Taduru Sreenath

ERG oncoprotein expression in prostate cancer: clonal progression of ERG-positive tumor cells and potential for ERG-based stratification

Gene fusions prevalent in prostate cancer (CaP) lead to the elevated expression of the ERG proto-oncogene. ERG activation present in 50–70% of prostate tumors underscores one of the most common oncogenic alterations in CaP. Despite numerous reports of gene fusions and mRNA expression, ERG oncoprotein status in CaP still remains to be defined. Furthermore, development of ERG protein-based assays may provide a new dimension to evaluation of gene fusions involving diverse androgen-regulated promoters and the ERG protein-coding sequence. Through exhaustive evaluations of 132 whole-mount prostates (261 tumor foci and over 200 000 benign glands) for the ERG oncoprotein nuclear expression, we demonstrated 99.9% specificity for detecting prostate tumor cells using a highly specific anti-ERG monoclonal antibody. The ERG oncoprotein expression correlated well with fusion transcript or gene fusion in randomly selected specimens. Strong concordance of ERG-positive foci of prostatic intraepithelial neoplasia (PIN) with ERG-positive carcinoma (82 out of 85 sections with PIN, 96.5%) affirms the biological role of ERG in clonal selection of prostate tumors in 65% (86 out of 132) of patients. Conversely, ERG negative PINs were associated with ERG-negative carcinoma. Taken together, the homogeneous and strong ERG expression detected in individual tumors establishes the potential for ERG oncoprotein-based stratification of CaP.

Dentin sialoprotein and dentin phosphoprotein have distinct roles in dentin mineralization.

Dentin sialophosphoprotein (DSPP), a major non-collagenous matrix protein of odontoblasts, is proteolytically cleaved into dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). Our previous studies revealed that DSPP null mice display a phenotype similar to human autosomal dominant dentinogenesis imperfecta, in which teeth have widened predentin and irregular dentin mineralization resulting in sporadic unmineralized areas in dentin and frequent pulp exposure. Earlier in vitro studies suggested that DPP, but not DSP, plays a significant role in initiation and maturation of dentin mineralization. However, the precise in vivo roles of DSP and DPP are far from clear. Here we report the generation of DPPcKO mice, in which only DSP is expressed in a DSPP null background, resulting in a conditional DPP knockout. DPPcKO teeth show a partial rescue of the DSPP null phenotype with the restored predentin width, an absence of irregular unmineralized areas in dentin, and less frequent pulp exposure. Micro-computed tomography (micro-CT) analysis of DPPcKO molars further confirmed this partial rescue with a significant recovery in the dentin volume, but not in the dentin mineral density. These results indicate distinct roles of DSP and DPP in dentin mineralization, with DSP regulating initiation of dentin mineralization, and DPP being involved in the maturation of mineralized dentin.

Delineation of TMPRSS2-ERG Splice Variants in Prostate Cancer

Purpose: The expression of the ETS-related gene (ERG) is low or undetectable in benign prostate epithelial cells. High prevalence of ERG overexpression in prostate cancer cells due to TMPRSS2-ERG fusions suggest for causal roles of ERG protein in the neoplastic process. TMPRSS2-ERG fusion junctions have been extensively studied in prostate cancer. However, virtually nothing is known about the nature of full-length transcripts and encoded proteins. This study focuses on qualitative and quantitative features of full-length TMPRSS2-ERG transcripts in prostate cancer. Experimental Design: Full-length TMPRSS2-ERG transcripts were cloned and sequenced from a cDNA library generated from pooled RNA of six TMPRSS2-ERG fusion–positive prostate tumors. The encoded ERG proteins were analyzed in HEK293 cells. Copy numbers of TMPRSS2-ERG splice variants were determined by quantitative reverse transcription-PCR in laser capture microdissected prostate cancer cells. Results: Two types of TMPRSS2-ERG cDNAs were identified: type I, which encodes full-length prototypical ERG protein (ERG1, ERG2, ERG3), and type II, encoding truncated ERG proteins lacking the ETS domain (ERG8 and a new variant, TEPC). In microdissected prostate tumor cells from 122 patients, relative abundance of these variants was in the following order: ERG8 > TEPC > ERG 3 > ERG1/2 with combined overexpression rate of 62.3% in prostate cancer. Increased ratio of type I over type II splice forms showed a trend of correlation with less favorable pathology and outcome. Conclusions: Qualitative and quantitative features of specific ERG splice variants defined here promise to enhance the utility of ERG as a biomarker and therapeutic target in prostate cancer.

