Exploring the association between COVID-19 and male genital cancer risk in European population: evidence from mendelian randomization analysis.

BACKGROUND: Recently accumulated evidence indicates a potential association between COVID-19 and elevated susceptibility to cancer, including male genital cancer. However, the causal nature of this relationship remains unclear. METHODS: In this Mendelian randomization (MR) study, we investigated the potential causal relationship between COVID-19 and male genital cancer using genetic variants as instrumental variables. We utilized summary statistics from two large-scale genome-wide association studies of COVID-19 hospitalized Vs. controls, as well as data from a population-based male genital cancer database based on European ancestry. We applied stringent quality control measures to select instrumental variables, including checking for linkage disequilibrium, removing low-quality variants, and assessing the strength of the instruments using the F-statistic. We conducted the MR  analysis using the inverse-variance weighted method and several sensitivity analyses (including MR Egger and Weighted Median MR analysis) to test the robustness of our results. RESULTS: Our MR analysis revealed no causal associations between COVID-19 hospitalization and the incidence of male genital cancer. In the inverse-variance weighted analysis, no causal associations were observed between patients with COVID-19 hospitalization and the incidence of male genital cancer (odds ratio = 1.000 and 95% confidence interval = 0.998-1.001, p = 0.668). The estimated causal effect was consistent across all sensitivity analyses (including the Weighted Median, the MR Egger analysis, and the MR PROSSO analysis). The leave-one-out analysis showed that there was no any sing Single-nucleotide polymorphism significantly influencing our results. CONCLUSIONS: Our study provides evidence that there is no causal association between COVID-19 hospitalization and male genital cancer.

Genetically predicted obesity and risk of hip osteoarthritis.

OBJECTIVES: To determine the causal association between genetically predicted obesity and the risk of hip osteoarthritis. METHODS: We performed two-sample Mendelian randomization (MR) analysis to analyze the association between body mass index (BMI) and hip osteoarthritis using pooled-level genome-wide association study (GWAS) data. The inverse variance weighted (IVW), MR‒Egger, and weighted median methods were used to estimate the causal association. In addition, we applied the MR Steiger filtering method, MR robust adjusted profile score (MR.RAPS) methods, and the MR Pleiotropy RESidual Sum and Outlier (MR-PRESSO) global test to examine and address potential horizontal pleiotropy. RESULTS: We found a causal relationship between genetically predicted BMI and the risk of hip osteoarthritis by the IVW method [OR = 1.45, 95% confidence interval (CI) = 1.04-2.00, P = 0.02]. In the sensitivity analysis, the results of the MR‒Egger and weighted median methods revealed similar estimations but with a wide CI with lower precision. The funnel plot, MR-Egger intercept, and MR-PRESSO all indicated the absence of a directional pleiotropic effect. In addition, no heterogeneity was observed in the present analysis. Therefore, the result of IVW is most suitable and reliable for the present MR analysis. CONCLUSION: There is a causal relationship between obesity and a higher risk of hip osteoarthritis, suggesting that weight management may be an intervention for the prevention and management of hip osteoarthritis. LEVEL OF EVIDENCE: Bioinformatics, Basic science.

PTEN loss promotes Warburg effect and prostate cancer cell growth by inducing FBP1 degradation.

Rationale: Fructose-1,6-bisphosphatase (FBP1) is a tumor suppressor and a key enzyme negatively regulating Warburg effect in cancer. However, regulation of FBP1 protein expression and its exact role in prostate cancer (PCa) is largely unclear. Phosphatase and tensin homolog (PTEN) is one of the most frequently deleted tumor suppressor genes in human PCa. However, the role of PTEN loss in aberrant Warburg effect in cancer remains poorly understood. Methods: Expression of PTEN and FBP1 was analyzed in several PCa cell lines and prostate tumor tissues in mice. Western blot (WB) and RT-PCR approaches were used to examine how PTEN regulates FBP1 expression. Co-immunoprecipitation (co-IP) and in vivo ubiquitination assays were used to define the regulatory mechanisms. A PCa xenograft model was employed to determine the impact of PTEN regulation of FBP1 on PCa growth in vivo. Result: We demonstrated that in a manner dependent of PI3K/AKT signal pathway PTEN regulated FBP1 expression in various PCa cell lines and tumors in mice. We confirmed that this regulation took place at the protein level and was mediated by SKP2 E3 ubiquitin ligase. Mechanistically, we showed that serine 271 phosphorylation of FBP1 by cyclin-dependent kinases (CDKs) was essential for SKP2-mediated degradation of FBP1 protein induced by PTEN loss. Most importantly, we further showed that loss of PTEN expression enhanced Warburg effect and PCa growth in mice in a manner dependent, at least partially on FBP1 protein degradation. Conclusions: Our results reveal a novel tumor-suppressive feature of PTEN in restraining FBP1 degradation and the Warburg effect. These results also suggest that prohibiting FBP1 protein degradation could be a viable therapeutic strategy for PTEN-deficient PCa.

