SDPR functions as a metastasis suppressor in breast cancer by promoting apoptosis.

Metastatic dissemination of breast cancer cells represents a significant clinical obstacle to curative therapy. The loss of function of metastasis suppressor genes is a major rate-limiting step in breast cancer progression that prevents the formation of new colonies at distal sites. However, the discovery of new metastasis suppressor genes in breast cancer using genomic efforts has been slow, potentially due to their primary regulation by epigenetic mechanisms. Here, we report the use of model cell lines with the same genetic lineage for the identification of a novel metastasis suppressor gene, serum deprivation response (SDPR), localized to 2q32-33, a region reported to be associated with significant loss of heterozygosity in breast cancer. In silico metaanalysis of publicly available gene expression datasets suggests that the loss of expression of SDPR correlates with significantly reduced distant-metastasis-free and relapse-free survival of breast cancer patients who underwent therapy. Furthermore, we found that stable SDPR overexpression in highly metastatic breast cancer model cell lines inhibited prosurvival pathways, shifted the balance of Bcl-2 family proteins in favor of apoptosis, and decreased migration and intravasation/extravasation potential, with a corresponding drastic suppression of metastatic nodule formation in the lungs of NOD/SCID mice. Moreover, SDPR expression is silenced by promoter DNA methylation, and as such it exemplifies epigenetic regulation of metastatic breast cancer progression. These observations highlight SDPR as a potential prognostic biomarker and a target for future therapeutic applications.

Blocking peptides and molecular mimicry as treatment for kidney disease.

Protein mimotopes, or blocking peptides, are small therapeutic peptides that prevent protein-protein interactions by selectively mimicking a native binding domain. Inexpensive technology facilitates straightforward design and production of blocking peptides in sufficient quantities to allow preventive and therapeutic trials in both in vitro and in vivo experimental disease models. The kidney is an ideal peptide target, since small molecules undergo rapid filtration and efficient bulk absorption by tubular epithelial cells. Because the half-life of peptides is markedly prolonged in the kidneys compared with the bloodstream, blocking peptides are an attractive tool for treating diverse renal diseases, including ischemia, proteinuric states, such as membranous nephropathy and focal and segmental glomerulosclerosis, and renal cell carcinoma. Therapeutic peptides represent one of the fastest-growing reagent classes for novel drug development in human disease, partly because of their ease of administration, high binding affinity, and minimal off-target effects. This review introduces the concepts of blocking peptide design, production, and administration and highlights the potential use of therapeutic peptides to prevent or treat specific renal diseases.

Polycystin-1 regulates the stability and ubiquitination of transcription factor Jade-1.

Autosomal-dominant polycystic kidney disease (ADPKD) and von Hippel-Lindau (VHL) disease lead to large kidney cysts that share pathogenetic features. The polycystin-1 (PC1) and pVHL proteins may therefore participate in the same key signaling pathways. Jade-1 is a pro-apoptotic and growth suppressive ubiquitin ligase for beta-catenin and transcriptional coactivator associated with histone acetyltransferase activity that is stabilized by pVHL in a manner that correlates with risk of VHL renal disease. Thus, a relationship between Jade-1 and PC1 was sought. Full-length PC1 bound, stabilized and colocalized with Jade-1 and inhibited Jade-1 ubiquitination. In contrast, the cytoplasmic tail or the naturally occurring C-terminal fragment of PC1 (PC1-CTF) promoted Jade-1 ubiquitination and degradation, suggesting a dominant-negative mechanism. ADPKD-associated PC1 mutants failed to regulate Jade-1, indicating a potential disease link. Jade-1 ubiquitination was mediated by Siah-1, an E3 ligase that binds PC1. By controlling Jade-1 abundance, PC1 and the PC1-CTF differentially regulate Jade-1-mediated transcriptional activity. A key target of PC1, the cyclin-dependent kinase inhibitor p21, is also up-regulated by Jade-1. Through Jade-1, PC1 and PC1 cleaved forms may exert fine control of beta-catenin and canonical Wnt signaling, a critical pathway in cystic renal disease. Thus, Jade-1 is a transcription factor and ubiquitin ligase whose activity is regulated by PC1 in a manner that is physiologic and may correlate with disease. Jade-1 may be an important therapeutic target in renal cystogenesis.

