Optimizing the aryl-triazole of cjoc42 for enhanced gankyrin binding and anti-cancer activity

Gankyrin is an oncoprotein overexpressed in numerous cancer types and appears to play a key role in regulating cell proliferation, cell growth, and cell migration. These roles are largely due to gankyrin’s protein-protein interaction with the 26S proteasome. We previously published a study exploring the aryl sulfonate ester of cjoc42 in an effort to enhance gankyrin binding and inhibit cancer cell proliferation. In order to further improve the gankyrin binding ability of the cjoc42 scaffold, an extensive SAR for the aryl-triazole moiety of cjoc42 was developed. Our cjoc42 derivatives exhibited enhanced gankyrin binding, as well as enhanced antiproliferative activity against Hep3B, HepG2, A549, and MDA-MB-231 cancer cell lines.     

Overexpression of gankyrin induces liver steatosis in zebrafish (Danio rerio)

Gankyrin is a small ankyrin-repeat protein that previous research has confirmed to be overexpressed in hepatocellular carcinoma (HCC). Although relevant literature has reported on gankyrin functions in cellular proliferation and tumorigenesis, the exact role of gankyrin is poorly understood in animal model systems. This study analyzed hepatic lipid accumulation in gankyrin transgenic (GK) zebrafish. Bromodeoxyuridine (BrdU)-positive cells were predominantly increased in the liver bud of GK larvae, indicating that gankyrin functionally promoted cell proliferation at the larval stage in GK fish. However, over 90% of the viable GK adults showed an increased lipid content, leading in turn to liver steatosis. Liver histology and oil red O staining also indicated the accumulation of fatty droplets in GK fish, consistent with the specific pathological features of severe steatosis. Molecular analysis revealed that gankyrin overexpression induced hepatic steatosis and modulated the expression profiles of four hepatic microRNAs, miR-16, miR-27b, miR-122, and miR-126, and 22 genes involved in lipid metabolism. Moreover, significantly increased hepatic cell apoptosis resulted in liver damage in GK adults, leading to liver failure and death after approximately 10months. This study is the first to report gankyrin as a potential link between microRNAs and liver steatosis in zebrafish.     

Novel insights into the INK4-CDK4/6-Rb pathway: counter action of gankyrin against INK4 proteins regulates the CDK4-mediated phosphorylation of Rb

The newly discovered oncogenic protein gankyrin, which contains six ankyrin repeats, has been reported to be involved in the phosphorylation and degradation of the retinoblastoma gene product, Rb. Using in vitro systems, we have identified a peptide fragment of gankyrin, 176LHLACDEERN185, which is responsible for binding of gankyrin to Rb. We further demonstrated a different mechanism for gankyrin to facilitate the phosphorylation of Rb, by binding with cyclin-dependent kinase 4 (CDK4). This binding does not inhibit the Rb-phosphorylating kinase activity of CDK4, but it competes with p16 binding to CDK4 and counteracts the inhibitory function of p16. We then showed that binding of gankyrin to CDK4 and the consequent counter action of p16 function were not affected by the Rb-binding peptide 176LHLACDEERN185, indicating that the two mechanisms are independent. They also involve different structural regions of gankyrin. While the Rb-binding motif is located at the fifth ankyrin repeat, truncation mutants of gankyrin, with the first three or four ankyrin repeats remaining, are sufficient for binding to CDK4 and for counteracting the inhibitory function of p16. These results demonstrate the potential importance of gankyrin in cell cycle control and tumorigenesis and suggest an expanded INK4-CDK4/6-Rb pathway.     