SPOP mutation induces DNA methylation via stabilizing GLP/G9a.

Mutations in SPOP E3 ligase gene are reportedly associated with genome-wide DNA hypermethylation in prostate cancer (PCa) although the underlying mechanisms remain elusive. Here, we demonstrate that SPOP binds and promotes polyubiquitination and degradation of histone methyltransferase and DNMT interactor GLP. SPOP mutation induces stabilization of GLP and its partner protein G9a and aberrant upregulation of global DNA hypermethylation in cultured PCa cells and primary PCa specimens. Genome-wide DNA methylome analysis shows that a subset of tumor suppressor genes (TSGs) including FOXO3, GATA5, and NDRG1, are hypermethylated and downregulated in SPOP-mutated PCa cells. DNA methylation inhibitor 5-azacytidine effectively reverses expression of the TSGs examined, inhibits SPOP-mutated PCa cell growth in vitro and in mice, and enhances docetaxel anti-cancer efficacy. Our findings reveal the GLP/G9a-DNMT module as a mediator of DNA hypermethylation in SPOP-mutated PCa. They suggest that SPOP mutation could be a biomarker for effective treatment of PCa with DNA methylation inhibitor alone or in combination with taxane chemotherapeutics.

Destruction of DNA-Binding Proteins by Programmable Oligonucleotide PROTAC (O'PROTAC): Effective Targeting of LEF1 and ERG.

DNA-binding proteins, including transcription factors (TFs), play essential roles in various cellular processes and pathogenesis of diseases, deeming to be potential therapeutic targets. However, these proteins are generally considered undruggable as they lack an enzymatic catalytic site or a ligand-binding pocket. Proteolysis-targeting chimera (PROTAC) technology has been developed by engineering a bifunctional molecule chimera to bring a protein of interest (POI) to the proximity of an E3 ubiquitin ligase, thus inducing the ubiquitination of POI and further degradation through the proteasome pathway. Here, the development of oligonucleotide-based PROTAC (O'PROTACs), a class of noncanonical PROTACs in which a TF-recognizing double-stranded oligonucleotide is incorporated as a binding moiety of POI is reported. It is demonstrated that O'PROTACs of lymphoid enhancer-binding factor 1 (LEF1) and ETS-related gene (ERG), two highly cancer-related transcription factors, successfully promote degradation of these proteins, impede their transcriptional activity, and inhibit cancer cell growth in vitro and in vivo. The programmable nature of O'PROTACs indicates that this approach is also applicable to destruct other TFs. O'PROTACs not only can serve as a research tool but also can be harnessed as a therapeutic arsenal to target DNA binding proteins for effective treatment of diseases such as cancer.

ATM-phosphorylated SPOP contributes to 53BP1 exclusion from chromatin during DNA replication.

53BP1 activates nonhomologous end joining (NHEJ) and inhibits homologous recombination (HR) repair of DNA double-strand breaks (DSBs). Dissociation of 53BP1 from DSBs and consequent activation of HR, a less error-prone pathway than NHEJ, helps maintain genome integrity during DNA replication; however, the underlying mechanisms are not fully understood. Here, we demonstrate that E3 ubiquitin ligase SPOP promotes HR during S phase of the cell cycle by excluding 53BP1 from DSBs. In response to DNA damage, ATM kinase-catalyzed phosphorylation of SPOP causes a conformational change in SPOP, revealed by x-ray crystal structures, that stabilizes its interaction with 53BP1. 53BP1-bound SPOP induces polyubiquitination of 53BP1, eliciting 53BP1 extraction from chromatin by a valosin-containing protein/p97 segregase complex. Our work shows that SPOP facilitates HR repair over NHEJ during DNA replication by contributing to 53BP1 removal from chromatin. Cancer-derived SPOP mutations block SPOP interaction with 53BP1, inducing HR defects and chromosomal instability.

