Renal Tumor Suppressor Research Articles

Characterization of renal cell carcinoma‐associated constitutional chromosome abnormalities by genome sequencing

Constitutional translocations, typically involving chromosome 3, have been recognized as a rare cause of inherited predisposition to renal cell carcinoma (RCC) for four decades. However, knowledge of the molecular basis of this association is limited. We have characterized the breakpoints by genome sequencing (GS) of constitutional chromosome abnormalities in five individuals who presented with RCC. In one individual with constitutional t(10;17)(q11.21;p11.2), the translocation breakpoint disrupted two genes: the known renal tumor suppressor gene (TSG) FLCN (and clinical features of Birt‐Hogg‐Dubé syndrome were detected) and RASGEF1A. In four cases, the rearrangement breakpoints did not disrupt known inherited RCC genes. In the second case without chromosome 3 involvement, the translocation breakpoint in an individual with a constitutional t(2;17)(q21.1;q11.2) mapped 12 Kb upstream of NLK. Interestingly, NLK has been reported to interact indirectly with FBXW7 and a previously reported RCC‐associated translocation breakpoint disrupted FBXW7. In two cases of constitutional chromosome 3 translocations, no candidate TSGs were identified in the vicinity of the breakpoints. However, in an individual with a constitutional chromosome 3 inversion, the 3p breakpoint disrupted the FHIT TSG (which has been reported previously to be disrupted in two apparently unrelated families with an RCC‐associated t(3;8)(p14.2;q24.1). These findings (a) expand the range of constitutional chromosome rearrangements that may be associated with predisposition to RCC, (b) confirm that chromosome rearrangements not involving chromosome 3 can predispose to RCC, (c) suggest that a variety of molecular mechanisms are involved the pathogenesis of translocation‐associated RCC, and (d) demonstrate the utility of GS for investigating such cases.

Theoretical studies of new Schiff base ligand derived from 1,3‐diaminopropane and 2‐acetyl ferrocene and studying some applications of its metal complexes

A novel bidentate Schiff base ligand (L) and some d‐transition metal chelates (Cr (III), Mn (II), Fe (III), Co (II), Ni (II), Cu (II), Zn (II) and Cd (II)) were synthesized and characterized using various physicochemical and spectroscopic techniques like elemental analysis, IR, mass, UV–visible and thermal analysis. The spectroscopic data suggested that the parent Schiff base ligand coordinated to the metal ions through both imine nitrogen atoms. The molecular and electronic structure of the free ligand was optimized theoretically, and the quantum chemical parameters were calculated. The molecular structure can be used to investigate the coordination sites and the total charge density around each atom. The free ligand and its complexes were screened for their antimicrobial activities for various pathogenic bacteria and fungi. The anticancer activities of the free ligand, Cr (III), Mn (II) and Fe (III) complexes were screened against MCF‐7 cell line and found that Mn (II) complex has the lowest IC50 (15.90 μg/ml). Molecular docking was used to predict the binding between the free ligand with receptor of mutant human androgen (ARccr) derived from androgen‐independent prostate cancer (1GS4), crystal structure of yeast‐specific serine/threonine protein phosphatase (ppz1) of Candida Albicans (5JPE) and crystal structure of renal tumor suppressor protein, folliculin (3 V42) and to identify the binding mode and the crucial functional groups interacting with the three proteins.

Abstract 1767: Rho-GTPase inhibits claudin-2 expression to promote proximal tubular epithelial cell plasticity and renal cell carcinoma

