Acyl-coenzyme A: cholesterol acyltransferase inhibitor avasimibe suppresses tumorigenesis and induces G1-phase cell-cycle arrest by activating PPARγ signaling pathway in bladder cancer.

Reprogramming of energy metabolism is one of the most important characteristics of tumors. Bladder cancer (BLCA) cells contain higher levels of cholesterol content compared to normal cells, and acyl-coenzyme A (CoA): cholesterol acyltransferase-1 (ACAT1) plays a crucial role in the esterification of cholesterol. Avasimibe is a drug that has been used in the treatment of atherosclerosis, and it can effectively inhibit ACAT1. We observed that ACAT1 was significantly up-regulated in BLCA and positively correlated with tumor grade. By avasimibe administration, the proliferation and migration ability of BLCA cells were reduced, while the production of ROS was strongly increased, accompanied by the up-regulated expression of ROS metabolism-related proteins SOD2 and catalase. Furthermore, BLCA cell cycle was arrested at the G1 phase, accompanied by the downregulation of cell cycle-related proteins (CCNA1/2, CCND1, CDK2 and CDK4), while the PPARγ was found to be up-regulated at both transcriptional and protein levels after avasimibe treatment. Then we found that the PPARγ antagonist GW9662 could reverse the effect of avasimibe on the cell cycle. Moreover, xenograft and pulmonary metastasis models further demonstrated that avasimibe could inhibit tumor cell growth and metastasis in vivo. Taken together, our results for the first time revealed that avasimibe can inhibit BLCA progression and metastasis, and PPARγ signaling pathway may play a key role in regulation of cell cycle distribution induced by avasimibe.

PRR11 promotes ccRCC tumorigenesis by regulating E2F1 stability.

Proline rich 11 (PRR11), a novel tumor-related gene, has been identified in different tumors. However, the relevant biological functions of PRR11 in human clear cell renal cell carcinoma (ccRCC) have not been studied. In this study, we first identified PRR11 as a biomarker of ccRCC and predictor of poor prognosis by bioinformatics. Then, we showed that PRR11 silencing substantially reduced ccRCC cell proliferation and migration in vitro and in vivo. Importantly, we found that PRR11 induced the degradation of the E2F1 protein through its interaction with E2F1, and PRR11 reduced the stability of the E2F1 protein in ccRCC cells, thereby affecting cell cycle progression. Further results indicated that the downregulation of E2F1 expression partially reversed the changes in ccRCC cell biology caused by PRR11 deletion. In addition, we showed that PRR11 was a target gene of c-Myc. The transcription factor c-Myc may have promoted the expression of PRR11 in ccRCC cells by binding to the PRR11 promoter region, thereby accelerating the progression of ccRCC. In summary, we found that PRR11 served as an oncogene in ccRCC, and PRR11 reduced the protein stability of E2F1 and could be activated by c-Myc.

The cholesterol esterification inhibitor avasimibe suppresses tumour proliferation and metastasis via the E2F-1 signalling pathway in prostate cancer.

BACKGROUND: New effective drugs for prostate cancer (PCa) treatment are urgently needed. Avasimibe was recently identified as a promising drug for anticancer therapies. The main purpose of this study was to explore the effects and the underlying mechanisms of avasimibe in prostate cancer. METHODS: In this study, MTT and clonogenic survival assays were performed to detect cell proliferation after avasimibe treatment. The effect of avasimibe on cell migration was measured by wound healing and transwell migration assays. Cell cycle distribution and apoptosis were detected by flow cytometry. Immunofluorescence staining and western blot analysis were used to detect the expression of cell cycle-related proteins and epithelial-mesenchymal transition (EMT)-related proteins. In vivo, the antitumour effects of avasimibe were evaluated using a xenograft model and pulmonary metastasis model. RESULTS: The study found that avasimibe suppresses tumour growth and triggers G1 phase arrest. Moreover, the expression of the cell cycle-related proteins CDK2/4/6, Cyclin D1 and Cyclin A1 + A2 was significantly increased and p21 expression was decreased after avasimibe treatment. The migration of PCa cells was attenuated after treatment with avasimibe, followed by the downregulation of the expression of the EMT-related proteins N-cadherin, ß-catenin, vimentin, Snail and MMP9 and upregulation of E-cadherin expression. Moreover, E2F-1 was elevated after treatment with avasimibe. After knockdown of E2F-1 expression, the inhibition of cell proliferation and migration caused by avasimibe was significantly recovered. The results of the xenograft model showed that avasimibe suppressed tumour growth in vivo. Immunofluorescence staining revealed lower levels of Ki67 and higher levels of E2F-1 in tumour tissues of the avasimibe group than those of the control group. A pulmonary metastasis model also confirmed the inhibition of PCa metastasis by avasimibe. The number of lung metastatic foci in the avasimibe group was significantly decreased compared with that in the control group. CONCLUSIONS: Our results suggest that avasimibe can suppress tumour proliferation and metastasis via the E2F-1 signalling pathway. These findings demonstrate the potential of avasimibe as a new effective drug for PCa treatment.

