Structural modification of the aryl sulfonate ester of cjoc42 for enhanced gankyrin binding and anti-cancer activity

Gankyrin is an oncogenic protein involved in various biological processes, such as cellular growth and proliferation. Its overexpression in certain cancers results in an increase of gankyrin-mediated protein-protein interactions (PPIs), leading to cancer proliferation. To date, only one small molecule (cjoc42) has been identified to bind gankyrin, which simultaneously inhibits its interaction with the 26S proteasome. Despite this advance, 2nd generation inhibitors are needed to improve gankyrin binding and cellular efficacy. To this end, an extensive SAR for the aryl sulfonate ester moiety of the cjoc42 scaffold was explored, and showed that substitutions at the 2-, 3-, and 4-positions manifested significant increases in gankyrin binding, resulting in the most potent binders of gankyrin to date. Subsequent cell-based assay evaluation of our derivatives demonstrated antiproliferative activity against pediatric liver cancer cell lines Hep3B and HepG2, which was not previously observed for cjoc42.     

NDRG1 regulates Filopodia-induced Colorectal Cancer invasiveness via modulating CDC42 activity

N-myc downstream regulated gene-1 (NDRG1) has been identified as a putative metastasis suppressor gene and proved to be a key player in cancer spreading and proliferation in our previous work. However, the effects of NDRG1 on tumor invasion and the mechanisms behind it are rarely understood. Here we provided in silico evidence that NDRG1 plays a crucial role in actin reorganization in colorectal cancer (CRC). Through in vitro experiments, we next observed filopodia formation was altered in NDRG1-modified cell lines, while cell division cycle-42 (CDC42) displayed excessive activation in NDRG1-silenced cells. Mechanistically, NDRG1 loss disrupts the binding between RhoGDIα and CDC42 and triggers the activation of CDC42 and the downstream cascades PAK1/Cofilin, thereby promotes the formation of filopodia and invasiveness of CRC. The knockdown of NDRG1 led to enhanced dissemination of CRC cells in vivo and correlates with active CDC42 expression. Using clinical sample analysis, we found an elevated level of active CDC42 in patients with advanced T stage, and it was negatively related to NDRG1 expression. In sum, these results uncover a mechanism utilized by NDRG1 to regulate CDC42 activity in coordinating cytoskeleton reorganization, which was crucial in cancer invasion.     

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.     

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.     

Crystal structure of the homolog of the oncoprotein gankyrin, an interactor of Rb and CDK4/6

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.     

Laminin 511-E8 Fragment Offers Superior Adhesion Properties for Gastric Cancer Cells Compared with Full-Length Laminin 511

Simple SummaryNumerous studies over the past few decades have revealed that the interactions of gastric cancer cells with laminins through integrins play important roles in tumor cell proliferation, infiltration, and metastasis. However, the association between gastric cancer cells and the laminin E8 fragment, which is the smallest integrin-binding component, has not been investigated. In this study, we revealed that the laminin 511-E8 fragment had a greater impact on the adhesion, morphology, and proliferation of gastric cancer cells than full-length laminin 511. Thus, the laminin 511-E8 fragment is considered to be suitable for investigating the interaction between gastric cancer cells and extracellular matrices in tumor invasion and metastasis. Further, the involvement of Cdc42 in the laminin 511-E8 fragment-induced enhanced adhesion of gastric cancer cells was suggested.AbstractBackground: The interaction between cancer cells and laminin (Ln) is a key event in tumor invasion and metastasis. Previously, we determined the effect of full-length Ln511 on gastric cancer cells. However, the interactions between the Ln511-E8 fragment, a truncated protein of Ln511, and gastric cancer cells have not been investigated. Methods: We investigated the adhesion properties of gastric cancer cells to full-length Ln511 and Ln511-E8 fragments. Results: The proliferation of four gastric cancer cell lines (SH-10-TC, MKN74, SC-6-JCK, and MKN45) was highest on the Ln511-E8 fragment. Further, a larger cytoplasm was observed in SH-10-TC and MKN74 cells cultured on full-length Ln511 or Ln511-E8 fragments. The percentage of adhesive cells was highest on the Ln511-E8 fragment in all four cell lines. Moreover, adhesion of the gastric cancer cells to Ln511-E8 fragment-coated plates was reduced by the Cdc42 GTPase inhibitor in a dose-dependent manner, suggesting the involvement of Cdc42 in the Ln511-E8 fragment-induced enhanced adhesion of gastric cancer cells. Conclusions: The Ln511-E8 fragment had a greater impact on the adhesion, morphology, and proliferation of gastric cancer cells than full-length laminin. Thus, the Ln511-E8 fragment is suitable for investigating the interaction between gastric cancer cells and extracellular matrices in tumor invasion and metastasis.     

