Oncogene database

5716

PSMD10

proteasome (prosome, macropain) 26S subunit, non-ATPase, 10;PSMD10;proteasome (prosome, macropain) 26S subunit, non-ATPase, 10

26S proteasome non-ATPase regulatory subunit 10|26S proteasome regulatory subunit p28|ankyrin repeat protein|gankyrin|hepatocellular carcinoma-associated protein p28-II

Xq22.3

protein-coding

Hepatocellular carcinoma (HCC) is one of the most common cancers in Asia and Africa, where hepatitis virus infection and exposure to specific liver carcinogens are prevalent. Although inactivation of some tumor suppressor genes such as p53 and p16INK4Ahas been identified, no known oncogene is commonly activated in hepatocellular carcinomas. Here we have isolated genes overexpressed in hepatocellular carcinomas by cDNA subtractive hybridization, and identified an oncoprotein consisting of six ankyrin repeats (gankyrin). The expression of gankyrin was increased in ALL 34 hepatocellular carcinomas studied. Gankyrin induced anchorage-independent growth and tumorigenicity in NIH/3T3 cells. Gankyrin bound to the product of the retinoblastoma gene (RB1), increasing its phosphorylation and releasing the activity of the transcription factor E2F-1. Gankyrin accelerated the degradation of RB1 in vitro and in vivo, and was identical to or interacted with a subunit of the 26S proteasome. These results demonstrate the importance of ubiquitin-proteasome pathway in the regulation of cell growth and oncogenic transformation, and indicate that gankyrin overexpression contributes to hepatocarcinogenesis by destabilizing RB1.

The oncoprotein gankyrin plays a central role in tumorigenesis and cell proliferation. Gankyrin interacts with the retinoblastoma tumor suppressor (Rb) and cyclin-dependent kinase 4/6 (CDK4/6), increases phosphorylation at specific residues of Rb by CDK4/6 in vivo, and promotes tumorigenesis. The phosphorylation of Rb by CDK4/6 leads to the deregulation of the cell cycle during G1/S transition. Although how phosphorylation occurs on Rb has been studied extensively, the mechanism of site-specific phosphorylation of Rb remains unclear due to a lack of information on the structural arrangement of Rb and CDK4/6. Here, we have determined and refined to 2.3-A resolution the crystal structure of a gankyrin homolog, the non-ATPase subunit 6 (Nas6p) of the proteasome from yeast. The crystal structure reveals that Nas6p contains seven ankyrin repeats. The number of the repeats is different from that predicted from the primary structure. Nas6p also possesses an unusual curved structure with two acidic regions at the N- and C-terminal regions separated by one basic region, suggesting that it has at least two functional surfaces. The tertiary structure of Nas6p, together with the previous biochemical studies, indicates that the CDK4/6 and Rb binding surfaces of gankyrin are located at the N- and C-terminal regions, respectively, and face the same side of gankyrin. These observations suggest that gankyrin brings Rb and CDK4/6 together through gankyrin-Rb and gankyrin-CDK4/6 interactions and determines the relative positioning of the substrate (Rb) and the enzyme (CDK4/6). Our findings provide mechanistic insight into site-specific phosphorylation of Rb caused by CDK4/6.

