified seven whereas AFP, AFP-L3, and PIVKA-II identified only three, one, and one, respectively. One of the 11 peaks in the SELDI-TOF MS 11-peak predictor from SELDI-TOF MS was identified as cystatin C. Conclusions: SELDI-TOF MS accurately distinguished patients with HCC from those with hepatitis C virus cirrhosis, was more accurate than traditional biomarkers in identifying small tumors, and should be further evaluated.

ESE-3, a Novel Member of an Epithelium-specific Ets Transcription Factor Subfamily, Demonstrates Different Target Gene Specificity from ESE-1

Most cancers originate as a result of aberrant gene expression in mainly glandular epithelial tissues leading to defects in epithelial cell differentiation. The latter is governed by distinct sets of transcriptional regulators. Here we report the characterization of epithelium-specific Ets factor, family member 3 (ESE-3), a novel member of the ESE subfamily of Ets transcription factors. ESE-3 shows highest homology to two other epithelium restricted Ets factors, ESE-1 and ESE-2. ESE-3, like ESE-1 and ESE-2, is exclusively expressed in a subset of epithelial cells with highest expression in glandular epithelium such as prostate, pancreas, salivary gland, and trachea. A potential role in branching morphogenesis is suggested, since ESE-3 transactivates the c-MET promoter via three high affinity binding sites. Additionally, ESE-3 binding to DNA sequences in the promoters of several glandular epithelium-specific genes suggests a role for ESE-3 in later stages of glandular epithelium differentiation. Although ESE-3 and ESE-1 bind with similar affinity to various Ets binding sites, ESE-3 and ESE-1 differ significantly in their ability to transactivate the promoters containing these sites. Our results support the notion that ESE-1, ESE-2, and ESE-3 represent a unique epithelium-specific subfamily of Ets factors that have critical but distinct functions in epithelial cell differentiation and proliferation.

Reduced PDEF Expression Increases Invasion and Expression of Mesenchymal Genes in Prostate Cancer Cells

The epithelium-specific Ets transcription factor, PDEF, plays a role in prostate and breast cancer, although its precise function has not been established. In prostate cancer, PDEF is involved in regulating prostate-specific antigen expression via interaction with the androgen receptor and NKX3.1, and down-regulation of PDEF by antiproliferative agents has been associated with reduced PDEF expression. We now report that reduced expression of PDEF leads to a morphologic change, increased migration and invasiveness in prostate cancer cells, reminiscent of transforming growth factor beta (TGFbeta) function and epithelial-to-mesenchymal transition. Indeed, inhibition of PDEF expression triggers a transcriptional program of genes involved in the TGFbeta pathway, migration, invasion, adhesion, and epithelial dedifferentiation. Our results establish PDEF as a critical regulator of genes involved in cell motility, invasion, and adhesion of prostate cancer cells.

Serum proteome profiling detects myelodysplastic syndromes and identifies CXC chemokine ligands 4 and 7 as markers for advanced disease

Myelodysplastic syndromes (MDS) are among the most frequent hematologic malignancies. Patients have a short survival and often progress to acute myeloid leukemia. The diagnosis of MDS can be difficult; there is a paucity of molecular markers, and the pathophysiology is largely unknown. Therefore, we conducted a multicenter study investigating whether serum proteome profiling may serve as a noninvasive platform to discover novel molecular markers for MDS. We generated serum proteome profiles from 218 individuals by MS and identified a profile that distinguishes MDS from non-MDS cytopenias in a learning sample set. This profile was validated by testing its ability to predict MDS in a first independent validation set and a second, prospectively collected, independent validation set run 5 months apart. Accuracy was 80.5% in the first and 79.0% in the second validation set. Peptide mass fingerprinting and quadrupole TOF MS identified two differential proteins: CXC chemokine ligands 4 (CXCL4) and 7 (CXCL7), both of which had significantly decreased serum levels in MDS, as confirmed with independent antibody assays. Western blot analyses of platelet lysates for these two platelet-derived molecules revealed a lack of CXCL4 and CXCL7 in MDS. Subtype analyses revealed that these two proteins have decreased serum levels in advanced MDS, suggesting the possibility of a concerted disturbance of transcription or translation of these chemokines in advanced MDS.