Discovery of multiple interacting partners of gankyrin,a proteasomal chaperone and an oncoprotein—Evidence for a common hot spot site at the interface and its functional relevance

Gankyrin, a non‐ATPase component of the proteasome and a chaperone of proteasome assembly, is also an oncoprotein. Gankyrin regulates a variety of oncogenic signaling pathways in cancer cells and accelerates degradation of tumor suppressor proteins p53 and Rb. Therefore gankyrin may be a unique hub integrating signaling networks with the degradation pathway. To identify new interactions that may be crucial in consolidating its role as an oncogenic hub, crystal structure of gankyrin‐proteasome ATPase complex was used to predict novel interacting partners. EEVD, a four amino acid linear sequence seems a hot spot site at this interface. By searching for EEVD in exposed regions of human proteins in PDB database, we predicted 34 novel interactions. Eight proteins were tested and seven of them were found to interact with gankyrin. Affinity of four interactions is high enough for endogenous detection. Others require gankyrin overexpression in HEK 293 cells or occur endogenously in breast cancer cell line‐ MDA‐MB‐435, reflecting lower affinity or presence of a deregulated network. Mutagenesis and peptide inhibition confirm that EEVD is the common hot spot site at these interfaces and therefore a potential polypharmacological drug target. In MDA‐MB‐231 cells in which the endogenous CLIC1 is silenced, trans‐expression of Wt protein (CLIC1_EEVD) and not the hot spot site mutant (CLIC1_AAVA) resulted in significant rescue of the migratory potential. Our approach can be extended to identify novel functionally relevant protein‐protein interactions, in expansion of oncogenic networks and in identifying potential therapeutic targets. Proteins 2014; 82:1283–1300. © 2013 Wiley Periodicals, Inc.     

Gankyrin is an ankyrin-repeat oncoprotein that interacts with CDK4 kinase and the S6 ATPase of the 26 S proteasome.

A yeast two-hybrid screen with the human S6 (TBP7, RPT3) ATPase of the 26 S proteasome has identified gankyrin, a liver oncoprotein, as an interacting protein. Gankyrin interacts with both free and regulatory complex-associated S6 ATPase and is not stably associated with the 26 S particle. Deletional mutagenesis shows that the C-terminal 78 amino acids of the S6 ATPase are necessary and sufficient to mediate the interaction with gankyrin. Deletion of an orthologous gene in Saccharomyces cerevisiae suggests that it is dispensable for cell growth and viability. Overexpression and precipitation of tagged gankyrin from cultured cells detects a complex containing co-transfected tagged S6 ATPase (or endogenous S6) and endogenous cyclin D-dependent kinase CDK4. The proteasomal ATPases are part of the AAA (ATPases associated with diverse cellular activities) family, members of which are molecular chaperones; gankyrin complexes may therefore influence CDK4 function during oncogenesis.     

Sclerotium rolfsii Lectin Induces Stronger Inhibition of Proliferation in Human Breast Cancer Cells than Normal Human Mammary Epithelial Cells by Induction of Cell Apoptosis

Sclerotium rolfsii lectin (SRL) isolated from the phytopathogenic fungus Sclerotium rolfsii has exquisite binding specificity towards O-linked, Thomsen-Freidenreich (Galβ1-3GalNAcα1-Ser/Thr, TF) associated glycans. This study investigated the influence of SRL on proliferation of human breast cancer cells (MCF-7 and ZR-75), non-tumorigenic breast epithelial cells (MCF-10A) and normal mammary epithelial cells (HMECs). SRL caused marked, dose-dependent, inhibition of proliferation of MCF-7 and ZR-75 cells but only weak inhibition of proliferation of non-tumorigenic MCF-10A and HMEC cells. The inhibitory effect of SRL on cancer cell proliferation was shown to be a consequence of SRL cell surface binding and subsequent induction of cellular apoptosis, an effect that was largely prevented by the presence of inhibitors against caspases -3, -8, or -9. Lectin histochemistry using biotin-labelled SRL showed little binding of SRL to normal human breast tissue but intense binding to cancerous tissues. In conclusion, SRL inhibits the growth of human breast cancer cells via induction of cell apoptosis but has substantially less effect on normal epithelial cells. As a lectin that binds specifically to a cancer-associated glycan, has potential to be developed as an anti-cancer agent.     