BACKGROUND: Aberrance of retinoblastoma protein (RB) signal pathway is known to play an important role in the carcinogenesis of human hepatocellular carcinoma (HCC). p28GANK, originally purified from human 26S proteasome as a non-ATPase subunit, was recently found in HCC and shown to interact with RB. The aim of this study was to investigate the expression profile of p28GANK and its correlation with RB in HCC. METHODS: The expression of p28GANK was evaluated in 55 surgically resected HCCs by immunohistochemistry (IHC), and the associations were explored between p28GANK level and clinicopathologic features as well as tumor suppressor RB. Western blotting was performed to determine p28GANK expression level in 12 HCCs. Immunofluorescence stainings of p28GANK and RB in U2-OS cells were examined by confocal microscopy. RESULTS: Positive p28GANK cytoplasmic staining was recognized in 55 HCCs. Nuclear positive occurrence of p28(GANK) in HCCs was more frequent than paracancerous hepatic tissues (P < 0.05). The overexpression probability of p28GANK was inversely associated with Edmonson s grade: overexpression occurred in nine out of 11 (81.8%), 22 out of 35 (62.9%) and two out of nine (22.2%) in I-II, III and IV graded cases, respectively (P = 0.004). Total cellular expression of p28GANK had curvilinear correlation with the nuclear expression of RB (r = 0.475, P = 0.019), while the nuclear expression of p28GANK had not. Western blot analysis showed that up-regulation of p28GANK expression was found in nine out of 12 HCCs compared with paracancerous liver tissues. Exogenously expressed p28GANK colocalized with RB in cytoplasm of U2-OS cells. CONCLUSIONS: These results confirm the role of p28GANK as a highly expressed oncoprotein in HCC by in situ examination. Its overexpression correlates with the differentiation status of HCC. The whole cellular p28GANK activation, not nuclear portion only, influences the alteration of RB. Underlying nuclear translocation of p28GANK may contribute to the counteraction against RB through a feed back loop. These data provide new evidence for p28GANK to be used as a promising drug target of a therapeutic agent against HCC.

BACKGROUND & AIMS: RNA interference (RNAi) is a powerful tool to silence gene expression. The adenoviral vector expressing small interfering RNA (siRNA) is highly effective in mammalian cells. However, its potential use as a therapeutic tool to target an oncogene specifically remains to be seen. We applied the adenovirus-delivered siRNA (AdSiRNA) to inhibit a hepatocellular carcinoma (HCC) oncogene, p28GANK, in HCC cell lines and investigated its antitumor effects. METHODS: The T7-RNA polymerase system was used to screen the specific target site. Double-strand oligonucleotide for transcription of short hairpin RNA was constructed into the adenoviral vector. Four HCC cell lines were infected with the RNAi-containing adenovirus. The RNAi effects on HCC were studied in cultured cells as well as in animal models. RESULTS: p28GANK expression was suppressed by up to 80% in HCC cells. Depletion of p28GANK inhibited HCC cell growth and tumorigenesis, enhanced dephosphorylation of RB1, and decreased transcription activity of E2F-1 in HuH-7 cells. Furthermore, depletion of p28GANK induced caspase-8- and caspase-9-mediated apoptosis of HCC cells. Finally, targeting p28GANK by adenovirus injection inhibited the growth of established tumors in nude mice. CONCLUSIONS: This study shows that the T7-system screening-based AdSiRNA can be used successfully to silence an oncogene. We proved the therapeutic potential of AdSiRNA on the treatment of HCC by targeting p28GANK. Our results indicate that p28GANK may serve as a novel therapeutic target for treating HCC.

Ubiquitin-dependent proteolysis mediates selective destruction of various cell cycle regulators, transcription factors and tumor suppressors. Gankyrin, a seven ankyrin-repeat protein, was originally identified as an oncoprotein commonly overexpressed in hepatocellular carcinomas and independently as a protein associated with the 19S regulatory complex of the 26S proteasome. Gankyrin also binds to CDK4 and the tumor suppressor RB, and accelerates phosphorylation and proteasomal degradation of RB. Recently, we have shown that gankyrin has an anti-apoptotic activity in cells exposed to DNA-damaging agents. Gankyrin binds to MDM2, a major E3 ubiquitin ligase for p53, and increases ubiquitylation and degradation of p53. Gankyrin increases activities of CDK4 and MDM2, and facilitates targeting of polyubiquitylated proteins to the 26S proteasome. Furthermore, inhibition of gankyrin induces apoptosis in cancer cells. Therefore, gankyrin is a promising target for potential anticancer therapeutic agents.

Gankyrin is an oncoprotein commonly overexpressed in most hepatocellular carcinomas. Gankyrin interacts with S6 ATPase of the 19S regulatory particle of the 26S proteasome and enhances the degradation of the tumor suppressors pRb and p53. Here, we report the structure of gankyrin in complex with the C-terminal domain of S6 ATPase. Almost ALL of the seven ankyrin repeats of gankyrin interact, through its concave region, with the C-terminal domain of S6 ATPase. The intermolecular interactions occur through the complementary charged residues between gankyrin and S6 ATPase. Biochemical studies based on the structure of the complex revealed that gankyrin interacts with pRb in both the presence and absence of S6 ATPase; however, the E182 residue in gankyrin is essential for the pRb interaction. These results provide a structural basis for the involvement of gankyrin in the pRb degradation pathway, through its association with S6 ATPase of the 26S proteasome.