Abstract The clear-cell renal cell carcinoma (ccRCC) is the most prevalent and malignant type of kidney cancer, for which there are no effective treatment. Moreover, the incidence of ccRCC has been increasing steadily. Disruption of epithelial cell polarity, assisted by tight junction (TJ) deregulation, facilitates rewiring of oncogene and tumor suppressor signaling pathways. In this regard, proximal tubular epithelium (PTE), where ccRCC originates is the sole nephron segment that expresses claudin-2 (hereon Cldn2), a TJ- protein. However, role of Cldn2 in ccRCC remains unknown despite well-recognized role of Cldn2 in regulating other cancers. We here report a novel role for Cldn2 in maintaining epithelial differentiation among PTE cells as its loss induces mesenchymal traits and associates with ccRCC progression. In specific, we found a specific (among claudin proteins) loss of Cldn2 expression in PTE cells subjected to stimuli, like EGF, TGF-ß and hypoxia, known to induce EMT in PTE cells as well as in a murine model of spontaneous renal tumorigenesis. Further analysis of a large renal cancer patient cohort revealed a significant decrease in Cldn2 levels in the ccRCC, and its positive association with cancer metastasis. In vitro, genetic ablation of claudin-2 expression in HK-2 or MDCK-II cells potentiated mesenchymal traits (upregulated Vimentin and Fibronectin; p<0.002) and cell motility (p<0.0002 versus control). In contrast, forced claudin-2 expression in RCC-derived (and claudin-2 low) Caki-2 cells (P<0.002) promoted epithelial differentiation (downregulation of Vimentin and Fibronectin; P<0.0012& P<0.00025), inhibited cell invasion (P<0.0001) and proliferation (P<0.001). Overall, our findings support a novel and tissue-specific role of claudin-2 in maintaining epithelial differentiation among renal proximal tubular epithelium and tumor suppressor. Further analysis identified a key role of Rho-GTPase in regulating renal claudin-2 expression as ROCK inhibitor (Y-27632) not only reverted EMT-associated loss of Cldn2 but also in Caci-2 cells. An ELISA assay further confirmed an early induction of Rho-GTPase activity in PTE cells subjected to the EMT. Of note, inhibiting Rho-GTPase not only improves renal fibrosis but also inhibits ccRCC. Taken together, we here report a novel role of claudin-2 in maintaining epithelial phenotype in PTE cells with potential clinical significance in acute and chronic renal diseases, and ccRCC progression. Citation Format: Balawant Kumar, Rizwan Ahmad, Pinelopi Kapitsino, Giovanna A. Giannico, Roy Zent, Raymond Clement Harris, Peter Clark, Punita Dhawan, Amar B. Singh. Rho-GTPase inhibits claudin-2 expression to promote proximal tubular epithelial cell plasticity and renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1767.

Structural basis for renal cancer by the dynamics of pVHL-dependent JADE1 stabilization and β-catenin regulation

Renal cancer is the major cause of mortality due to abnormal functioning of von Hippel-Lindau (pVHL) and Jade Family PHD Finger 1 (JADE1) complex. E3 ubiquitin ligase JADE1 is stabilized by pVHL interaction through its plant homeodomains (PHDs). JADE1 acts as a renal tumor suppressor that promotes the ubiquitination and degradation of β-catenin by inhibiting canonical WNT signalling. Current study focuses on the structural characterization of reported missense mutations in pVHL through in silico approaches. The predicted 3-dimensional structures of pVHLWT, pVHLY98H, pVHLY112H, pVHLL118P and pVHLR167W were subjected to binding analysis against JADE1 through molecular docking and simulation assays. In all cases, JADE1 binding was observed at the β-domain, except pVHLL118P that exhibited binding with JADE1 through its α-domain. Our results signify that JADE1 stabilization is induced by pVHL α-domain, while β-domain is required for JADE1 binding. pVHL binding was mediated through β1 and β2-strands against the concave surface of the JADE1-PHD domain. The pVHL-JADE1 complex was evaluated to scrutinize the β-catenin-binding interface, which suggested the contribution of both α and β-domains of pVHL in β-catenin binding. The eleven-residue (Tyr30-Thr40) β-catenin segment exhibited association in a bipartite manner with pVHL-JADE1 complex. The presented model depicts a pVHL-JADE1 interface for the cooperative regulation of β-catenin binding. We propose that reduced JADE1 stabilization in case of pVHLL118P and pVHLR167W may correlate with the increased activity of β-catenin that may lead to renal cancer through β-catenin de-repression. Overall, β-catenin targeting mechanism coupled with the structural knowledge of JADE1-pVHL complex will provide better understanding of renal cancer pathogenesis.

Formation of Renal Cysts and Tumors in Vhl/Trp53-Deficient Mice Requires HIF1α and HIF2α.

