A Mutation in the Catalytic Loop of Hsp90 Specifically Impairs ATPase Stimulation by Aha1p,But Not Hch1p

Heat shock protein 90 (Hsp90) is a molecular chaperone that plays a central role in maintaining cellular homeostasis by facilitating activation of a large number of client proteins. ATP-dependent client activation by Hsp90 is tightly regulated by a host of co-chaperone proteins that control progression through the activation cycle. ATPase stimulation of Hsp90 by Aha1p requires a conserved RKxK motif that interacts with the catalytic loop of Hsp90. In this study, we explore the role of this RKxK motif in the biological and biochemical properties of Hch1p. We found that this motif is required for Hch1p-mediated ATPase stimulation in vitro, but mutations that block stimulation do not impair the action of Hch1p in vivo. This suggests that the biological function of Hch1p is not directly linked to ATPase stimulation. Moreover, a mutation in the catalytic loop of Hsp90 specifically impairs ATPase stimulation by Aha1p but not by Hch1p. Our work here suggests that both Hch1p and Aha1p regulate Hsp90 function through interaction with the catalytic loop but do so in different ways.     

The Imprinted Gene PEG3 Inhibits Wnt Signaling and Regulates Glioma Growth

The imprinted gene PEG3 confers parenting and sexual behaviors, alters growth and development, and regulates apoptosis. However, a molecular mechanism that can account for the diverse functions of Peg3/Pw1 is not known. To elucidate Peg3-regulated pathways, we performed a functional screen in zebrafish. Enforced overexpression of PEG3 mRNA during zebrafish embryogenesis decreased β-catenin protein expression and inhibited Wnt-dependent tail development. Peg3/Pw1 also inhibited Wnt signaling in human cells by binding to β-catenin and promoting its degradation via a p53/Siah1-dependent, GSK3β-independent proteasomal pathway. The inhibition of the Wnt pathway by Peg3/Pw1 suggested a role in tumor suppression. Hypermethylation of the PEG3 promoter in primary human gliomas led to a loss of imprinting and decreased PEG3 mRNA expression that correlated with tumor grade. The decrease in Peg3/Pw1 protein expression increased β-catenin, promoted proliferation, and inhibited p53-dependent apoptosis in human CD133⁺ glioma stem cells. Thus, mammalian imprinting utilizes Peg3/Pw1 to co-opt the Wnt pathway, thereby regulating development and glioma growth.     

Receptor Tyrosine Kinase-like Orphan Receptor 2 (Ror2) Expression Creates a Poised State of Wnt Signaling in Renal Cancer

Expression of the receptor tyrosine kinase-like orphan receptor 2 (Ror2) has been identified in an increasing array of tumor types and is known to play a role as an important mediator of Wnt signaling cascades. In this study, we aimed to clarify Ror2 interactions with the Wnt pathways within the context of renal cell carcinoma (RCC). An examination of Ror2 expression in primary human RCC tumors showed a significant correlation with several Wnt signaling genes, including the classical feedback target gene Axin2. We provide evidence that Ror2 expression results in a partially activated state for canonical Wnt signaling through an increased signaling pool of β-catenin, leading to an enhancement of downstream target genes following Wnt3a stimulation in both renal and renal carcinoma-derived cells. Additionally, inhibition of low-density lipoprotein receptor-related protein 6 (LRP6) with either siRNA or dickkopf decreased the response to Wnt3a stimulation, but no change was seen in the increased β-catenin pool associated with Ror2 expression, suggesting that LRP6 cofactor recruitment is necessary for a Wnt3a-induced signal but that it does not participate in the Ror2 effect on β-catenin signaling. These results highlight a new role for Ror2 in conveying a tonic signal to stabilize soluble β-catenin and create a poised state of enhanced responsiveness to Wnt3a exogenous signals in RCC.     