Urine proteomic profiling of pediatric nephrotic syndrome

The prognosis of pediatric nephrotic syndrome (NS) correlates with the responsiveness to glucocorticoid therapy. Steroid-resistant NS (SRNS) patients progress to end-stage renal disease, while steroid-sensitive NS (SSNS) and steroid-dependent (SDNS) patients do not. We have performed proteomic profiling of urine samples from a cross section of pediatric and adolescent subjects with SSNS, SRNS, and orthostatic proteinuria (OP) to identify urinary biomarkers of steroid resistance. We performed surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF MS) on urine from 19 subjects with SSNS/SDNS in remission, 14 with SSNS/SDNS in relapse, 5 with SRNS in relapse, and 6 with OP. Genetic algorithm search of principal component space revealed a group of five peaks distinguishing SRNS subjects, with mass/charge (m/z) values of 3,917.07, 4,155.53, 6,329.68, 7,036.96, and 11,117.4. Our analyses identified the peak at m/z 11,117.4 with an accuracy of 95% for classifying SRNS. Multidimensional protein fractionation and mass spectrometric analysis of SRNS urine samples combined with immunodepletion identified the 11,117.4 protein as β2-microglobulin (B2M). Using an unbiased protein profiling approach, we have validated previously reported findings of B2M as a biomarker associated with SRNS. Prospective studies are warranted to establish additional biomarkers that would be predictive of SRNS.

The Ets transcription factor ESE‐1 mediates induction of the COX‐2 gene by LPS in monocytes

Cyclooxygenase‐2 (COX‐2) is a key enzyme in the production of prostaglandins that are major inflammatory agents. COX‐2 production is triggered by exposure to various cytokines and to bacterial endotoxins. We present here a novel role for the Ets transcription factor ESE‐1 in regulating the COX‐2 gene in response to endotoxin and other pro‐inflammatory stimuli. We report that the induction of COX‐2 expression by lipopolysaccharide (LPS) and pro‐inflammatory cytokines correlates with ESE‐1 induction in monocyte/macrophages. ESE‐1, in turn, binds to several E26 transformation specific (Ets) sites on the COX‐2 promoter. In vitro analysis demonstrates that ESE‐1 binds to and activates the COX‐2 promoter to levels comparable to LPS‐mediated induction. Moreover, we provide results showing that the induction of COX‐2 by LPS may require ESE‐1, as the mutation of the Ets sites in the COX‐2 promoter or overexpression of a dominant‐negative form of ESE‐1 inhibits LPS‐mediated COX‐2 induction. The effect of ESE‐1 on the COX‐2 promoter is further enhanced by cooperation with other transcription factors such as nuclear factor‐κB and nuclear factor of activated T cells. Neutralization of COX‐2 is the goal of many anti‐inflammatory drugs. As an activator of COX‐2 induction, ESE‐1 may become a target for such therapeutics as well. Together with our previous reports of the role of ESE‐1 as an inducer of nitric oxide synthase in endothelial cells and as a mediator of pro‐inflammatory cytokines in vascular and connective tissue cells, these results establish ESE‐1 as an important player in the regulation of inflammation.

ESE-1 is a potent repressor of type II collagen gene (COL2A1) transcription in human chondrocytes.