Gankyrin promotes osteosarcoma tumorigenesis by forming a positive feedback loop with YAP

BackgroundAlthough gankyrin has been identified as a vital regulator of tumorigenesis, its role and regulatory mechanism in osteosarcoma (OS) remain unclear.MethodsQRT-PCR, western blot and IHC staining were conducted to detect the expression of gankyrin in OS. Pearson’s χ² test was adopted to examine the associations between gankyrin expression and clinicopathologic characteristics. Kaplan-Meier method was used to investigate the relationship between gankyrin expression and overall survival of patients with OS. Next, a series of in vitro and in vivo assays were performed to determine the positive feedback loop between gankyrin and YAP in OS.ResultsWe first reported that gankyrin is upregulated in human OS specimens and cell lines and predicts OS progression and poor prognosis. Furthermore, we demonstrated that gankyrin protects miR-200a-mediated yes-associated protein (YAP) downregulation through p53 and establishes a positive feedback loop to regulate YAP signaling in U2OS and MG63 cells. Intriguingly, gankyrin interacts with YAP to promote OS cell growth in vitro. In addition, our results showed that gankyrin promotes OS tumor growth and regulates YAP levels in vivo. Notably, we also observed a positive correlation between gankyrin and YAP expression in human OS tissues, and co-upregulation of gankyrin and YAP indicated a poor prognosis.ConclusionsOur results identify that gankyrin acts as an oncogene in OS by forming a positive feedback loop with YAP, and disrupting the gankyrin-YAP regulation may be beneficial for controlling OS tumorigenesis.     

Expression of the 11beta-hydroxysteroid dehydrogenase type II enzyme in breast tumors and modulation of activity and cell growth in PMC42 cells

Manipulating the metabolism of glucocorticoids may serve as a useful adjunct in the treatment of breast cancer. The 11β-hydroxysteroid dehydrogenase type 2 enzyme (11βHSD2) potently inactivates glucocorticoids thereby protecting the non-selective mineralocorticoid receptor (MR) in fluid transporting tissues. In the present study, Western blot analysis showed the presence of 11βHSD2 in 66% of the breast tumor samples. The 11βHSD2 and MR are also present in the breast tumor cell line PMC42. Glycyrrhetinic acid abolished glucocorticoid metabolism and inhibited cell growth by 40%, the latter at concentrations consistent with glucocorticoid receptor (GR) and MR binding studies. Metabolism was increased by glucocorticoids, the anti-glucocorticoid RU 38486 and anti-mineralocorticoid spironolactone, while aldosterone had no effect. Neither cortisol nor aldosterone affected cell proliferation, but both RU 38486 and spironolactone caused a significant decrease in cell number. The effects of RU 38486 were only observed at micromolar concentrations and are inconsistent with an action via GR or progesterone receptor (PR). This study shows that 11βHSD2 activity and cell proliferation of PMC42 cells can be modulated via steroid receptors.     

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.     

Thyroid hormone is a MAPK-dependent growth factor for thyroid cancer cells and is anti-apoptotic

Thyroid hormone (l-thyroxine, T(4), or 3,5,3'-triiodo-l-thyronine, T(3)) treatment of human papillary and follicular thyroid cancer cell lines resulted in enhanced cell proliferation, measured by proliferating cell nuclear antigen (PCNA). Thyroid hormone also induced activation of the Ras/MAPK (ERK1/2) signal transduction pathway. ERK1/2 activation and cell proliferation caused by thyroid hormone were blocked by an iodothyronine analogue, tetraiodothyroacetic acid (tetrac), that inhibits binding of iodothyronines to the cell surface receptor for thyroid hormone on integrin alphaVbeta3. A MAPK cascade inhibitor at MEK, PD 98059, also blocked hormone-induced cell proliferation. We then assessed the possibility that thyroid hormone is anti-apoptotic. We first established that resveratrol (10 microM), a pro-apoptotic agent in other cancer cells, induced p53-dependent apoptosis and c-fos, c-jun and p21 gene expression in both papillary and follicular thyroid cancer cells. Induction of apoptosis by the stilbene required Ser-15 phosphorylation of p53. Resveratrol-induced gene expression and apoptosis were inhibited more than 50% by physiological concentrations of T(4). T(4) activated MAPK in the absence of resveratrol, caused minimal Ser-15 phosphorylation of p53 and did not affect c-fos, c-jun and p21 mRNA abundance. Thus, plasma membrane-initiated activation of the MAPK cascade by thyroid hormone promotes papillary and follicular thyroid cancer cell proliferation in vitro.     