Casein Kinase 2 (CK2)-mediated Phosphorylation of Hsp90β as a Novel Mechanism of Rifampin-induced MDR1 Expression

The P-glycoprotein (P-gp) encoded by the MDR1 gene is a drug-exporting transporter located in the cellular membrane. P-gp induction is regarded as one of the main mechanisms underlying drug-induced resistance. Although there is great interest in the regulation of P-gp expression, little is known about its underlying regulatory mechanisms. In this study, we demonstrate that casein kinase 2 (CK2)-mediated phosphorylation of heat shock protein 90β (Hsp90β) and subsequent stabilization of PXR is a key mechanism in the regulation of MDR1 expression. Furthermore, we show that CK2 is directly activated by rifampin. Upon exposure to rifampin, CK2 catalyzes the phosphorylation of Hsp90β at the Ser-225/254 residues. Phosphorylated Hsp90β then interacts with PXR, causing a subsequent increase in its stability, leading to the induction of P-gp expression. In addition, inhibition of CK2 and Hsp90β enhances the down-regulation of PXR and P-gp expression. The results of this study may facilitate the development of new strategies to prevent multidrug resistance and provide a plausible mechanism for acquired drug resistance by CK2-mediated regulation of P-gp expression.     

Inhibition of nonsense-mediated decay rescues p53β/γ isoform expression and activates the p53 pathway in MDM2-overexpressing and select p53-mutant cancers

Inactivation of p53 is present in almost every tumor, and hence, p53-reactivation strategies are an important aspect of cancer therapy. Common mechanisms for p53 loss in cancer include expression of p53-negative regulators such as MDM2, which mediate the degradation of wildtype p53 (p53α), and inactivating mutations in the TP53 gene. Currently, approaches to overcome p53 deficiency in these cancers are limited. Here, using non–small cell lung cancer and glioblastoma multiforme cell line models, we show that two alternatively spliced, functional truncated isoforms of p53 (p53β and p53γ, comprising exons 1 to 9β or 9γ, respectively) and that lack the C-terminal MDM2-binding domain have markedly reduced susceptibility to MDM2-mediated degradation but are highly susceptible to nonsense-mediated decay (NMD), a regulator of aberrant mRNA stability. In cancer cells harboring MDM2 overexpression or TP53 mutations downstream of exon 9, NMD inhibition markedly upregulates p53β and p53γ and restores activation of the p53 pathway. Consistent with p53 pathway activation, NMD inhibition induces tumor suppressive activities such as apoptosis, reduced cell viability, and enhanced tumor radiosensitivity, in a relatively p53-dependent manner. In addition, NMD inhibition also inhibits tumor growth in a MDM2-overexpressing xenograft tumor model. These results identify NMD inhibition as a novel therapeutic strategy for restoration of p53 function in p53-deficient tumors bearing MDM2 overexpression or p53 mutations downstream of exon 9, subgroups that comprise approximately 6% of all cancers.     

A Novel C-Terminal Homologue of Aha1 Co-Chaperone Binds to Heat Shock Protein 90 and Stimulates Its ATPase Activity in Entamoeba histolytica

Cytosolic heat shock protein 90 (Hsp90) has been shown to be essential for many infectious pathogens and is considered a potential target for drug development. In this study, we have carried out biochemical characterization of Hsp90 from a poorly studied protozoan parasite of clinical importance, Entamoeba histolytica. We have shown that Entamoeba Hsp90 can bind to both ATP and its pharmacological inhibitor, 17-AAG (17-allylamino-17-demethoxygeldanamycin), with K_d values of 365.2 and 10.77 μM, respectively, and it has a weak ATPase activity with a catalytic efficiency of 4.12 × 10^− 4 min^− 1 μM^− 1. Using inhibitor 17-AAG, we have shown dependence of Entamoeba on Hsp90 for its growth and survival. Hsp90 function is regulated by various co-chaperones. Previous studies suggest a lack of several important co-chaperones in E. histolytica. In this study, we describe the presence of a novel homologue of co-chaperone Aha1 (activator of Hsp90 ATPase), EhAha1c, lacking a canonical Aha1 N-terminal domain. We also show that EhAha1c is capable of binding and stimulating ATPase activity of EhHsp90. In addition to highlighting the potential of Hsp90 inhibitors as drugs against amoebiasis, our study highlights the importance of E. histolytica in understanding the evolution of Hsp90 and its co-chaperone repertoire.     