The epithelium‐specific ETS (ESE)‐1 transcription factor is induced in chondrocytes by interleukin‐1β (IL‐1β). We reported previously that early activation of EGR‐1 by IL‐1β results in suppression of the proximal COL2A1 promoter activity by displacement of Sp1 from GC boxes. Here we report that ESE‐1 is a potent transcriptional suppressor of COL2A1 promoter activity in chondrocytes and accounts for the sustained, NF‐κB‐dependent inhibition by IL‐1β. Of the ETS factors tested, this response was specific to ESE‐1, since ESE‐3, which was also induced by IL‐1β, suppressed COL2A1 promoter activity only weakly. In contrast, overexpression of ETS‐1 increased COL2A1 promoter activity and blocked the inhibition by IL‐1β. These responses to ESE‐1 and ETS‐1 were confirmed using siRNA‐ESE1 and siRNA‐ETS1. In transient cotransfections, the inhibitory responses to ESE‐1 and IL‐1β colocalized in the −577/−132 bp promoter region, ESE‐1 bound specifically to tandem ETS sites at −403/−381 bp, and IL‐1‐induced binding of ESE‐1 to the COL2A1 promoter was confirmed in vivo by ChIP. Our results indicate that ESE‐1 serves a potent repressor function by interacting with at least two sites in the COL2A1 promoter. However, the endogenous response may depend upon the balance of other ETS factors such as ETS‐1, and other IL‐1‐induced factors, including EGR‐1 at any given time. Intracellular ESE‐1 staining in chondrocytes in cartilage from patients with osteoarthritis (OA), but not in normal cartilage, further suggests a fundamental role for ESE‐1 in cartilage degeneration and suppression of repair. J. Cell. Physiol. 215: 562–573, 2008.

Optimization and evaluation of surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) with reversed-phase protein arrays for protein profiling.

Abstract Surface-enhanced laser desorption/ionization (SELDI) time-of-flight mass spectrometry with protein arrays has facilitated the discovery of disease-specific protein profiles in serum. Such results raise hopes that protein profiles may become a powerful diagnostic tool. To this end, reliable and reproducible protein profiles need to be generated from many samples, accurate mass peak heights are necessary, and the experimental variation of the profiles must be known. We adapted the entire processing of protein arrays to a robotics system, thus improving the intra-assay coefficients of variation (CVs) from 45.1% to 27.8% (p<0.001). In addition, we assessed up to 16 technical replicates, and demonstrated that analysis of 2–4 replicates significantly increases the reliability of the protein profiles. A recent report on limited long-term reproducibility seemed to concord with our initial inter-assay CVs, which varied widely and reached up to 56.7%. However, we discovered that the inter-assay CV is strongly dependent on the drying time before application of the matrix molecule. Therefore, we devised a standardized drying process and demonstrated that our optimized SELDI procedure generates reliable and long-term reproducible protein profiles with CVs ranging from 25.7% to 32.6%, depending on the signal-to-noise ratio threshold used.

Differential gene expression of bone marrow-derived CD34+ cells is associated with survival of patients suffering from myelodysplastic syndrome

One feature of the molecular pathology of myelodysplastic syndromes (MDS) is aberrant gene expression. Such aberrations may be related to patient survival, and may indicate to novel diagnostic and therapeutic targets. Therefore, we aimed at identifying aberrant gene expression that is associated with MDS and patient survival. Bone marrow-derived CD34+ hematopoietic progenitor cells from six healthy persons and 16 patients with MDS were analyzed on cDNA macroarrays comprising 1,185 genes. Thereafter, our patients were followed-up for 54 months. We found differential expression of genes that were hitherto unrecognized in the context of MDS. Differential expression of 10 genes was confirmed by quantitative real-time RT-PCR. Hierarchical cluster analysis facilitated the separation of CD34+ cells of normal donors from patients with MDS. More importantly, it also distinguished MDS-patients with short and long survival. Scrutinizing our cDNA macroarray data for genes that are associated with short survival, we found, among others, increased expression of six different genes that encode the proteasome subunits. On the other hand, the most differentially down-regulated gene was IEX-1, which encodes an anti-apoptotic protein. We confirmed its decreased expression on RNA and protein level in an independent validation set of patient samples. The presented data broadens our notion about the molecular pathology of MDS and may lend itself to better identify patients with short survival. Furthermore, our findings may help to define new molecular targets for drug development and therapeutic approaches for patients with poor prognosis.