Tumor suppressor von Hippel-Lindau (VHL) stabilization of Jade-1 protein occurs through plant homeodomains and is VHL mutation dependent.

The von Hippel-Lindau (VHL) gene is the major renal cancer gene in adults. The mechanism of renal tumor suppression by VHL protein is only partly elucidated. VHL loss increases expression of the hypoxia-inducible factor alpha transcription factors. However, clinical and biochemical data indicate that the hypoxia-inducible factors are necessary but not sufficient for renal tumorigenesis, which suggests other VHL effector pathways are involved. Jade-1 protein interacts strongly with VHL and is most highly expressed in renal proximal tubules, precursor cells of renal cancer. Short-lived Jade-1 protein contains plant homeodomain (PHD) and candidate PEST degradation motifs and is substantially stabilized by VHL. The effect of VHL on Jade-1 protein abundance and relative protein stability was further examined in immunoblots and metabolic labeling experiments using two time points. VHL-Jade-1 binding was tested in coimmunoprecipitations. In cotransfection studies with wild-type VHL, the Jade-1 PHD-extended PHD module, not the candidate PEST domain, was required for full VHL-mediated stabilization. This module is also found in leukemia transcription factors AF10 and AF17, as well as closely related Jade-like proteins, which suggests all might be VHL regulated. Intriguingly, naturally occurring truncations and mutations of VHL affected wild-type Jade-1 binding and stabilization. Although the VHL beta domain was sufficient for Jade-1 binding, both the alpha and beta domains were required for Jade-1 stabilization. Thus, truncating VHL mutations, which are severe and associated with renal cancer development, prevented Jade-1 stabilization. Moreover, well-controlled cotransfection and metabolic labeling experiments revealed that VHL missense mutations that cause VHL disease without renal cancer, such as Tyr98His and Tyr112His, stabilized Jade-1 fully. In contrast, like the VHL truncations, VHL missense mutations commonly associated with renal cancer, such as Leu118Pro or Arg167Trp, did not stabilize Jade-1 fully. Therefore, loss of Jade-1 stability may correlate with renal cancer risk. Endogenous Jade-1 in stable renal cancer lines also exhibited VHL mutation-dependent regulation. As in the cotransfections, VHL truncations did not increase endogenous Jade-1 abundance, whereas the VHL missense mutations tested partially increased Jade-1 expression. Additional studies with non-PHD proteins indicated that Jade-1 stabilization by VHL is highly specific. Fibronectin was not stabilized like Jade-1 by VHL, nor were candidate VHL interactors from a yeast screen. Thus, protein stabilization likely reflects the biological activity of largely intact VHL protein on the PHD-extended PHD module of Jade-1. Dysregulation of the VHL protein stabilization pathway or of Jade-1 itself may therefore contribute to VHL renal disease and renal cancer pathogenesis.

Previously unidentified changes in renal cell carcinoma gene expression identified by parametric analysis of microarray data.

BACKGROUND: Renal cell carcinoma is a common malignancy that often presents as a metastatic-disease for which there are no effective treatments. To gain insights into the mechanism of renal cell carcinogenesis, a number of genome-wide expression profiling studies have been performed. Surprisingly, there is very poor agreement among these studies as to which genes are differentially regulated. To better understand this lack of agreement we profiled renal cell tumor gene expression using genome-wide microarrays (45,000 probe sets) and compare our analysis to previous microarray studies. METHODS: We hybridized total RNA isolated from renal cell tumors and adjacent normal tissue to Affymetrix U133A and U133B arrays. We removed samples with technical defects and removed probesets that failed to exhibit sequence-specific hybridization in any of the samples. We detected differential gene expression in the resulting dataset with parametric methods and identified keywords that are overrepresented in the differentially expressed genes with the Fisher-exact test. RESULTS: We identify 1,234 genes that are more than three-fold changed in renal tumors by t-test, 800 of which have not been previously reported to be altered in renal cell tumors. Of the only 37 genes that have been identified as being differentially expressed in three or more of five previous microarray studies of renal tumor gene expression, our analysis finds 33 of these genes (89%). A key to the sensitivity and power of our analysis is filtering out defective samples and genes that are not reliably detected. CONCLUSIONS: The widespread use of sample-wise voting schemes for detecting differential expression that do not control for false positives likely account for the poor overlap among previous studies. Among the many genes we identified using parametric methods that were not previously reported as being differentially expressed in renal cell tumors are several oncogenes and tumor suppressor genes that likely play important roles in renal cell carcinogenesis. This highlights the need for rigorous statistical approaches in microarray studies.