TAp73 Protein Stability Is Controlled by Histone Deacetylase 1 via Regulation of Hsp90 Chaperone Function

Histone deacetylases (HDACs) play important roles in fundamental cellular processes, and HDAC inhibitors are emerging as promising cancer therapeutics. p73, a member of the p53 family, plays a critical role in tumor suppression and neural development. Interestingly, p73 produces two classes of proteins with opposing functions: the full-length TAp73 and the N-terminally truncated ΔNp73. In the current study, we sought to characterize the potential regulation of p73 by HDACs and found that histone deacetylase 1 (HDAC1) is a key regulator of TAp73 protein stability. Specifically, we showed that HDAC1 inhibition by HDAC inhibitors or by siRNA shortened the half-life of TAp73 protein and subsequently decreased TAp73 expression under normal and DNA damage-induced conditions. Mechanistically, we found that HDAC1 knockdown resulted in hyperacetylation and inactivation of heat shock protein 90, which disrupted the interaction between heat shock protein 90 and TAp73 and thus promoted the proteasomal degradation of TAp73. Functionally, we found that down-regulation of TAp73 was required for the enhanced cell migration mediated by HDAC1 knockdown. Together, we uncover a novel regulation of TAp73 protein stability by HDAC1-heat shock protein 90 chaperone complex, and our data suggest that TAp73 is a critical downstream mediator of HDAC1-regulated cell migration.     

Pantothenate Kinase 1 Inhibits the Progression of Hepatocellular Carcinoma by Negatively Regulating Wnt/β-catenin Signaling

Hyperactivation of Wnt/β-catenin signaling has been reported in hepatocellular carcinoma (HCC). However, the mechanisms underlying the hyperactivation of Wnt/β-catenin signaling are incompletely understood. In this study, Pantothenate kinase 1 (PANK1) is shown to be a negative regulator of Wnt/β-catenin signaling. Downregulation of PANK1 in HCC correlates with clinical features. Knockdown of PANK1 promotes the proliferation, growth and invasion of HCC cells, while overexpression of PANK1 inhibits the proliferation, growth, invasion and tumorigenicity of HCC cells. Mechanistically, PANK1 binds to CK1α, exerts protein kinase activity and cooperates with CK1α to phosphorylate N-terminal serine and threonine residues in β-catenin both in vitro and in vivo. Additionally, the expression levels of PANK1 and β-catenin can be used to predict the prognosis of HCC. Collectively, the results of this study highlight the crucial roles of PANK1 protein kinase activity in inhibiting Wnt/β-catenin signaling, suggesting that PANK1 is a potential therapeutic target for HCC.     

GALNT1 Enhances Malignant Phenotype of Gastric Cancer via Modulating CD44 Glycosylation to Activate the Wnt/β-catenin Signaling Pathway

O-glycosylation is a widespread post-translational modification of proteins. Aberrant O-glycosylation is a hallmark of cancer. Here, we show that the polypeptide N-acetylgalactosamine-transferase 1 (GALNT1) is frequently upregulated in gastric cancer and is correlated with poor survival. Overexpression of GALNT1 promoted, whereas knockdown suppressed proliferation, migration, and invasion of gastric cancer cells in vitro and in vivo. Mechanistically, GALNT1 enhances aberrant initiation of O-glycosylation and results in CD44 glycoproteins modified with abundant Tn antigens, thereby activating the Wnt/β-catenin signaling pathway. Collectively, this study demonstrates that GALNT1 overexpression in gastric cancer promotes the Wnt/β-catenin signaling pathway via abnormal O-glycosylation of CD44 to enhance malignancy, providing a novel strategy for the development of therapeutic reagents against gastric cancer.     