Erythropoietin enhancement of rat pancreatic tumor cell proliferation requires the activation of ERK and JNK signals

Erythropoietin (EPO) regulates the proliferation and differentiation of erythroid cells by binding to its specific transmembrane receptor EPOR. Recent studies, however, have shown that the EPOR is additionally present in various cancer cells and EPO induces the proliferation of these cells, suggesting a different function for EPO other than erythropoiesis. Therefore, the purpose of the present study was to examine EPOR expression and the role of EPO in the proliferation and signaling cascades involved in this process, using the rat pancreatic tumor cell line AR42J. Our results showed that AR42J cells expressed EPOR, and EPO significantly enhanced their proliferation. Cell cycle analysis of EPO-treated cells indicated an increased percentage of cells in the S phase, whereas cell numbers in G0/G1 phase were significantly reduced. Phosphorylation of extracellular regulatory kinase 1/2 (ERK1/2) and c-Jun NH₂ terminal kinase 1/2 (JNK1/2) was rapidly stimulated and sustained after EPO addition. Treatment of cells with mitogen-activated protein/ERK kinase (MEK) inhibitor PD98059 or JNK inhibitor SP600125 significantly inhibited EPO-enhanced proliferation and also increased the fraction of cells in G0/G1 phase. Furthermore, the inhibition of JNK using small interference RNA (siRNA) suppressed EPO-enhanced proliferation of AR42J cells. Taken together, our results indicate that AR42J cells express EPOR and that the activation of both ERK1/2 and JNK1/2 by EPO is essential in regulating proliferation and the cell cycle. Thus both appear to play a key role in EPO-enhanced proliferation and suggest that the presence of both is required for EPO-mediated proliferation of AR42J cells. erythropoietin receptor; cell signaling; mitogen-activated protein kinase induction     

CD44+ fibroblasts increases breast cancer cell survival and drug resistance via IGF2BP3‐CD44‐IGF2 signalling

CD44, a cell adhesion protein, involves in various process in cancer such as cell survival and metastasis. Most researches on CD44 in cancer focus on cancer cells. Recently, it is found that CD44 expression is high in fibroblasts of tumour microenvironment. However, its role in communication between fibroblasts and breast cancer cells is seldom known. In this study, CD44‐positive (CD44⁺Fbs) and CD44‐negative carcinoma‐associated fibroblasts (CD44^?Fbs) were isolated and cocultured with breast cancer cells for analysis of cell survival and drug resistance. We found that CD44⁺Fbs promoted breast cancer cell survival and paclitaxel resistance and inhibited paclitaxel‐induced apoptosis. Our further research for the molecular mechanism showed that IGF2BP3 bound to CD44 mRNA and enhanced CD44 expression, which increased IGF2 levels of fibroblasts and then stimulated breast cancer cell proliferation and drug resistance. IGF2 was found to activate Hedgehog signal pathway in breast cancer cells. In conclusion, the results illustrated that in CD44⁺Fbs, binding of IGF2BP3 and CD44 promotes IGF2 expression and then accelerates breast cancer cell proliferation, survival and induced chemotherapy resistance likely by activating Hedgehog signal pathways.     

Delivery of BR2‐SOX17 fusion protein can inhibit cell survival,proliferation, and invasion in gastric cancer cells through regulating Klotho gene expression

The prognosis of advanced gastric cancer is poor and understanding the biology and subsequent development of new targeting therapy is still an urgent need. This study was conducted to explore the effect of BR2 (a 17‐amino acid peptide)‐SOX17 (human sex determining region Y (SRY)‐related high‐mobility group (HMG) box protein family member 17) fusion protein on Klotho gene expression in gastric cancer cells. The regulatory effects of SOX17 on Klotho gene in gastric cancer cells were tested using dual‐luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay. The therapeutic effects of BR2‐SOX17 were evaluated by proliferation, colony formation, invasion assay, and cell apoptosis analysis. Results showed that SOX17 enhanced Klotho gene expression in gastric adenocarcinoma cells through binding to the promoter of Klotho gene. BR2‐SOX17 fusion protein was effective in delivering SOX17 into gastric cancer cells and subsequently inhibited the cell proliferation, colony formation, and invasion, increased E‐cadherin protein expression, decreased vimentin protein expression, as well as induced apoptosis. Our findings suggested SOX17 can bind to the promoter of Klotho gene to enhance Klotho gene expression in gastric cancer cells. The fused BR2‐SOX17 protein is an effective agent for targeting therapy of gastric cancer.