Transcriptome-based analysis of kidney gene expression changes associated with diabetes in OVE26 mice, in the presence and absence of losartan treatment.

Diabetes is among the most common causes of end-stage renal disease, although its pathophysiology is incompletely understood. We performed next-generation sequencing-based transcriptome analysis of renal gene expression changes in the OVE26 murine model of diabetes (age 15 weeks), relative to non-diabetic control, in the presence and absence of short-term (seven-day) treatment with the angiotensin receptor blocker, losartan (n = 3-6 biological replicates per condition). We detected 1438 statistically significant changes in gene expression across conditions. Of the 638 genes dysregulated in diabetes relative to the non-diabetic state, >70% were downregulation events. Unbiased functional annotation of genes up- and down-regulated by diabetes strongly associated (p<1 × 10(-8)) with terms for oxidative stress and for endoplasmic reticulum stress/protein folding. Most of the individual gene products up- or down-regulated with diabetes were unaffected by losartan treatment; however, of the gene products dysregulated in diabetes and influenced by losartan treatment, the vast majority of changes were in the direction of amelioration rather than exacerbation of the diabetic dysregulation. This group of losartan-protected genes associated strongly with annotation terms for endoplasmic reticulum stress, heat shock proteins, and chaperone function, but not oxidative stress; therefore, the losartan-unaffected genes suggest avenues for additional therapeutic opportunity in diabetes. Interestingly, the gene product most highly upregulated by diabetes (>52-fold), encoded by the cationic amino acid transporter Slc7a12, and the gene product most highly downregulated by diabetes (>99%)--encoded by the "pseudogene" Gm6300--are adjacent in the murine genome, are members of the SLC7 gene family, and are likely paralogous. Therefore, diabetes activates a near-total genetic switch between these two paralogs. Other individual-level changes in gene expression are potentially relevant to diabetic pathophysiology, and novel pathways are suggested. Genes unaffected by diabetes alone but exhibiting increased renal expression with losartan produced a signature consistent with malignant potential.

Candidate tumor suppressor and pVHL partner Jade-1 binds and inhibits AKT in renal cell carcinoma.

The von Hippel-Lindau (VHL) tumor suppressor pVHL is lost in the majority of clear-cell renal cell carcinomas (RCC). Activation of the PI3K/AKT/mTOR pathway is also common in RCC, with PTEN loss occurring in approximately 30% of the cases, but other mechanisms responsible for activating AKT at a wider level in this setting are undefined. Plant homeodomain protein Jade-1 (PHF17) is a candidate renal tumor suppressor stabilized by pVHL. Here, using kinase arrays, we identified phospho-AKT1 as an important target of Jade-1. Overexpressing or silencing Jade-1 in RCC cells increased or decreased levels of endogenous phospho-AKT/AKT1. Furthermore, reintroducing pVHL into RCC cells increased endogenous Jade-1 and suppressed endogenous levels of phospho-AKT, which colocalized with and bound to Jade-1. The N-terminus of Jade-1 bound both the catalytic domain and the C-terminal regulatory tail of AKT, suggesting a mechanism through which Jade-1 inhibited AKT kinase activity. Intriguingly, RCC precursor cells where Jade-1 was silenced exhibited an increased capacity for AKT-dependent anchorage-independent growth, in support of a tumor suppressor function for Jade-1 in RCC. In support of this concept, an in silico expression analysis suggested that reduced Jade-1 expression is a poor prognostic factor in clear-cell RCC that is associated with activation of an AKT1 target gene signature. Taken together, our results identify 2 mechanisms for Jade-1 fine control of AKT/AKT1 in RCC, through loss of pVHL, which decreases Jade-1 protein, or through attenuation in Jade-1 expression. These findings help explain the pathologic cooperativity in clear-cell RCC between PTEN inactivation and pVHL loss, which leads to decreased Jade-1 levels that superactivate AKT. In addition, they prompt further investigation of Jade-1 as a candidate biomarker and tumor suppressor in clear-cell RCC.