ZMYND8 preferentially binds phosphorylated EZH2 to promote a PRC2-dependent to -independent function switch in hypoxia-inducible factor-activated cancer.

Both gene repressor (Polycomb-dependent) and activator (Polycomb-independent) functions of the Polycomb protein enhancer of zeste homolog 2 (EZH2) are implicated in cancer progression. EZH2 protein can be phosphorylated at various residues, such as threonine 487 (T487), by CDK1 kinase, and such phosphorylation acts as a Polycomb repressive complex 2 (PRC2) suppression "code" to mediate the gene repressor-to-activator switch of EZH2 functions. Here we demonstrate that the histone reader protein ZMYND8 is overexpressed in human clear cell renal cell carcinoma (ccRCC). ZMYND8 binds to EZH2, and their interaction is largely enhanced by CDK1 phosphorylation of EZH2 at T487. ZMYND8 depletion not only enhances Polycomb-dependent function of EZH2 in hypoxia-exposed breast cancer cells or von Hippel-Lindau (VHL)-deficient ccRCC cells, but also suppresses the FOXM1 transcription program. We further show that ZMYND8 is required for EZH2-FOXM1 interaction and is important for FOXM1-dependent matrix metalloproteinase (MMP) gene expression and EZH2-mediated migration and invasion of VHL-deficient ccRCC cells. Our results identify a previously uncharacterized role of the chromatin reader ZMYND8 in recognizing the PRC2-inhibitory phosphorylation "code" essential for the Polycomb-dependent to -independent switch of EZH2 functions. They also reveal an oncogenic pathway driving cell migration and invasion in hypoxia-inducible factor-activated (hypoxia or VHL-deficient) cancer.

An acetyl-histone vulnerability in PI3K/AKT inhibition-resistant cancers is targetable by both BET and HDAC inhibitors.

Acquisition of resistance to phosphatidylinositol 3-kinase (PI3K)/AKT-targeted monotherapy implies the existence of common resistance mechanisms independent of cancer type. Here, we demonstrate that PI3K/AKT inhibitors cause glycolytic crisis, acetyl-coenzyme A (CoA) shortage, and a global decrease in histone acetylation. In addition, PI3K/AKT inhibitors induce drug resistance by selectively augmenting histone H3 lysine 27 acetylation (H3K27ac) and binding of CBP/p300 and BRD4 proteins at a subset of growth factor and receptor (GF/R) gene loci. BRD4 occupation at these loci and drug-resistant cell growth are vulnerable to both bromodomain and histone deacetylase (HDAC) inhibitors. Little or no occupation of HDAC proteins at the GF/R gene loci underscores the paradox that cells respond equivalently to the two classes of inhibitors with opposite modes of action. Targeting this unique acetyl-histone-related vulnerability offers two clinically viable strategies to overcome PI3K/AKT inhibitor resistance in different cancers.

DNA Damage Promotes TMPRSS2-ERG Oncoprotein Destruction and Prostate Cancer Suppression via Signaling Converged by GSK3ß and WEE1.

TMPRSS2-ERG gene fusion occurs in approximately 50% of cases of prostate cancer (PCa), and the fusion product is a key driver of prostate oncogenesis. However, how to leverage cellular signaling to ablate TMPRSS2-ERG oncoprotein for PCa treatment remains elusive. Here, we demonstrate that DNA damage induces proteasomal degradation of wild-type ERG and TMPRSS2-ERG oncoprotein through ERG threonine-187 and tyrosine-190 phosphorylation mediated by GSK3ß and WEE1, respectively. The dual phosphorylation triggers ERG recognition and degradation by the E3 ubiquitin ligase FBW7 in a manner independent of a canonical degron. DNA damage-induced TMPRSS2-ERG degradation was abolished by cancer-associated PTEN deletion or GSK3ß inactivation. Blockade of DNA damage-induced TMPRSS2-ERG oncoprotein degradation causes chemotherapy-resistant growth of fusion-positive PCa cells in culture and in mice. Our findings uncover a previously unrecognized TMPRSS2-ERG protein destruction mechanism and demonstrate that intact PTEN and GSK3ß signaling are essential for effective targeting of ERG protein by genotoxic therapeutics in fusion-positive PCa.