Regulation and function of the TAZ transcription co-activator

TAZ (WWTR1), identified as a 14-3-3 binding protein with a PDZ binding motif, is implicated in mesenchymal stem cell differentiation. TAZ has been shown to be negatively regulated by phosphorylation–dependent and phosphorylation-independent mechanisms. Coupled with ASPP2, PP1 dephosphorylates TAZ to activate TAZ. TEADs mediate TAZ function in promoting cell proliferation and epithelial-mesenchymal transition (EMT). TAZ senses different cellular signals such as cell density and the extracellular matrix stiffness. Significantly, TAZ is overexpressed in breast cancer samples and papillary thyroid carcinoma tissues. These results indicate that TAZ plays an important role in cancer development and presents a novel target for TAZ overexpressed cancer therapy.     

Role of the insulin-like growth factor system on an estrogen-dependent cancer phenotype in the MCF-7 human breast cancer cell line

We previously established that exposure of the estrogen receptor (ER) positive MCF-7 human breast cancer cell line to 17-β-estradiol (E₂) results in the post-confluent development of multilayered cellular aggregates (foci) which is consistent with the in vivo cancer phenotype of uncontrolled cellular proliferation. In this investigation, the interaction between the insulin-like growth factor receptor (IGF-IR) and ER-signaling systems in regard to post-confluent focus development was studied. We demonstrated that focus development requires the presence of E₂ and insulin-like growth factor I (IGF-I) or insulin-like growth factor II (IGF-II), as well as intact ER and IGF-IR.

Focus development in MCF-7 cultures, which occurs only after formation of a confluent monolayer, coincides with E₂ regulation of key members of the IGF-signaling system such as IGF-IR, IGF-II, insulin receptor substrate 1 (IRS-1), and insulin-like growth factor binding protein 3 (IGFBP-3), as demonstrated by real-time polymerase chain reaction (PCR). To establish the relevancy of an intact IGF-signaling system for foci formation, we generated stable clones from MCF-7 with IGF-IR suppressed by siRNA. Results from these studies implicate signaling through the IGF-IR to be an integral requirement for E₂-dependent post-confluent proliferation and focus formation. In summary, these studies establish the interactive roles of IGFs and E₂ in the post-confluent development of foci, and will allow subsequent identification of targets for therapeutic intervention in the control and treatment of estrogen-dependent breast cancer.     

Inhibition of thioredoxin reductase by a novel series of bis-1,2-benzisoselenazol-3(2H)-ones: Organoselenium compounds for cancer therapy

Thioredoxin reductase (TrxR) is critical for cellular redox regulation and is involved in tumor proliferation, apoptosis and metastasis. Its C-terminal redox-active center contains a cysteine (Cys497) and a unique selenocysteine (Sec498), which are exposed to solvent and easily accessible. Thus, it is becoming an important target for anticancer drugs. Selective inhibition of TrxR by 1,2-(bis-1,2-benzisoselenazol-3(2H)-one)ethane (4a) prevents proliferation of several cancer cell lines both in vivo and in vitro. Using the structure of 4a as a starting point, a series of novel bis-1,2-benzisoselenazol-3(2H)-ones was designed, prepared and tested to explore the structure-activity relationships (SARs) for this class of inhibitor and to improve their potency. Notably, 1,2-(5,5'-dimethoxybis(1,2-benzisoselenazol-3(2H)-one))ethane (12) was found to be more potent than 4a in both in vitro and in vivo evaluation. Its binding sites were confirmed by biotin-conjugated iodoacetamide assay and a SAR model was generated to guide further structural modification.     