Wild-type IDH1 inhibits the tumor growth through degrading HIF-α in renal cell carcinoma.

The purpose of our study was to explore the effect and intrinsic mechanism of wild-type IDH1 and its substrate α-KG on renal cell carcinoma (RCC). IDH1 was observed lower expression in RCC cell lines. Phenotype experiment was carried out in the wild-type IDH1 and mutant IDH1R132H plasmid treated cell line. The results showed that the wild-type IDH1 could significantly inhibit the proliferation, migration and promote the apoptosis of RCC cell lines, which were consistent with the IDH1's substrate α-KG. The mutant IDH1R132H was found to lose this biological function of IDH1. Moreover, we verified the proliferation inhibition of IDH1 in vivo. In addition, we verified the correlation between IDH1 and hypoxia signal-related proteins in vitro and in vivo, specifically, IDH1 overexpression could significantly reduce the expression of HIF-1α and HIF-2α proteins and its downstream proteins (VEGF, TGF-α). Furthermore, we preliminarily verified the possibility of α-KG in the RCC's treatment by injecting α-KG into the xenograft model. α-KG significantly reduced tumor size and weight in tumor-bearing mice. This study provided a new therapeutic target and small molecule for the study of the treatment and mechanism of RCC.

KNSTRN promotes tumorigenesis and gemcitabine resistance by activating AKT in bladder cancer.

KNSTRN is a component of the mitotic spindle, which was rarely investigated in tumorigenesis. AKT plays an essential role in tumorigenesis by modulating the phosphorylation of various substrates. The activation of AKT is regulated by PTEN and PIP3. Here, we prove KNSTRN is positively correlated with malignancy of bladder cancer and KNSTRN activates AKT phosphorylation at Thr308 and Ser473. More importantly, our study reveals that both KNSTRN and PTEN interact with PH domain of AKT at cell membrane. The amount of KNSTRN interacted with AKT is negatively related to PTEN. Furthermore, PIP3 pull-down assay proves that KNSTRN promoted AKT movement to PIP3. These data suggest KNSTRN may activate AKT phosphorylation by promoting AKT movement to PIP3 and alleviating PTEN suppression. Based on the activation of AKT phosphorylation, our study demonstrates that KNSTRN promotes bladder cancer metastasis and gemcitabine resistance in vitro and in vivo. Meanwhile, the effect of KNSTRN on tumorigenesis and gemcitabine resistance could be restored by AKT specific inhibitor MK2206 or AKT overexpression. In conclusion, we identify an oncogene KNSTRN that promotes tumorigenesis and gemcitabine resistance by activating AKT phosphorylation and may serve as a therapeutic target in bladder cancer.

TRPM8 Inhibition Regulates the Proliferation, Migration and ROS Metabolism of Bladder Cancer Cells.

INTRODUCTION: Based on accumulating evidence, transient receptor potential (TRP) ion channels may play important roles in the occurrence and the progression of cancer. TRP melastatin 8 (TRPM8), a member of the TRP family, functions as a Ca2+-permeable channel and regulates various physiological and pathological processes. However, the effects of TRPM8 on bladder cancer (BCa) and its underlying mechanisms have not been elucidated. METHODS: BCa tissues and matched noncancerous tissues were collected to examine the expression of the TRPM8 mRNA and protein using qRT-PCR, Western blotting and immunofluorescence staining. Meanwhile, the effect of knockdown or inhibition of the activity of the TRPM8 protein on the proliferation, migration and ROS metabolism of bladder cancer cells was detected using the MTT assay, clonogenic survival assay, Transwell chamber migration assay, and reactive oxygen species (ROS) detection, respectively. Furthermore, a mouse model transplanted with BCa cells was established to assess tumor growth after TRPM8 expression was inhibited in vivo. RESULTS: Compared with the noncancerous tissues, the levels of TRPM8 in BCa tissues were significantly increased. Knockdown or inhibition of the activity of the TRPM8 protein in BCa cells reduced cell proliferation and migration. Moreover, the production of ROS was increased in cells treated with siTRPM8, which was accompanied by increased levels of Catalase, HO-1 and SOD2. Furthermore, a mouse model transplanted with the stable TRPM8-deficient T24 cell line was established, demonstrating that knockdown of TRPM8 delayed tumor growth in vivo. DISCUSSION: TRPM8 might play an essential for BCa tumor progression and metastasis by interfering with BCa cell proliferation, motility, ROS metabolism and migration.