Overexpression of vascular endothelial growth factor and the development of post-transplantation cancer.

Cancer is an increasing and major problem after solid organ transplantation. In part, the increased cancer risk is associated with the use of immunosuppressive agents, especially calcineurin inhibitors. We propose that the effect of calcineurin inhibitors on the expression of vascular endothelial growth factor (VEGF) leads to an angiogenic milieu that favors tumor growth. Here, we used 786-0 human renal cancer cells to investigate the effect of cyclosporine (CsA) on VEGF expression. Using a full-length VEGF promoter-luciferase construct, we found that CsA markedly induced VEGF transcriptional activation through the protein kinase C (PKC) signaling pathway, specifically involving PKC zeta and PKC delta isoforms. Moreover, CsA promoted the association of PKC zeta and PKC delta with the transcription factor Sp1 as observed by immunoprecipitation assays. Using promoter deletion constructs, we found that CsA-mediated VEGF transcription was primarily Sp1 dependent. Furthermore, CsA-induced and PKC-Sp1-mediated VEGF transcriptional activation was partially inhibited by von Hippel-Lindau protein. CsA also promoted the progression of human renal tumors in vivo, wherein VEGF is overexpressed. Finally, to evaluate the in vivo significance of CsA-induced VEGF overexpression in terms of post-transplantation tumor development, we injected CT26 murine carcinoma cells (known to form angiogenic tumors) into mice with fully MHC mismatched cardiac transplants. We observed that therapeutic doses of CsA increased tumor size and VEGF mRNA expression and also enhanced tumor angiogenesis. However, coadministration of a blocking anti-VEGF antibody inhibited this CsA-mediated tumor growth. Collectively, these findings define PKC-mediated VEGF transcriptional activation as a key component in the progression of CsA-induced post-transplantation cancer.

Role of Jade-1 in the histone acetyltransferase (HAT) HBO1 complex.

Regulation of global chromatin acetylation is important for chromatin remodeling. A small family of Jade proteins includes Jade-1L, Jade-2, and Jade-3, each bearing two mid-molecule tandem plant homology domain (PHD) zinc fingers. We previously demonstrated that the short isoform of Jade-1L protein, Jade-1, is associated with endogenous histone acetyltransferase (HAT) activity. It has been found that Jade-1L/2/3 proteins co-purify with a novel HAT complex, consisting of HBO1, ING4/5, and Eaf6. We investigated a role for Jade-1/1L in the HBO1 complex. When overexpressed individually, neither Jade-1/1L nor HBO1 affected histone acetylation. However, co-expression of Jade-1/1L and HBO1 increased acetylation of the bulk of endogenous histone H4 in epithelial cells in a synergistic manner, suggesting that Jade1/1L positively regulates HBO1 HAT activity. Conversely, small interfering RNA-mediated depletion of endogenous Jade resulted in reduced levels of H4 acetylation. Moreover, HBO1-mediated H4 acetylation activity was enhanced severalfold by the presence of Jade-1/1L in vitro. The removal of PHD fingers affected neither binding nor mutual Jade-1-HBO1 stabilization but completely abrogated the synergistic Jade-1/1L- and HBO1-mediated histone H4 acetylation in live cells and in vitro with reconstituted oligonucleosome substrates. Therefore, PHDs are necessary for Jade-1/1L-induced acetylation of nucleosomal histones by HBO1. In contrast to Jade-1/1L, the PHD zinc finger protein ING4/5 failed to synergize with HBO1 to promote histone acetylation. The physical interaction of ING4/5 with HBO1 occurred in the presence of Jade-1L or Jade-3 but not with the Jade-1 short isoform. In summary, this study demonstrates that Jade-1/1L are crucial co-factors for HBO1-mediated histone H4 acetylation.

Jade-1 inhibits Wnt signalling by ubiquitylating beta-catenin and mediates Wnt pathway inhibition by pVHL.