To elucidate the possible involvement of gankyrin in ESCC progression and the effect of its down-regulation in ESCC, we investigated the expression of gankyrin in ESCC tissues comparing it with the corresponding normal esophageal epithelia and tested a short-hairpin RNA (shRNA) expression vector for gankyrin in ESCC cell lines. Gankyrin protein expression in 11 ESCC cell lines (KYSE series) was examined by RT-PCR and western blot. The expression of gankyrin mRNA in 30 ESCC tissues was compared with the corresponding normal epithelia by Real-time PCR. expression of gankyrin protein was immunohistochemically analyzed in the ESCC of 103 patients. A gankyrin-shRNA vector was stably transfected into KYSE 170 cells to assess the role of gankyrin in cell motility, invasion and proliferation in vitro and tumor formation in vivo. Gankyrin expression increased in ALL 11 ESCC cell lines. Real-time PCR revealed that gankyrin expression was higher in the cancerous tissue for ALL 30 patients. In immunohistochemistry, gankyrin overexpression was correlated with lower survival rate (p = 0.0001), extent of the primary tumor, lymph node metastasis, distant lymph node metastasis and stage (p = 0.0072, p = 0.0004, p = 0.0172 and p = 0.0002, respectively). A shRNA vector against gankyrin repressed growth, cell motility, invasiveness in vitro and tumor formation in vivo. Gankyrin overexpression is associated with poor prognosis. It may play an important role in ESCC tumor progression and could be a potentially important therapeutic gene target in ESCC.

A single heat shock factor (HSF), mediating the heat shock response, exists from yeast to Drosophila, whereas several related HSFs have been found in mammals. This raises the question of the specific or redundant functions of the different members of the HSF family and in particular of HSF1 and HSF2, which are both ubiquitously expressed. Using immortalized mouse embryonic fibroblasts (iMEFs) derived from wild-type, Hsf1(-/-), Hsf2(-/-) or double-mutant mice, we observed the distinctive behaviors of these mutants with respect to proteasome inhibition. This proteotoxic stress reduces to the same extent the viability of Hsf1(-/-)- and Hsf2(-/-)-deficient cells, but through different underlying mechanisms. Contrary to Hsf2(-/-) cells, Hsf1(-/-) cells are unable to induce pro-survival heat shock protein expression. Conversely, proteasome activity is lower in Hsf2(-/-) cells and the expression of some proteasome subunits, such as Psmb5 and gankyrin, is decreased. As gankyrin is an oncoprotein involved in p53 degradation, we analyzed the status of p53 in HSF-deficient iMEFs and observed that it was strongly stabilized in Hsf2(-/-) cells. This study points a new role for HSF2 in the regulation of protein degradation and suggests that pan-HSF inhibitors could be valuable tools to reduce chemoresistance to proteasome inhibition observed in cancer therapy.

p28(GANK) (also known as PSMD10 or gankyrin) is a novel oncoprotein that is highly expressed in hepatocellular carcinoma (HCC). Through its interaction with various proteins, p28(GANK) mediates the degradation of the tumor suppressor proteins Rb and p53. Although p53 was reported to downregulate beta-catenin, whether p28(GANK) is involved in the regulation of beta-catenin remains uncertain. Here we report that both growth factors and Ras upregulate p28(GANK) expression through the activation of the phosphoinositide 3-kinase-AKT pathway. Upregulation of p28(GANK) expression subsequently enhanced the transcription activity of beta-catenin. This effect was observed in p53-deficient cells, suggesting a p53-independent mechanism for the p28(GANK)-mediated activation of beta-catenin. p28(GANK) overexpression also reduced E-cadherin protein levels, leading to increased release of free beta-catenin into the cytoplasm from the cadherin-bound pool. Interestingly, exogenous expression of p28(GANK) resulted in elevated expression of the endogenous protein. We also observed that both beta-catenin and c-Myc were transcriptional activators of p28(GANK), and a correlation between p28(GANK) overexpression and c-Myc, cyclin D1 and beta-catenin activation in primary human HCC. Together, these results suggest that p28(GANK) expression is regulated by a positive feedback loop involving beta-catenin, which may play a critical role in tumorigenesis and the progression of HCC.