A novel germline EGFR variant p.R831H causes predisposition to familial CDK12-mutant prostate cancer with tandem duplicator phenotype.

5-10% of total prostate cancer (PCa) cases are hereditary. Particularly, immune checkpoint inhibitor-sensitive tandem duplicator phenotype (TDP) accounts for 6.9% of PCa cases, whereas genetic susceptibility genes remain completely unknown. We identified a Chinese family with two PCa patients, in which the PCa phenotype co-segregated with a rare germline variant EGFRR831H. Patient-derived conditionally reprogrammed cells (CRC) exhibited increased EGFR and AKT phosphorylation, and a sensitivity to EGFR antagonist Afatinib in migration assays, suggesting the EGFR allele was constitutively active. Both EGFRR831H-mutant tumours contained biallelic CDK12 inactivation, together with prominent tandem duplication across the genome. These somatic mutations could be detected in urine before surgery. Analysis of public databases showed a significant correlation between the mutation status of EGFR and CDK12. Taken together, our genetic and functional analyses identified a previously undescribed link between EGFR and PCa.

BORA regulates cell proliferation and migration in bladder cancer.

BACKGROUND: Bladder cancer is having a gradually increasing incidence in China. Except for the traditional chemotherapy drugs, there are no emerging new drugs for almost 30 years in bladder cancer. New potential therapeutic targets and biomarkers are urgently needed. METHODS: BORA is the activator of kinase Aurora A and plays an important role in cell cycle progression. To investigate the function of BORA in BCa, we established BORA knockdown and overexpression cell models for in vitro studies, xenograft and pulmonary metastasis mouse models for in vivo studies. RESULTS: Our results indicated that BORA was upregulated in human bladder cancer (BCa) compared to the normal bladder and paracancerous tissues at transcriptional and translational levels. We found that BORA was positively related to BCa cell proliferation. Furthermore, BORA knockdown induced cell cycle arrest in G2/M phase while BORA overexpression decreased the proportion of cells in G2/M, associated with PLK1-CDC25C-CDK1 alteration. Interestingly, we observed that knockdown of BORA inhibited BCa cell migration and invasion, accompanied with alterations of epithelial-mesenchymal transition (EMT) pathway related proteins. In vivo studies confirmed the inhibition effect of BORA knockdown on BCa cell growth and migration. CONCLUSIONS: Our study indicates that BORA regulates BCa cell cycle and growth, meanwhile influences cell motility by EMT, and could be a novel biomarker and potential therapeutic target in BCa.

WFDC2 suppresses prostate cancer metastasis by modulating EGFR signaling inactivation.

WAP four-disulfide core domain 2 (WFDC2) is a small secretory protein that has been widely studied in ovarian cancer. It has been proven that WFDC2 promotes proliferation and metastasis in ovarian cancer, and serves as a diagnostic biomarker. However, the specific function of WFDC2 in prostate cancer has not been reported. Here, we first screened the diagnostic marker and favorable prognostic factor WFDC2 in prostate cancer by bioinformatics. WFDC2 expression was negatively correlated with Gleason score and metastasis in prostate cancer. Then, we revealed that overexpression of WFDC2, and addition of recombinant protein HE4 can significantly inhibit prostate cancer metastasis in vivo and in vitro. By co-immunoprecipitation and co-localization assays, we proved that WFDC2 binds to the extracellular domain of epidermal growth factor receptor (EGFR). Immunoblot showed that WFDC2 overexpression and recombinant protein HE4 addition inactivated the EGFR/AKT/GSK3B/Snail signaling pathway, and then restrained the progression of epithelial-mesenchymal transition. In conclusion, our study identified that the tumor suppressor WFDC2 can suppress prostate cancer metastasis by inactivating EGFR signaling.

The prediction value of PI-RADS v2 score in high-grade Prostate Cancer: a multicenter retrospective study.