Neuronal Cyclin-Dependent Kinase 5 Activity Is Critical for Survival

Cyclin-dependent kinase 5 (Cdk5) null mice exhibit a unique phenotype characterized by perinatal mortality, disrupted cerebral cortical layering attributable to abnormal neuronal migration, lack of cerebellar foliation, and chromatolytic changes of neurons in the brainstem and the spinal cord. Because Cdk5 is expressed in both neurons and astrocytes, it has been unclear whether this phenotype is primarily attributable to defects in neurons or in astrocytes. Herein we report reconstitution of Cdk5 expression in neurons in Cdk5 null mice and its effect on the null phenotype. Unlike the Cdk5 null mice, the reconstituted Cdk5 null mice that express the Cdk5 transgene under the p35 promoter (TgKO mice) were viable and fertile. Because Cdk5 expression is mainly limited to neurons in these mice and rescues the defects in the nervous system of the Cdk5 null phenotype, it clearly demonstrates that Cdk5 activity is necessary for normal development and survival of p35-expressing neurons.

DSPP effects on in vivo bone mineralization

Dentin sialophosphoprotein has been implicated in the mineralization process based on the defective dentin formation in Dspp null mice (Dspp-/-). Dspp is expressed at low levels in bone and Dspp-/- femurs assessed by quantitative micro-computed tomography (micro-CT) and Fourier transform infrared spectroscopic imaging (FTIRI) exhibit some mineral and matrix property differences from wildtype femurs in both developing and mature mice. Compared to wildtype, Dspp-/- mice initially (5 weeks) and at 7 months had significantly higher trabecular bone volume fractions and lower trabecular separation, while at 9 months, bone volume fraction and trabecular number were lower. Cortical bone mineral density, area, and moments of inertia in Dspp-/- were reduced at 9 months. By FTIRI, Dspp-/- animals initially (5 months) contained more stoichiometric bone apatite with higher crystallinity (crystal size/perfection) and lower carbonate substitution. This difference progressively reversed with age (significantly decreased crystallinity and increased acid phosphate content in Dspp-/- cortical bone by 9 months of age). Mineral density as determined in 3D micro-CT and mineral-to-matrix ratios as determined by 2D FTIRI in individual cortical and trabecular bones were correlated (r(2)=0.6, p<0.04). From the matrix analysis, the collagen maturity of both cortical and trabecular bones was greater in Dspp-/- than controls at 5 weeks; by 9 months this difference in cross-linking pattern did not exist. Variations in mineral and matrix properties observed at different ages are attributable, in part, to the ability of the Dspp gene products to regulate both initial mineralization and remodeling, implying an effect of Dspp on bone turnover.

Androgen-independent expression of hoxb-13 in the mouse prostate.

Hox genes encode transcriptional regulatory proteins that are largely responsible for establishing the body plan of all metazoan organisms. A subset of Hox genes is expressed during the period of organogenesis and into adulthood. hoxb‐13 is a recently‐described member of the Hox gene family that is expressed in the spinal cord, hindgut, and urogenital sinus during embryogenesis.

Amelogenin-mediated Regulation of Osteoclastogenesis, and Periodontal Cell Proliferation and Migration

We previously reported that amelogenin isoforms M180 and leucine-rich amelogenin peptide (LRAP) are expressed in the periodontal region, and that their absence is associated with increased cementum defects in amelogenin-knockout (KO) mice. The aim of the present study was to characterize the functions of these isoforms in osteoclastogenesis and in the proliferation and migration of cementoblast/periodontal ligament cells. The co-cultures of wild-type (WT) osteoclast progenitor and KO cementoblast/periodontal ligament cells displayed more tartrate-resistant acid phosphatase (TRAP)-positive cells than the co-cultures of WT cells. The addition of LRAP to both co-cultures significantly reduced RANKL expression and the TRAP-positive cells. Proliferation and migration rates of the KO cementoblast/periodontal ligament cells were lower than those of WT cells and increased with the addition of either LRAP or P172 (a porcine homolog of mouse M180). Thus, we demonstrate the regulation of osteoclastogenesis by LRAP, and the proliferation and migration of cementoblast/periodontal ligament cells by LRAP and P172.