Inhibition of autophagy aggravates DNA damage response and gastric tumorigenesis via Rad51 ubiquitination in response to H. pylori infection.

Helicobacter pylori (H. pylori) infection is the strongest known risk factor for the development of gastric cancer. DNA damage response (DDR) and autophagy play key roles in tumorigenic transformation. However, it remains unclear how H. pylori modulate DDR and autophagy in gastric carcinogenesis. Here we report that H. pylori infection promotes DNA damage via suppression of Rad51 expression through inhibition of autophagy and accumulation of p62 in gastric carcinogenesis. We find that H. pylori activated DNA damage pathway in concert with downregulation of repair protein Rad51 in gastric cells, C57BL/6 mice and Mongolian gerbils. In addition, autophagy was increased early and then decreased gradually during the duration of H. pylori infection in vitro in a CagA-dependent manner. Moreover, loss of autophagy led to promotion of DNA damage in H. pylori-infected cells. Furthermore, knockdown of autophagic substrate p62 upregulated Rad51 expression, and p62 promoted Rad51 ubiquitination via the direct interaction of its UBA domain. Finally, H. pylori infection was associated with elevated levels of p62 in gastric intestinal metaplasia and decreased levels of Rad51 in dysplasia compared to their H. pylori- counterparts. Our findings provide a novel mechanism into the linkage of H. pylori infection, autophagy, DNA damage and gastric tumorigenesis.

The novel BET-CBP/p300 dual inhibitor NEO2734 is active in SPOP mutant and wild-type prostate cancer.

CULLIN3-based E3 ubiquitin ligase substrate-binding adaptor gene SPOP is frequently mutated in prostate cancer (PCa). PCa harboring SPOP hotspot mutants (e.g., F133V) are resistant to BET inhibitors because of aberrant elevation of BET proteins. Here, we identified a previously unrecognized mutation Q165P at the edge of SPOP MATH domain in primary and metastatic PCa of a patient. The Q165P mutation causes structural changes in the MATH domain and impairs SPOP dimerization and substrate degradation. Different from F133V hotspot mutant tumors, Q165P mutant patient-derived xenografts (PDXs) and organoids were modestly sensitive to the BET inhibitor JQ1. Accordingly, protein levels of AR, BRD4 and downstream effectors such as RAC1 and phosphorylated AKT were not robustly elevated in Q165P mutant cells as in F133V mutant cells. However, NEO2734, a novel dual inhibitor of BET and CBP/p300, is active in both hotspot mutant (F133V) and non-hotspot mutant (Q165P) PCa cells in vitro and in vivo. These data provide a strong rationale to clinically investigate the anti-cancer efficacy of NEO2734 in SPOP-mutated PCa patients.

EZH2 cooperates with gain-of-function p53 mutants to promote cancer growth and metastasis.

In light of the increasing number of identified cancer-driven gain-of-function (GOF) mutants of p53, it is important to define a common mechanism to systematically target several mutants, rather than developing strategies tailored to inhibit each mutant individually. Here, using RNA immunoprecipitation-sequencing (RIP-seq), we identified the Polycomb-group histone methyltransferase EZH2 as a p53 mRNA-binding protein. EZH2 bound to an internal ribosome entry site (IRES) in the 5'UTR of p53 mRNA and enhanced p53 protein translation in a methyltransferase-independent manner. EZH2 augmented p53 GOF mutant-mediated cancer growth and metastasis by increasing protein levels of mutant p53. EZH2 overexpression was associated with worsened outcome selectively in patients with p53-mutated cancer. Depletion of EZH2 by antisense oligonucleotides inhibited p53 GOF mutant-mediated cancer growth. Our findings reveal a non-methyltransferase function of EZH2 that controls protein translation of p53 GOF mutants, inhibition of which causes synthetic lethality in cancer cells expressing p53 GOF mutants.