Background: To explore the prediction value of PI-RADS v2 in high-grade prostate cancer and establish a prediction model combined with related variables of prostate cancer. Material and Methods: A total of 316 patients with newly discovered prostate cancer at Zhongnan Hospital of Wuhan University and Renmin Hospital of Wuhan University from December 2017 to August 2019 were enrolled in this study. The clinic information as age, tPSA, fPSA, prostate volume, Gleason score and PI-RADS v2 score have been collected. Univariate analysis was performed based on every variable to investigate the risk factors of high-grade prostate cancer. ROC curves were generated for the risk factors to distinguish the cut-off points. Logistic regression analyses were used to investigate the independent risk factors of high-grade prostate cancer. Nomogram prediction model was generated based on multivariate logistic regression analysis. The calibration curve, ROC curve, leave-one-out cross validation and independent external validation were performed to evaluate the discriminative ability, accuracy and stability of the nomogram prediction model. Results: Of 316 patients, a total of 187 patients were diagnosed as high-grade prostate cancer. Univariate analysis showed tPSA, fPSA, prostate volume, PSAD and PI-RADS v2 score were significantly different between the high- and low-grade prostate cancer patients. Univariate and multivariate logistic regression analyses showed only tPSA, prostate volume and PI-RADS v2 score were the independent risk factors of high-grade prostate cancer. The nomogram could predict the probability of high-grade prostate cancer, with a sensitivity of 79.4% and a specificity of 77.6%. The calibration curve displayed good agreement of the predicted probability with the actual observed probability. AUC of the ROC curve was 0.840 (0.797-0.884). Leave-one-out cross validation indicated the nomogram prediction model could classify 81.4% cases accurately. External data validation was performed with a sensitivity of 80.6% and a specificity of 77.3%, the Kappa value was 0.5755. Conclusions: PI-RADS v2 score had the value in predicting high-grade prostate cancer and the nomogram prediction model may help early diagnose the high risk prostate cancer.

The role and function of PPARγ in bladder cancer.

Peroxisome proliferator-activated receptor gamma (PPARγ), a member of the nuclear receptor superfamily, participates in multiple physiological and pathological processes. Extensive studies have revealed the relationship between PPARγ and various tumors. However, the expression and function of PPARγ in bladder cancer seem to be controversial. It has been demonstrated that PPARγ affects the occurrence and progression of bladder cancer by regulating proliferation, apoptosis, metastasis, and reactive oxygen species (ROS) and lipid metabolism, probably through PPARγ-SIRT1 feedback loops, the PI3K-Akt signaling pathway, and the WNT/ß-catenin signaling pathway. Considering the frequent relapses after chemotherapy, some researchers have focused on the relationship between PPARγ and chemotherapy sensitivity in bladder cancer. Moreover, the feasibility of PPARγ ligands as potential therapeutic targets for bladder cancer has been uncovered. Taken together, this review summarizes the relevant literature and our findings to explore the complicated role and function of PPARγ in bladder cancer.

Establishing the prediction models for recurrence and progression of T1G3 bladder urothelial carcinoma.

We aim to determine clinical recurrence and progression risk factors of T1G3 bladder cancer (BCa), and to establish recurrence and progression prediction models. 5-year follow-up records of 106 T1G3 BCa patients from January 2012 to December 2016 were analyzed for recurrence and progression. Two-sample T-test, Chi-square test, Mann-Whitney test, Kaplan-Meier curves, Cox univariate and multivariate analyses were performed to determine the independent risk factors. Effective prognostic nomograms were established to provide individualized prediction, and the calibration curves were founded to evaluate the agreements of the predicted probability with the actual observed probability. Receiver operating characteristic (ROC) curves were generated for the recurrence and progression prediction models. The stability of prediction models was validated with an external cohort included 61 T1G3 BCa patients. Of the 106 T1G3 BCa patients, 77 were males (72.6%) and 29 were females (27.4%), with median age 70 years. Within 5 years, recurrence was identified in 67 cases (63.2%), and progression was identified in 31 cases (29.2%). The results showed that large size of tumor, multifocal tumors, recrudescent tumor, non-BCG perfusion therapy were the independent risk factors for recurrence, and large size of tumor, multifocal tumors, recrudescent tumor, concomitant carcinoma in situ (CIS) were the independent risk factors for progression. However, no evidence shown that tumor location or operative method was independent risk factors for recurrence and progression. Based on the results of Cox regression analyses, the independent risk factors were used to establish the prediction nomograms to calculate the recurrence and progression probability of each T1G3 BCa patient. Calibration curves, ROC curves and external validation displayed that the nomograms had great value of prediction.

ACAT1 and Metabolism-Related Pathways Are Essential for the Progression of Clear Cell Renal Cell Carcinoma (ccRCC), as Determined by Co-expression Network Analysis.