Isoquercitrin Suppresses Colon Cancer Cell Growth in Vitro by Targeting the Wnt/β-Catenin Signaling Pathway

Flavonoids are plant-derived polyphenolic molecules that have potential biological effects including anti-oxidative, anti-inflammatory, anti-viral, and anti-tumoral effects. These effects are related to the ability of flavonoids to modulate signaling pathways, such as the canonical Wnt signaling pathway. This pathway controls many aspects of embryonic development and tissue maintenance and has been found to be deregulated in a range of human cancers. We performed several in vivo assays in Xenopus embryos, a functional model of canonical Wnt signaling studies, and also used in vitro models, to investigate whether isoquercitrin affects Wnt/β-catenin signaling. Our data provide strong support for an inhibitory effect of isoquercitrin on Wnt/β-catenin, where the flavonoid acts downstream of β-catenin translocation to the nuclei. Isoquercitrin affects Xenopus axis establishment, reverses double axes and the LiCl hyperdorsalization phenotype, and reduces Xnr3 expression. In addition, this flavonoid shows anti-tumoral effects on colon cancer cells (SW480, DLD-1, and HCT116), whereas exerting no significant effect on non-tumor colon cell (IEC-18), suggesting a specific effect in tumor cells in vitro. Taken together, our data indicate that isoquercitrin is an inhibitor of Wnt/β-catenin and should be further investigated as a potential novel anti-tumoral agent.     

PRMT5 acts as a tumor suppressor by inhibiting Wnt/β-catenin signaling in murine gastric tumorigenesis

Previous studies have demonstrated the in vitro oncogenic role of protein arginine methyltransferase 5 (PRMT5) in gastric cancer cell lines. The in vivo function of PRMT5 in gastric tumorigenesis, however, is still unexplored. Here, we showed that Prmt5 deletion in mouse gastric epithelium resulted in spontaneous tumorigenesis in gastric antrum. All Prmt5-deficient mice displayed intestinal-type gastric cancer within 4 months of age. Of note, 20% (2/10) of Prmt5 mutants finally developed into invasive gastric cancer by 8 months of age. Gastric cancer caused by PRMT5 loss exhibited the increase in Lgr5⁺ stem cells, which are proposed to contribute to both the gastric tumorigenesis and progression in mouse models. Consistent with the notion that Lgr5 is the target of Wnt/β-catenin signaling, whose activation is the most predominant driver for gastric tumorigenesis, Prmt5 mutant gastric cancer showed the activation of Wnt/β-Catenin signaling. Furthermore, in human gastric cancer samples, PRMT5 deletion and downregulation were frequently observed and associated with the poor prognosis. We propose that as opposed to the tumor-promoting role of PRMT5 well-established in the progression of various cancer types, PRMT5 functions as a tumor suppressor in vivo, at least during gastric tumor formation.     

β-Site Amyloid Precursor Protein Cleaving Enzyme 1(BACE1) Regulates Notch Signaling by Controlling the Cleavage of Jagged 1 (Jag1) and Jagged 2 (Jag2) Proteins

BACE1 is a type I transmembrane aspartyl protease that cleaves amyloid precursor protein at the β-secretase site to initiate the release of β-amyloid peptide. As a secretase, BACE1 also cleaves additional membrane-bound molecules by exerting various cellular functions. In this study, we showed that BACE1 can effectively shed the membrane-anchored signaling molecule Jagged 1 (Jag1). We also mapped the cleavage sites of Jag1 by ADAM10 and ADAM17. Although Jag1 shares a high degree of homology with Jag2 in the ectodomain region, BACE1 fails to cleave Jag2 effectively, indicating a selective cleavage of Jag1. Abolished cleavage of Jag1 in BACE1-null mice leads to enhanced astrogenesis and, concomitantly, reduced neurogenesis. This characterization provides biochemical evidence that the Jag1-Notch pathway is under the control of BACE1 activity.     