Non-invasive cardiac output measurement with electrical velocimetry in patients undergoing liver transplantation: comparison of an invasive method with pulmonary thermodilution.

BACKGROUND: The goal of this study was to evaluate the accuracy and interchangeability between continuous cardiac output (CO) measured by electrical velocimetry (COEv) and continuous cardiac output obtained using the pulmonary thermodilution method (COPAC) during living donor liver transplantation (LDLT). METHOD: Twenty-three patients were enrolled in this prospective observational study. CO was recorded by both two methods and compared at nine specific time points. The data were analyzed using correlation coefficients, Bland-Altman analysis for the percentage errors, and the concordance rate for trend analysis using a four-quadrant plot. RESULTS: In total, 207 paired datasets were recorded during LDLT. CO data were in the range of 2.8-12.7 L/min measured by PAC and 3.4-14.9 L/min derived from the EV machine. The correction coefficient between COPAC and COEv was 0.415 with p < 0.01. The 95% limitation agreement was - 5.9 to 3.4 L/min and the percentage error was 60%. The concordance rate was 56.5%. CONCLUSIONS: The Aesculon™ monitor is not yet interchangeable with continuous thermodilution CO monitoring during LDLT. TRIAL REGISTRATION: The study was approved by the Institutional Review Board of Chang Gung Medical Foundation in Taiwan (registration number: 201600264B0 ).

DUB3 Promotes BET Inhibitor Resistance and Cancer Progression by Deubiquitinating BRD4.

The bromodomain and extra-terminal domain (BET) protein BRD4 is emerging as a promising anticancer therapeutic target. However, resistance to BET inhibitors often occurs, and it has been linked to aberrant degradation of BRD4 protein in cancer. Here, we demonstrate that the deubiquitinase DUB3 binds to BRD4 and promotes its deubiquitination and stabilization. Expression of DUB3 is transcriptionally repressed by the NCOR2-HDAC10 complex. The NCOR2 gene is frequently deleted in castration-resistant prostate cancer patient specimens, and loss of NCOR2 induces elevation of DUB3 and BRD4 proteins in cancer cells. DUB3-proficient prostate cancer cells are resistant to the BET inhibitor JQ1 in vitro and in mice, but this effect is diminished by DUB3 inhibitory agents such as CDK4/6 inhibitor in a RB-independent manner. Our findings identify a previously unrecognized mechanism causing BRD4 upregulation and drug resistance, suggesting that DUB3 is a viable therapeutic target to overcome BET inhibitor resistance in cancer.

Dual inhibition of AKT-mTOR and AR signaling by targeting HDAC3 in PTEN- or SPOP-mutated prostate cancer.

AKT-mTOR and androgen receptor (AR) signaling pathways are aberrantly activated in prostate cancer due to frequent PTEN deletions or SPOP mutations. A clinical barrier is that targeting one of them often activates the other. Here, we demonstrate that HDAC3 augments AKT phosphorylation in prostate cancer cells and its overexpression correlates with AKT phosphorylation in patient samples. HDAC3 facilitates lysine-63-chain polyubiquitination and phosphorylation of AKT, and this effect is mediated by AKT deacetylation at lysine 14 and 20 residues and HDAC3 interaction with the scaffold protein APPL1. Conditional homozygous deletion of Hdac3 suppresses prostate tumorigenesis and progression by concomitant blockade of AKT and AR signaling in the Pten knockout mouse model. Pharmacological inhibition of HDAC3 using a selective HDAC3 inhibitor RGFP966 inhibits growth of both PTEN-deficient and SPOP-mutated prostate cancer cells in culture, patient-derived organoids and xenografts in mice. Our study identifies HDAC3 as a common upstream activator of AKT and AR signaling and reveals that dual inhibition of AKT and AR pathways is achievable by single-agent targeting of HDAC3 in prostate cancer.