Kidney cancer ranks as one of the top 10 causes of cancer death; this cancer is difficult to detect, difficult to treat, and poorly understood. The most common subtype of kidney cancer is clear cell renal cell carcinoma (ccRCC) and its progression is influenced by complex gene interactions. Few clinical studies have investigated the molecular markers associated with the progression of ccRCC. In this study, we collected microarray profiles of 72 ccRCCs and matched normal samples to identify differentially expressed genes (DEGs). Then a weighted gene co-expression network analysis (WGCNA) was conducted to identify co-expressed gene modules. By relating all co-expressed modules to clinical features, we found that the brown module and Fuhrman grade had the highest correlation (r = -0.8, p = 1e-09). Thus, the brown module was regarded as a clinically significant module and subsequently analyzed. Functional annotation showed that the brown module focused on metabolism-related biological processes and pathways, such as fatty acid oxidation and amino acid metabolism. We then performed a protein-protein interaction (PPI) network to identify the hub nodes in the brown module. It is worth noting that only one candidate, acetyl-CoA acetyltransferase (ACAT1), was considered to be the final target most relevant to the Fuhrman grade of ccRCC, by applying the intersection of hub genes in the co-expressed network and the PPI network. ACAT1 was subsequently validated using another two external microarray datasets and the TCGA dataset. Intriguingly, validation results indicated that ACAT1 was negatively correlated with four grades of ccRCC, which was also consistent with our results from qRT-PCR analysis and immunohistochemistry staining of clinical samples. Overexpression of ACAT1 inhibited the proliferation and migration of human ccRCC cells in vitro. In addition, the Kaplan-Meier survival curve showed that patients with a lower expression of ACAT1 showed a significantly lower overall survival rate and disease-free survival rate, indicating that ACAT1 could act as a prognostic and recurrence/progression biomarker of ccRCC. In summary, we found and confirmed that ACAT1 might help to identify the progression of ccRCC, which might have important clinical implications for enhancing risk stratification, therapeutic decision, and prognosis prediction in ccRCC patients.

Predicting recurrence of nonmuscle-invasive bladder cancer (Ta-T1): A study based on 477 patients.

The aim of this study was to determine clinical recrudescent risk factors of 477 patients with newly discovered nonmuscle-invasive bladder cancer (NMIBC) (Ta-T1) in our hospital, and based on these factors, to establish a recurrence risk prediction model of each NMIBC patient.This study included 477 patients with newly discovered NMIBC (Ta-T1) from January 2012 to December 2016; all patients were treated surgically by transurethral resection of bladder tumor (TURBT). The outcomes of patients were with or without recurrence within 2 years. The nomograms were based on Cox regression analyses, and the calibration curves were founded to evaluate the agreements of the predicted probability with the actual observed probability.Of the 477 patients with NMIBC, 392 were males (82.2%) and 85 were females (17.8%), with median age 64 years. Recurrence was identified in 327 cases (68.6%). The results showed that old age, female sex, smoking history, large size of tumor, multifocal tumors, high grade, and high stage are risk factors for NMIBC recurrence, whereas no significant association was seen between tumor location and recurrence in our study. Based on the results of Cox regression analyses, several independent risk factors, including smoking history, tumor size, multifocal, immediate infusion therapy, T stage, and tumor grade, were used to establish a nomogram to calculate the recurrence probability of each NMIBC patient, and the calibration curve displayed that this nomogram had a great value of prediction.Old age, female sex, smoking history, large size of tumor, multifocal tumors, high grade, and high stage are risk factors for NMIBC recurrence, whereas immediate infusion therapy is a protective factor. And a nomogram was established as a prediction model to calculate the recurrence probability of NMIBC patients.

Identification of several cell cycle relevant genes highly correlated with the progression and prognosis of human bladder urothelial tumor.

The incidence of bladder cancer (BCa) in China is the highest among genitourinary system tumors, and its progression is affected by multitudinous pathways, of which cell cycle progress plays an important role. This study screened and enriched differentially expressed genes (DEGs) from four gene expression profiles using bioinformatics analysis methods. The enrichment and analysis of gene function showed that these genes were highly correlated with cell cycle regulation. Identification of candidate small molecules was conducted to evaluate the application of clinical transformation in these DEGs. Prognostic and stage-related expression analysis further sorted five highly expressed genes associated with worse prognosis and higher stages in patients with BCa. Further analysis revealed their interaction in cell cycle regulation and genetical alteration. Meanwhile, we validated the elevated expression of these genes through transcription and translation levels. Taking the results together, we could infer that these five genes are valuable in diagnosis, prediction, and providing candidate therapeutic targets for patients with BCa in different stages.