The von Hippel-Lindau protein pVHL suppresses renal tumorigenesis in part by promoting the degradation of hypoxia-inducible HIF-alpha transcription factors; additional mechanisms have been proposed. pVHL also stabilizes the plant homeodomain protein Jade-1, which is a candidate renal tumour suppressor that may correlate with renal cancer risk. Here we show that Jade-1 binds the oncoprotein beta-catenin in Wnt-responsive fashion. Moreover, Jade-1 destabilizes wild-type beta-catenin but not a cancer-causing form of beta-catenin. Whereas the well-established beta-catenin E3 ubiquitin ligase component beta-TrCP ubiquitylates only phosphorylated beta-catenin, Jade-1 ubiquitylates both phosphorylated and non-phosphorylated beta-catenin and therefore regulates canonical Wnt signalling in both Wnt-off and Wnt-on phases. Thus, the different characteristics of beta-TrCP and Jade-1 may ensure optimal Wnt pathway regulation. Furthermore, pVHL downregulates beta-catenin in a Jade-1-dependent manner and inhibits Wnt signalling, supporting a role for Jade-1 and Wnt signalling in renal tumorigenesis. The pVHL tumour suppressor and the Wnt tumorigenesis pathway are therefore directly linked through Jade-1.

Estimated glomerular filtration rate in sickle cell anemia is associated with polymorphisms of bone morphogenetic protein receptor 1B.

Renal disease is common in sickle cell anemia. In this exploratory work, we used data from a longitudinal study of the natural history of sickle cell disease to examine the hypothesis that polymorphisms (SNPs) in selected candidate genes are associated with glomerular filtration rate (GFR). DNA samples and clinical and laboratory data were available for 1,140 patients with sickle cell anemia. GFR was estimated using the Cockcroft-Gault and Schwartz formulas for adults and children, respectively. We examined approximately 175 haplotype tagging (ht) SNPs in about 70 genes of the TGFbeta/BMP pathway for their association with GFR using linear regression. Four SNPs in BMPR1B, a bone morphogenetic protein (BMP) receptor gene, yielded statistically significant associations (P values ranging from 0.015 to 0.046). Three haplotypes in this gene were also associated with GFR. The TGF-beta/BMP pathway has been associated with the development of diabetic nephropathy, which has some features in common with sickle cell nephropathy. Our results suggest that, as with other subphenotypes of sickle cell disease, renal function may be genetically modulated.

Jade-1, a candidate renal tumor suppressor that promotes apoptosis.

Medical therapies are lacking for advanced renal cancer, so there is a great need to understand its pathogenesis. Most renal cancers have defects in the von Hippel-Lindau tumor suppressor pVHL. The mechanism by which pVHL protein functions in renal tumor suppression remains unclear. Jade-1 is a short-lived, kidney-enriched transcription factor that is stabilized by direct interaction with pVHL. Loss of Jade-1 stabilization by pVHL correlates with renal cancer risk, making the relationship between Jade-1 and renal cancer compelling. We report that Jade-1 expression was barely detectable in all tested renal cancer cell lines, regardless of VHL status. Strikingly, proteasome inhibitor treatment increased endogenous Jade-1 expression up to 10-fold. Jade-1 inhibited renal cancer cell growth, colony formation, and tumor formation in nude mice. Intriguingly, Jade-1 also affected the pattern of cell growth in monolayer culture and 3D culture. Jade-1 increased apoptosis by 40-50% and decreased levels of antiapoptotic Bcl-2. Antisense Jade-1-expressing cells confirmed these results. Therefore, Jade-1 may suppress renal cancer cell growth in part by increasing apoptosis. Jade-1 may represent a proapoptotic barrier to proliferation that must be overcome generally in renal cancer, perhaps initially by pVHL inactivation and subsequently by increased proteasomal activity. Therefore, Jade-1 may be a renal tumor suppressor.

EphA2: expression in the renal medulla and regulation by hypertonicity and urea stress in vitro and in vivo.