Flavonoid-Rich Ethanol Extract from the Leaves of Diospyros kaki Attenuates D-Galactose-Induced Oxidative Stress and Neuroinflammation-Mediated Brain Aging in Mice.

Aging is a major factor that contributes to neurological impairment and neuropathological changes, such as inflammation, oxidative stress, neuronal apoptosis, and synaptic dysfunction. Flavonoids act as protective antioxidant and anti-inflammatory agents against various age-related neurodegenerative diseases. Here, we investigated the protective effect and mechanisms of the flavonoid-rich ethanol extract from the leaves of Diospyros kaki (FELDK) in the cortex and hippocampus of D-galactose- (gal-) aged mice. Our results showed that FELDK treatment restored memory impairment in mice as determined by the Y-maze and Morris water maze tests. FELDK decreased oxidative stress levels via inhibiting reactive oxygen species (ROS) and malondialdehyde (MDA) production and elevating antioxidative enzymes. FELDK also alleviated D-gal-induced neuroinflammation via suppressing the expression of advanced glycation end products (AGEs) and receptor for AGEs (RAGE) and activating microgliosis and astrocytosis, nuclear factor kappa B (NF-κB) nuclear translocation, and downstream inflammatory mediators. Moreover, FELDK inhibited the phosphatidylinositol 3-kinase (PI3K)/Akt and C-jun N-terminal kinase (JNK) apoptotic signaling pathways and ameliorated the impairment of synapse-related proteins. Hence, these results indicate that FELDK exerts neuroprotective effects on D-gal-induced brain aging. Thus, FELDK may be a potential therapeutic strategy for preventing and treating age-related neurodegenerative diseases such as Alzheimer's disease.

Loss of FOXO1 Cooperates with TMPRSS2-ERG Overexpression to Promote Prostate Tumorigenesis and Cell Invasion.

E26 transformation-specific transcription factor ERG is aberrantly overexpressed in approximately 50% of all human prostate cancer due to TMPRSS2-ERG gene rearrangements. However, mice with prostate-specific transgenic expression of prostate cancer-associated ERG alone fail to develop prostate cancer, highlighting that ERG requires other lesions to drive prostate tumorigenesis. Forkhead box (FOXO) transcription factor FOXO1 is a tumor suppressor that is frequently inactivated in human prostate cancer. Here, we demonstrate that FOXO1, but not other FOXO proteins (FOXO3 and FOXO4), binds and inhibits the transcriptional activity of prostate cancer-associated ERG independently of FOXO1 transcriptional activity. Knockdown of endogenous FOXO1 increased invasion of TMPRSS2-ERG fusion-positive VCaP cells, an effect completely abolished by ERG knockdown. Patient specimen analysis demonstrated that FOXO1 and ERG protein expression inversely correlated in a subset of human prostate cancer. Although human ERG transgene expression or homozygous deletion of Foxo1 alone in the mouse prostate failed to promote tumorigenesis, concomitant ERG transgene expression and Foxo1 deletion resulted in upregulation of ERG target genes, increased cell proliferation, and formation of high-grade prostatic intraepithelial neoplasia. Overall, we provide biochemical and genetic evidence that aberrantly activated ERG cooperates with FOXO1 deficiency to promote prostate tumorigenesis and cell invasion. Our findings enhance understanding of prostate cancer etiology and suggest that the FOXO1-ERG signaling axis can be a potential target for treatment of prostate cancer. Cancer Res; 77(23); 6524-37. ©2017 AACR.

Intrinsic BET inhibitor resistance in SPOP-mutated prostate cancer is mediated by BET protein stabilization and AKT-mTORC1 activation.

Bromodomain and extraterminal domain (BET) protein inhibitors are emerging as promising anticancer therapies. The gene encoding the E3 ubiquitin ligase substrate-binding adaptor speckle-type POZ protein (SPOP) is the most frequently mutated in primary prostate cancer. Here we demonstrate that wild-type SPOP binds to and induces ubiquitination and proteasomal degradation of BET proteins (BRD2, BRD3 and BRD4) by recognizing a degron motif common among them. In contrast, prostate cancer-associated SPOP mutants show impaired binding to BET proteins, resulting in decreased proteasomal degradation and accumulation of these proteins in prostate cancer cell lines and patient specimens and causing resistance to BET inhibitors. Transcriptome and BRD4 cistrome analyses reveal enhanced expression of the GTPase RAC1 and cholesterol-biosynthesis-associated genes together with activation of AKT-mTORC1 signaling as a consequence of BRD4 stabilization. Our data show that resistance to BET inhibitors in SPOP-mutant prostate cancer can be overcome by combination with AKT inhibitors and further support the evaluation of SPOP mutations as biomarkers to guide BET-inhibitor-oriented therapy in patients with prostate cancer.