MicroRNA-200 Is Induced by Thioredoxin-interacting Protein and Regulates Zeb1 Protein Signaling and Beta Cell Apoptosis

Small noncoding microRNAs have emerged as important regulators of cellular processes, but their role in pancreatic beta cells has only started to be elucidated. Loss of pancreatic beta cells is a key factor in the pathogenesis of diabetes, and we have demonstrated that beta cell expression of thioredoxin-interacting protein (TXNIP) is increased in diabetes and causes beta cell apoptosis, whereas TXNIP deficiency is protective against diabetes. Recently, we found that TXNIP also impairs beta cell function by inducing microRNA (miR)-204. Interestingly, using INS-1 beta cells and primary islets, we have now discovered that expression of another microRNA, miR-200, is induced by TXNIP and by diabetes. Furthermore, we found that miR-200 targeted and decreased Zeb1 (zinc finger E-box-binding homeobox 1) and promoted beta cell apoptosis as measured by cleaved caspase-3 levels, Bax/Bcl2 ratio, and TUNEL. In addition, Zeb1 knockdown mimicked the miR-200 effects on beta cell apoptosis, suggesting that Zeb1 plays an important role in mediating miR-200 effects. Moreover, miR-200 increased beta cell expression of the epithelial marker E-cadherin, consistent with inhibition of epithelial-mesenchymal transition, a process thought to be involved in beta cell expansion. Thus, we have identified a novel TXNIP/miR-200/Zeb1/E-cadherin signaling pathway that, for the first time, links miR-200 to beta cell apoptosis and diabetes and also beta cell TXNIP to epithelial-mesenchymal transition. In addition, our results shed new light on the regulation and function of miR-200 in beta cells and show that TXNIP-induced microRNAs control various processes of beta cell biology.     

Plasminogen Activator Inhibitor-1 Is a Transcriptional Target of the Canonical Pathway of Wnt/β-Catenin Signaling

Plasminogen activator inhibitor-1 (PAI-1) is a multifunctional glycoprotein that plays a critical role in the pathogenesis of chronic kidney and cardiovascular diseases. Although transforming growth factor (TGF)-β1 is a known inducer of PAI-1, how it controls PAI-1 expression remains enigmatic. Here we investigated the mechanism underlying TGF-β1 regulation of PAI-1 in kidney tubular epithelial cells (HKC-8). Surprisingly, overexpression of Smad2 or Smad3 in HKC-8 cells blocked PAI-1 induction by TGF-β1, whereas knockdown of them sensitized the cells to TGF-β1 stimulation, suggesting that Smad signaling is not responsible for PAI-1 induction. Blockade of several TGF-β1 downstream pathways such as p38 MAPK or JNK, but not phosphatidylinositol 3-kinase/Akt and ERK1/2, only partially inhibited PAI-1 expression. TGF-β1 stimulated β-catenin activation in tubular epithelial cells, and ectopic expression of β-catenin induced PAI-1 expression, whereas inhibition of β-catenin abolished its induction. A functional T cell factor/lymphoid enhancer-binding factor-binding site was identified in the promoter region of the PAI-1 gene, which interacted with T cell factor upon β-catenin activation. Deletion or site-directed mutation of this site abolished PAI-1 response to β-catenin or TGF-β1 stimulation. Similarly, ectopic expression of Wnt1 also activated PAI-1 expression and promoter activity. In vivo, PAI-1 was induced in kidney tubular epithelia in obstructive nephropathy. Delivery of Wnt1 gene activated β-catenin and promoted PAI-1 expression after obstructive injury, whereas blockade of Wnt/β-catenin signaling by Dickkopf-1 gene inhibited PAI-1 induction. Collectively, these studies identify PAI-1 as a direct downstream target of Wnt/β-catenin signaling and demonstrate that PAI-1 induction could play a role in mediating the fibrogenic action of this signaling.