EphA2, a member of the large family of Eph receptor tyrosine kinases, is highly expressed in epithelial tissue and has been implicated in cell-cell and cell-matrix interactions, as well as cell growth and survival. Expression of EphA2 mRNA and protein was markedly upregulated by both hypertonic stress and by elevated urea concentrations in cells derived from the murine inner medullary collecting duct. This upregulation likely required transactivation of the epidermal growth factor (EGF) receptor tyrosine kinase and metalloproteinase-dependent ectodomain cleavage of an EGF receptor ligand, based on pharmacological inhibitor studies. A human EphA2 promoter fragment spanning nucleotides -4030 to +21 relative to the putative EphA2 transcriptional start site was responsive to tonicity but insensitive to urea. A promoter fragment spanning -1890 to +128 recapitulated both tonicity- and urea-dependent upregulation of expression, consistent with transcriptional activation. Neither the bona fide p53 response element at approximately -1.5 kb nor a pair of putative TonE elements at approximately -3 kb conferred the tonicity responsiveness. EphA2 mRNA and protein were expressed at low levels in rat renal cortex but at high levels in the collecting ducts of the renal medulla and papilla. Water deprivation in rats increased EphA2 expression in renal papilla, whereas dietary supplementation with 20% urea increased EphA2 expression in outer medulla. These data indicate that transcription and expression of the EphA2 receptor tyrosine kinase are regulated by tonicity and urea in vitro and suggest that this phenomenon is also operative in vivo. Renal medullary EphA2 expression may represent an adaptive response to medullary hypertonicity or urea exposure.

von Hippel-Lindau partner Jade-1 is a transcriptional co-activator associated with histone acetyltransferase activity.

Jade-1 was identified as a protein partner of the von Hippel-Lindau tumor suppressor pVHL. The interaction of Jade-1 and pVHL correlates with renal cancer risk. We have investigated the molecular function of Jade-1. Jade-1 has two zinc finger motifs called plant homeodomains (PHD). A line of evidence suggests that the PHD finger functions in chromatin remodeling and protein-protein interactions. We determined the cellular localization of Jade-1 and examined whether Jade-1 might have transcriptional and histone acetyltransferase (HAT) functions. Biochemical cell fractionation studies as well as confocal images of cells immunostained with a specific Jade-1 antibody revealed that endogenous Jade-1 is localized predominantly in the cell nucleus. Tethering of Gal4-Jade-1 fusion protein to Gal4-responsive promoters in co-transfection experiments activated transcription 5-6-fold, indicating that Jade-1 is a possible transcriptional activator. It was remarkable that overexpression of Jade-1 in cultured cells specifically increased levels of endogenous acetylated histone H4, but not histone H3, strongly suggesting that Jade-1 associates with HAT activity specific for histone H4. Deletion of the two PHD fingers completely abolished Jade-1 transcriptional and HAT activities, indicating that these domains are indispensable for Jade-1 nuclear functions. In addition, we demonstrated that TIP60, a known HAT with histone H4/H2A specificity, physically associates with Jade-1 and is able to augment Jade-1 HAT function in live cells, strongly suggesting that TIP60 might mediate Jade-1 HAT activity. Thus, Jade-1 is a novel candidate transcriptional co-activator associated with HAT activity and may play a key role in the pathogenesis of renal cancer and von Hippel-Lindau disease.

The von Hippel-Lindau tumor suppressor stabilizes novel plant homeodomain protein Jade-1.

The von Hippel-Lindau disease gene (VHL) is the causative gene for most adult renal cancers. However, the mechanism by which VHL protein functions as a renal tumor suppressor remains largely unknown. To identify low occupancy VHL protein partners with potential relevance to renal cancer, we screened a human kidney library against human VHL p30 using a yeast two-hybrid approach. Jade-1 (gene for Apoptosis and Differentiation in Epithelia) encodes a previously uncharacterized 64-kDa protein that interacts strongly with VHL protein and is most highly expressed in kidney. Jade-1 protein is short-lived and contains a candidate destabilizing (PEST) motif and plant homeodomains that are not required for the VHL interaction. Jade-1 is abundant in proximal tubule cells, which are clear-cell renal cancer precursors, and expression increases with differentiation. Jade-1 is expressed in cytoplasm and the nucleus diffusely and in speckles, where it partly colocalizes with VHL. VHL reintroduction into renal cancer cells increases endogenous Jade-1 protein abundance up to 10-fold. Furthermore, VHL increases Jade-1 protein half-life up to 3-fold. Thus, direct protein stabilization is identified as a new VHL function. Moreover, Jade-1 protein represents a novel candidate regulatory factor in VHL-mediated renal tumor suppression.