Interaction of the RAGE cytoplasmic domain with diaphanous-1 is required for ligand-stimulated cellular migration through activation of Rac1 and Cdc42

Cellular migration is a fundamental process linked to diverse pathological states such as diabetes and its complications, atherosclerosis, inflammation, and cancer. The receptor for advanced glycation end products (RAGE) is a multiligand cell surface macromolecule which binds distinct ligands that accumulate in these settings. RAGE-ligand interaction evokes central changes in key biological properties of cells, including proliferation, generation of inflammatory mediators, and migration. Although RAGE-dependent signal transduction is critically dependent on its short cytoplasmic domain, to date the proximate mechanism by which this RAGE domain engages and stimulates cytoplasmic signaling pathways has yet to be identified. Here we show that the RAGE cytoplasmic domain interacts with Diaphanous-1 (Dia-1) both in vitro and in vivo. We employed the human RAGE cytoplasmic domain as "bait" in the yeast two-hybrid assay and identified the formin homology (FH1) domain of Dia-1 as a potential binding partner of this RAGE domain. Immunoprecipitation studies revealed that the RAGE cytoplasmic domain interacts with the FH1 domain of Dia-1. Down-regulation of Dia-1 expression by RNA interference blocks RAGE-mediated activation of Rac-1 and Cdc42 and, in parallel, RAGE ligand-stimulated cellular migration. Taken together, these findings indicate that the interaction of the RAGE cytoplasmic domain with Dia-1 is required to transduce extracellular environmental cues evoked by binding of RAGE ligands to their cell surface receptor, a chief consequence of which is Rac-1 and Cdc42 activation and cellular migration. Because RAGE and Dia-1 are implicated in the regulation of inflammatory, vascular, and transformed cell migration, these findings highlight this interaction as a novel target for therapeutic intervention in inflammation, atherosclerosis, diabetes, and cancer.     

Gankyrin activates the hedgehog signalling to drive metastasis in osteosarcoma

Gankyrin is a regulatory subunit of the 26-kD proteasome complex and promotes the occurrence and progression of many malignancies. However, the role of gankyrin in osteosarcoma (OS) metastasis remains unclear. Hedgehog signalling has been shown to regulate stem cell homeostasis and cancer metastasis, but the mechanisms that activate this pathway in OS are still poorly understood. Here, a series of in vitro and in vivo assays were carried out to explore the function and mechanism of gankyrin regulating Hedgehog signalling in OS. We demonstrated that gankyrin promotes migration, invasion and regulates the expression of some stemness factors by up-regulating Gli1 in OS. Importantly, our data showed an interaction between gankyrin and Gli1. Moreover, gankyrin suppresses the ubiquitin-mediated degradation of Gli1 protein in OS. Gankyrin also significantly promotes the lung metastasis of OS in vivo. Our findings suggest that gankyrin drives metastasis and regulates the expression of some stemness factors in osteosarcoma by activating Hedgehog signalling, indicating that drug screening for compounds targeting gankyrin may contribute to the development of novel and effective therapies for OS.     

MicroRNA-490-3p inhibits proliferation of A549 lung cancer cells by targeting CCND1

MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate the translation of messenger RNAs by binding their 3′-untranslated region (3′UTR). In this study, we found that miR-490-3p is significantly down-regulated in A549 lung cancer cells compared with the normal bronchial epithelial cell line. To better characterize the role of miR-490-3p in A549 cells, we performed a gain-of-function analysis by transfecting the A549 cells with chemically synthesized miR-490-3P mimics. Overexpression of miR-490-3P evidently inhibits cell proliferation via G1-phase arrest. We also found that forced expression of miR-490-3P decreased both mRNA and protein levels of CCND1, which plays a key role in G1/S phase transition. In addition, the dual-luciferase reporter assays indicated that miR-490-3P directly targets CCND1 through binding its 3′UTR. These findings indicated miR-490-3P could be a potential suppressor of cellular proliferation.     