CDK5/FBW7-dependent ubiquitination and degradation of EZH2 inhibits pancreatic cancer cell migration and invasion.

Pancreatic cancer is one of the most lethal cancer types. Enhancer of zeste homolog 2 (EZH2) is an oncogenic protein overexpressed in pancreatic cancer, and EZH2 could be a potential therapeutic target for the treatment of pancreatic cancer. Although significant progress has been made toward understanding the function and deregulation of EZH2 in cancer cells, the posttranslational regulation of EZH2 in cancer cells is still unclear. F-box and WD repeat domain-containing 7 (FBW7) acts as a tumor suppressor by targeting multiple oncoprotein substrates for ubiquitination and degradation. Here we demonstrate that EZH2 is a bona fide substrate of FBW7 in pancreatic cancer cells. We provide evidence that the activated CDK5 kinase is involved in the EZH2 phosphorylation that is required for FBW7-mediated degradation. We further show that FBW7 suppresses EZH2 activity and inhibits tumor migration and invasion via degradation of EZH2 in pancreatic cancer cells. Furthermore, immunohistochemistry analysis revealed that expression of EZH2 protein negatively correlates with FBW7 protein levels in a cohort of human pancreatic cancer specimens. Collectively, our findings demonstrate that FBW7 is a novel E3 ligase of EZH2 that regulates the EZH2 protein level in pancreatic cancer and represents a viable strategy for effective treatment of pancreatic cancer.

Aberrant activation of hedgehog signaling promotes cell proliferation via the transcriptional activation of forkhead Box M1 in colorectal cancer cells.

BACKGROUND: Recent evidence suggests that the aberrant activation of Hedgehog (Hh) signaling by Gli transcription factors is characteristic of a variety of aggressive human carcinomas, including colorectal cancer (CRC). Forkhead box M1 (FoxM1) controls the expression of a number of cell cycle regulatory proteins, and FoxM1 expression is elevated in a broad range of human malignancies, which suggests that it plays a crucial role in tumorigenesis. However, the mechanisms underlying FoxM1 expression are not fully understood. Here, we aim to further investigate the molecular mechanism by which Gli1 regulates FoxM1 in CRC. METHODS: Western blotting and immunohistochemistry (IHC) were used to evaluate FoxM1 and Gli1 protein expression, respectively, in CRC tissues and matched adjacent normal mucosa. BrdU (5-bromo-2'-deoxyuridine) and clone formation assays were used to clarify the influence of FoxM1 on CRC cell growth and proliferation. Chromatin immunoprecipitation (ChIP) and luciferase experiments were performed to explore the potential mechanisms by which Gli1 regulates FoxM1. Additionally, the protein and mRNA expression levels of Gli1 and FoxM1 in six CRC cell lines were measured using Western blotting and real-time PCR. Finally, the effect of Hh signaling on the expression of FoxM1 was studied in cell biology experiments, and the effects of Hh signaling activation and FoxM1 inhibition on the distribution of CRC cells among cell cycle phases was assessed by flow cytometry. RESULTS: Gli1 and FoxM1 were abnormally elevated in human CRC tissues compared with matched adjacent normal mucosa samples, and FoxM1 is a downstream target gene of the transcription factor Gli1 in CRC and promoted CRC cell growth and proliferation. Moreover, the aberrant activation of Hh signaling promoted CRC cell proliferation by directly binding to the promoter of FoxM1 and transactivating the activity of FoxM1 in CRC cells. CONCLUSION: The dysregulation of the Hh-Gli1-FoxM1 axis is essential for the proliferation and growth of human CRC cells and offers a potent target for therapeutic intervention in CRC.