The von Hippel-Lindau (VHL) tumor suppressor gene is inactivated in the majority of clear cell renal cell carcinomas (ccRCC), but genetic ablation of Vhl alone in mouse models is insufficient to recapitulate human tumorigenesis. One function of pVHL is to regulate the stability of the hypoxia-inducible factors (HIF), which become constitutively activated in the absence of pVHL. In established ccRCC, HIF1α has been implicated as a renal tumor suppressor, whereas HIF2α is considered an oncoprotein. In this study, we investigated the contributions of HIF1α and HIF2α to ccRCC initiation in the context of Vhl deficiency. We found that deleting Vhl plus Hif1a or Hif2a specifically in the renal epithelium did not induce tumor formation. However, HIF1α and HIF2α differentially regulated cell proliferation, mitochondrial abundance and oxidative capacity, glycogen accumulation, and acquisition of a clear cell phenotype in Vhl-deficient renal epithelial cells. HIF1α, but not HIF2α, induced Warburg-like metabolism characterized by increased glycolysis, decreased oxygen consumption, and decreased ATP production in mouse embryonic fibroblasts, providing insights into the cellular changes potentially occurring in Vhl mutant renal cells before ccRCC formation. Importantly, deletion of either Hif1a or Hif2a completely prevented the formation of renal cysts and tumors in Vhl/Trp53 mutant mice. These findings argue that both HIF1α and HIF2α exert protumorigenic functions during the earliest stages of cyst and tumor formation in the kidney. Cancer Res; 76(7); 2025-36. ©2016 AACR.

Jade-1: its structure, regulation and functions in the renal cancer.

Jade-1 is originally identified by the yeast two-hybrid system as a protein partner of von Hippel-Lindau (pVHL) tumor suppressor, a well-known renal tumor suppressor. In cellular signaling pathways, many upstream Jade-1 regulators, such as pVHL, CK1α, PC1, and NPHP4, can control its activity by stabilization, phosphorylation, and nuclear translocation. Numerous downstream effectors, including β-catenin, AKT, p21, and Bcl-2, are well modulated by Jade-1, which mainly regulates cell proliferation and apoptosis. Jade-1 is also deemed to be a candidate of transcriptional co-activator associated with histone acetyltransferase (HAT) activity. This review focuses on the anticancer role of Jade-1 in clear cell renal carcinoma and the inhibitory effect of Jade-1 on cystic renal diseases. This review aims to provide a basis of disease prevention or therapy.

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.

NBP, a zebrafish homolog of human Kank3, is a novel Numb interactor essential for epidermal integrity and neurulation

Numb is an adaptor protein implicated in diverse basic cellular processes. Using the yeast-two hybrid system we isolated a novel Numb interactor in zebrafish called NBP which is an ortholog of human renal tumor suppressor Kank. NBP interacts with the PTB domain of Numb through a region well conserved among vertebrate Kanks containing the NGGY sequence. Similar NBP and Numb morphant phenotype such as impaired convergence and extension movements during gastrulation, neurulation and epidermis defects and enhanced phenotypic aberrations in double morphants suggest that the genes interact genetically. We demonstrate that the expression of NBP undergoes quantitative and qualitative changes during embryogenesis and that the protein accumulates at the cell periphery to sites of cell–cell contact during gastrulation and later in development it concentrates at the basal poles of differentiated cells. These findings imply a possible role of NBP in establishing and maintaining cell adhesion and tissue integrity.

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.

Hereditary renal carcinogenesis fitting Knudson's two-hit model: genotype, environment, and phenotype.

Cancer is a heritable disorder of somatic cells. Environment and heredity both operate in the origin of human cancer. The Eker (Tsc2 gene mutant) rat model of hereditary renal carcinoma (RC) is an example of Mendelian dominantly inherited predisposition to a specific cancer in an experimental animal. To the best of our knowledge, this was the first isolation of a Mendelian dominantly predisposing cancer gene in a naturally occurring animal model. Recently, we discovered a new hereditary renal carcinoma in the rat in Japan, and the rat was named the "Nihon" rat, and its predisposing (Nihon) gene could be a novel renal tumor suppressor gene. We present these unique models, comparing these two predisposing genes (both are located on rat chromosome 10), for the study of problems in carcinogenesis, for instance, species-specific difference in tumorigenesis, cell stage and tissue/cell-type specific tumorigenesis, multistep carcinogenesis, modifier gene(s) in renal carcinogenesis, cancer prevention, and the development of the therapeutic treatments that can be translated to human patients, as well as how environmental factors interact with cancer susceptibility gene(s).

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.