Solution structure of the human oncogenic protein gankyrin containing seven ankyrin repeats and analysis of its structure--function relationship

Human gankyrin (226 residues, 24.4 kDa) is a liver oncoprotein that plays an important role in the development of human hepatocellular carcinomas. In this paper, its solution structure is reported, which is the largest ankyrin protein ever determined by NMR. The highly degenerate primary sequences of the seven ankyrin repeats presented a major challenge, which was overcome by combined use of TROSY experiments, perdeuterated samples, isotope-filtered NMR experiments, and residual dipolar couplings. The final structure was of high quality, with atomic rmsds for the backbone (N, C', and C(alpha)) and all heavy atoms (residues 4-224) of 0.69 +/- 0.09 and 1.04 +/- 0.09 A, respectively. Detailed analyses of NMR data suggested that the conserved TPLH motifs play important structural roles in stabilizing the repeating ankyrin scaffold. Gankyrin is conformationally more stable than the tumor suppressor p16(INK4A), possibly due to the structural roles of conserved residues evidenced by slowly exchanging backbone amides as well as hydrogen bonding networks involving labile side chain protons. Structural comparison with p16(INK4A) identified several residues of gankyrin that are potentially important for CDK4 binding, whereas observation of the thiol proton of C180 indicated a well-structured Rb-binding site in the helical region of the sixth ankyrin repeat. Interestingly, the CDK4-binding site and Rb-binding site located in N- and C-terminal regions, respectively, are separated by comparatively more stable ankyrin repeats and highly condensed positive surface charge. These results and analyses will shed light on the structural basis of the function of human gankyrin.     

Role of insulin and IGF1 receptors in proliferation of cultured renal proximal tubule cells.

We have used a murine proximal tubule cell line (MCT cells) to determine the presence and binding characteristics of insulin and IGF1 receptors and to correlate these parameters with the concentration-response relationships for ligand-induced cellular proliferation. Separate insulin and IGF1 receptors were identified by equilibrium binding assays. Half-maximal displacement of either peptide occurred at 3-10 nM; crossover binding to the alternate receptor occurred with a 10- to 100-fold lower affinity. Peptide effects on cellular proliferation were determined by measuring [3H]thymidine incorporation. Both insulin and IGF1 stimulate thymidine incorporation in a dose-dependent manner with similar increases above the basal level. The estimated half-maximal stimulation (EC50) occurred at 4 nM for IGF1 and 8 nM for insulin. A comparison of the receptor binding affinities with the dose-response relationships for [3H]thymidine incorporation reveals that each growth factor appears to be exerting its effect via binding to its own receptor. Therefore, in this cell line, physiologic concentrations of either insulin or IGF1 can modulate cellular growth. To our knowledge this is the first demonstration of a mitogenic effect which may be modulated by ligand binding to the insulin receptor in proximal tubule epithelia.     

Regulation of circGOLPH3 and its binding protein CBX7 on the proliferation and apoptosis of prostate cancer cells

To clarify the mechanism of circGOLPH3 regulation on prostate cancer cells, we performed an overexpression and interference circGOLPH3 assay in prostate cancer cells PC-3 and then evaluated cellular viability, proliferation, cell cycle, and apoptosis of prostate cancer cells by MTT, CCK8, Edu stain, TUNEL stain, and flow cytometry. Binding proteins of CircGOLPH3 were identified by RNA pull-down, mass spectrometry, and RNA-binding protein immunoprecipitation (RIP) assays. The expressions of CircGOLPH3 and CBX7 were measured by qRT-PCR. The results showed that after overexpression of circGOLPH3, the proliferative capacity and the viability of PC-3cells were significantly improved, whereas apoptosis was inhibited. CircGOLPH3 could bind to the CBX7 protein that was highly expressed in the PC-3 cell. Additionally, a functional test on CBX7 showed that the CBX7 overexpression notably improved the proliferative capacity and the viability of PC-3 cells and decreased cellular apoptosis, which was consistent with the effects of circGOLPH3. The validated the present study that circGOLPH3 and its binding protein CBX7 can promote prostate cancer cell proliferation and inhibit apoptosis.