OBJECTIVE: The oncoprotein, gankyrin, is known to facilitate cell proliferation through phosphorylation and degradation of retinoblastoma protein. In the present study, we evaluated the expression of gankyrin and phosphorylated retinoblastoma protein in human testis and testicular germ cell tumors. METHODS: The effects of suppression of gankyrin by locked nucleic acid on phosphorylation status of retinoblastoma and cell proliferation were analyzed using western blot analysis and testicular tumor cell line NEC8. The expressions of gankyrin, retinoblastoma and retinoblastoma protein were analyzed in 93 testicular germ cell tumor samples and five normal human testis by immunohistochemistry. The retinoblastoma protein expression was determined using an antibody to retinoblastoma protein, Ser795. RESULTS: Gankyrin was expressed in NEC8 cells as well as a normal human testis and testicular tumors. Suppression of gankyrin by locked nucleic acid led to suppression of retinoblastoma protein and cell proliferation in NEC8 cells. Immunohistochemistry of normal testis showed that gankyrin is expressed dominantly in spermatocytes. In testicular germ cell tumors, high expressions of gankyrin and phosphorylated-retinoblastoma protein were observed in seminoma and embryonal carcinoma, whereas the expressions of both proteins were weak in histological subtypes of non-seminoma. Growing teratoma and testicular malignant transformation tissues expressed phosphorylated-retinoblastoma protein strongly, but gankyrin faintly. CONCLUSION: Gankyrin is dominantly expressed in normal spermatocytes and seminoma/embryonal carcinoma, and its expression correlates well with retinoblastoma protein expression except in the growing teratoma and testicular malignant transformation cases. These data provide new insights into the molecular mechanisms of normal spermatogenesis and pathogenesis of testicular germ cell tumors.

Increased cellular levels of protein-protein interactions involving the ankyrin repeat oncoprotein gankyrin are directly linked to aberrant cellular events and numerous cancers. Inhibition of these protein-protein interactions is thus an attractive therapeutic strategy. However, the relatively featureless topology of gankyrin s putative binding face and large surface areas involved in gankyrin-dependent protein-protein interactions present a dramatic challenge to small molecule discovery. The size, high folding energies, and well-defined surfaces present in many proteins overcome some of the challenges faced by small molecule discovery. We used split-superpositive Green Fluorescent Protein (split-spGFP) reassembly to screen a 5x10(9) library of resurfaced proteins that are shape complementary to the putative binding face of gankyrin and identified mutants that potently and selectively bind this oncoprotein in vitro and in living cells. Collectively, our findings represent the first synthetic proteins that bind gankyrin and may represent a general strategy for developing protein basic research tools and drug leads that bind disease-relevant ankyrin repeats.

A complex with the C-terminal portion of the proteosomal subunit S6 ATPase is the only available structure of a protein-protein interaction involving the oncoprotein gankyrin. However, difficulties associated with recombinant expression of S6 ATPase alone, or truncations thereof, have limited our understanding of this assembly. We replaced the C-terminal portion of FtsH from Escherichia coli with the structurally homologous C-terminal portion of S6 ATPase and used this grafted protein to characterize the gankyrin-S6 ATPase binding interaction by isothermal titration calorimetry.

Synthesis of peptides corresponding to the 201-210 and 200-206 sequences of p28sis oncoprotein is reported. This region of the oncoprotein was chosen as a probable epitope basing on the analysis of its secondary structure and hydrophilicity profile. The synthesis was carried out by classical method in solution using side-protecting groups of the benzyl type. Conjugates of the peptides with protein carriers and polyclonal antibodies to the peptides were obtained. Antiserum against the 201-210 peptide conjugate is shown to be specific to p28sis oncoprotein and its p56sis dimer.