Dph3, a Small Protein Required for Diphthamide Biosynthesis, Is Essential in Mouse Development

ABSTRACT The translation elongation factor 2 in eukaryotes (eEF-2) contains a unique posttranslationally modified histidine residue, termed diphthamide, which serves as the only target for diphtheria toxin and Pseudomonas aeruginosa exotoxin A. Diphthamide biosynthesis is carried out by five highly conserved proteins, Dph1 to Dph5, and an as-yet-unidentified amidating enzyme. The evolutionary conservation of the complex diphthamide biosynthesis pathway throughout eukaryotes implies a key role for diphthamide in normal cellular physiology. Of the proteins required for diphthamide synthesis, Dph3 is the smallest, containing only 82 residues. In addition to having a role in diphthamide biosynthesis, Dph3 is also involved in modulating the functions of the Elongator complex in yeast. To explore the physiological roles of Dph3 and to begin to investigate the function of diphthamide, we generated dph3 knockout mice and showed that dph3+/− mice are phenotypically normal, whereas dph3−/− mice, which lack the diphthamide modification on eEF-2, are embryonic lethal. Loss of both dph3 alleles causes a general delay in embryonic development accompanied by lack of allantois fusion to the chorion and increased degeneration and necrosis in neural tubes and is not compatible with life beyond embryonic day 11.5. The dph3−/− placentas also developed abnormally, showing a thinner labyrinth lacking embryonic erythrocytes and blood vessels. These results attest to the physiological importance of Dph3 in development. The biological roles of Dph3 are also discussed.

Spatial Expression of Cbfa1/Runx2 Isoforms in Teeth and Characterization of Binding Sites in the DSPP Gene

Cbfa1/Runx2 is an essential transcription factor for osteoblast and odontoblast differentiation. Heterogeneous mutations of Cbfa1 gene result in cleidocranial dysplasia, an autosomal dominant syndrome, characterized by abnormal skeletal genesis and dental disorders. Recently three Cbfa1/Runx isoforms (Pebp2 f A/type I, til-1/type II, and Osf2/type III) have been identified that differ in their amino-terminal sequences. The precise roles of Cbfa1/Runx2 isoforms in odontoblast development are not known. The purpose of this study was to determine and compare expression patterns of the three Cbfa1/Runx2 isoforms in newborn tooth organs. Toward this aim, we developed three probes: type I and type II, which specifically hybridize with Pebp2 f A and til-1, respectively, and type II/III, which hybridizes with osf2 and partially with til-1. In addition, Cbfa1/Runx2 binding sites were identified in the regulatory elements of mouse dentin sialophosphoprotein (mDSPP) gene, which encodes a matrix protein expressed during odontogenesis. In situ hybridization performed with the specific Cbfa1/Runx2 isoform probes demonstrated that all isoforms are expressed in teeth and bone. The type I isoform was expressed at higher levels than isoforms type II and type II/III in developing newborn mouse incisors. Genomic mDSPP clones were isolated and characterized containing ~2.6 kb of the promoter region. Computer analysis of the promoter segment and intron 1 revealed a number of potential transcriptional factor binding sites including five Cbfa1/Runx2 binding sites, three in the promoter region and two within intron 1. DNA-protein assay and antibody supershift experiments showed that these binding sites interact with nuclear extracts isolated from the mouse odontoblast cell line MO6-G3. Further characterization of the functional role of Cbfa1/Runx2 in the regulation of the mDSPP gene expression is being investigated.

Oncogenic activation of ERG: A predominant mechanism in prostate cancer

Prevalent gene fusions involving regulatory sequences of the androgen receptor (AR) regulated genes (primarily TMPRSS2) and protein coding sequences of nuclear transcription factors of the ETS gene family (predominantly ERG) result in unscheduled androgen dependent ERG expression in prostate cancer (CaP).Cumulative data from a large number of studies in the past six years accentuate ERG alterations in more than half of all CaP patients in Western countries. Studies underscore that ERG functions are involved in the biology of CaP. ERG expression in normal context is selective to endothelial cells, specific hematopoetic cells and pre-cartilage cells. Normal functions of ERG are highlighted in hematopoetic stem cells. Emerging data continues to unravel molecular and cellular mechanisms by which ERG may contribute to CaP. Herein, we focus on biological and clinical aspects of ERG oncogenic alterations, potential of ERG-based stratification of CaP and the possibilities of targeting the ERG network in developing new therapeutic strategies for the disease.