Glycyrrhetinic Acid as a Hepatocyte Targeting Ligand-Functionalized Platinum(IV) Complexes for Hepatocellular Carcinoma Therapy and Overcoming Multidrug Resistance.

Promising targeted therapy options to overcome drug resistance and side effects caused by platinum(II) drugs for treatment in hepatocellular carcinoma are urgently needed. Herein, six novel multifunctional platinum(IV) complexes through linking platinum(II) agents and glycyrrhetinic acid (GA) were designed and synthesized. Among them, complex 20 showed superior antitumor activity against tested cancer cells including cisplatin resistance cells than cisplatin and simultaneously displayed good liver-targeting ability. Moreover, complex 20 can significantly cause DNA damage and mitochondrial dysfunction, promote reactive oxygen species generation, activate endoplasmic reticulum stress, and eventually induce apoptosis. Additionally, complex 20 can effectively inhibit cell migration and invasion and trigger autophagy and ferroptosis in HepG-2 cells. More importantly, complex 20 demonstrated stronger tumor inhibition ability than cisplatin or the combo of cisplatin/GA with almost no systemic toxicity in HepG-2 or A549 xenograft models. Collectively, complex 20 could be developed as a potential anti-HCC agent for cancer treatment.

Predictive model and risk analysis for coronary heart disease in people living with HIV using machine learning.

OBJECTIVE: This study aimed to construct a coronary heart disease (CHD) risk-prediction model in people living with human immunodeficiency virus (PLHIV) with the help of machine learning (ML) per electronic medical records (EMRs). METHODS: Sixty-one medical characteristics (including demography information, laboratory measurements, and complicating disease) readily available from EMRs were retained for clinical analysis. These characteristics further aided the development of prediction models by using seven ML algorithms [light gradient-boosting machine (LightGBM), support vector machine (SVM), eXtreme gradient boosting (XGBoost), adaptive boosting (AdaBoost), decision tree, multilayer perceptron (MLP), and logistic regression]. The performance of this model was assessed using the area under the receiver operating characteristic curve (AUC). Shapley additive explanation (SHAP) was further applied to interpret the findings of the best-performing model. RESULTS: The LightGBM model exhibited the highest AUC (0.849; 95% CI, 0.814-0.883). Additionally, the SHAP plot per the LightGBM depicted that age, heart failure, hypertension, glucose, serum creatinine, indirect bilirubin, serum uric acid, and amylase can help identify PLHIV who were at a high or low risk of developing CHD. CONCLUSION: This study developed a CHD risk prediction model for PLHIV utilizing ML techniques and EMR data. The LightGBM model exhibited improved comprehensive performance and thus had higher reliability in assessing the risk predictors of CHD. Hence, it can potentially facilitate the development of clinical management techniques for PLHIV care in the era of EMRs.

Novel NF-κB Inhibitor-Conjugated Pt(IV) Prodrug to Enable Cancer Therapy through ROS/ER Stress and Mitochondrial Dysfunction and Overcome Multidrug Resistance.

Although cisplatin has been widely used for clinical purposes, its application is limited due to its obvious side effects. To mitigate the defects of cisplatin, here, six "multitarget prodrugs" were synthesized by linking cisplatin and NF-κB inhibitors. Notably, complex 9 demonstrated a 63-fold enhancement in the activity against A549/CDDP cells with lower toxicity toward normal LO2 cells compared to cisplatin. Additionally, complex 9 could effectively cause DNA damage, induce mitochondrial dysfunction, generate reactive oxygen species, and induce cell apoptosis through the mitochondrial pathway and ER stress. Remarkably, complex 9 effectively inhibited the NF-κB/MAPK signaling pathway and disrupted the PI3K/AKT signaling transduction. Importantly, complex 9 showed superior in vivo antitumor efficiency compared to cisplatin or the combination of cisplatin/4, without obvious systemic toxicity in A549 or A549/CDDP xenograft models. Our results demonstrated that the dual-acting mechanism endowed the complexes with high efficiency and low toxicity, which may represent an efficient strategy for cancer therapy.

HERVK-mediated regulation of neighboring genes: implications for breast cancer prognosis.

Human endogenous retroviruses (HERVs) are the remnants of ancient retroviral infections integrated into the human genome. Although most HERVs are silenced or rendered inactive by various regulatory mechanisms, they retain the potential to influence the nearby genes. We analyzed the regulatory map of 91 HERV-Ks on neighboring genes in human breast cancer and investigated the impact of HERV-Ks on the tumor microenvironment (TME) and prognosis of breast cancer. Nine RNA-seq datasets were obtained from GEO and NCBI SRA. Differentially expressed genes and HERV-Ks were analyzed using DESeq2. Validation of high-risk prognostic candidate genes using TCGA data. These included Overall survival (multivariate Cox regression model), immune infiltration analysis (TIMER), tumor mutation burden (maftools), and drug sensitivity analysis (GSCA). A total of 88 candidate genes related to breast cancer prognosis were screened, of which CD48, SLAMF7, SLAMF1, IGLL1, IGHA1, and LRRC8A were key genes. Functionally, these six key genes were significantly enriched in some immune function-related pathways, which may be associated with poor prognosis for breast cancer (p = 0.00016), and the expression levels of these genes were significantly correlated with the sensitivity of breast cancer treatment-related drugs. Mechanistically, they may influence breast cancer development by modulating the infiltration of various immune cells into the TME. We further experimentally validated these genes to confirm the results obtained from bioinformatics analysis. This study represents the first report on the regulatory potential of HERV-K in the neighboring breast cancer genome. We identified three key HERV-Ks and five neighboring genes that hold promise as novel targets for future interventions and treatments for breast cancer.

Dual-targeting tumor cells hybrids derived from Pt(IV) species and NF-κB inhibitors enables cancer therapy through mitochondrial dysfunction and ER stress and overcomes cisplatin resistance.

To ameliorate the defects including serious side effects and drug resistance of Pt(II) drugs (e.g., cisplatin and oxaliplatin), here a novel of "dual-prodrug" by containing Pt(II) drugs and NF-κB inhibitors were synthesized and characterized. Among them, Pt(IV) complex 11 exhibited better cytotoxic activity than other Pt(IV) complexes and the corresponding Pt(II) drugs, with IC50 values ranged from 0.31 to 0.91 µM, respectively, and also displayed low toxicity toward two normal cells HL-7702 and BEAS-2B. More importantly, complex 11 significantly reversed cisplatin resistance in A549/CDDP cells, indicating that complex 11 was able to overcome multidrug resistance. Following mechanism studies demonstrated that complex 11 significantly induced DNA damage and ROS generation, arrest the cell cycle at the G2/M stage, suppressed cell migration and intrusion, and induced cell apoptosis through activated ER stress and mitochondrial apoptosis pathway in A549 cells. Moreover, complex 11 effectively suppressed the IKKß phosphorylation, IκBα phosphorylation and NF-κB p65 phosphorylation and nuclear translocation, leading to blocked the NF-κB signal pathway in A549 cells. In vivo tests showed that the inhibitory rate in the complex 11 reached 69.2 %, which was much higher than that of oxaliplatin (55.6 %), 1a (39.7 %) and the combination of oxaliplatin/1a (65.1 %), without causing loss in the body weight.

GABBR2 as a Downstream Effector of the Androgen Receptor Induces Cisplatin Resistance in Bladder Cancer.

The precise molecular mechanisms responsible for resistance to cisplatin-based chemotherapy in patients with bladder cancer remain elusive, while we have indicated that androgen receptor (AR) activity in urothelial cancer is associated with its sensitivity. Our DNA microarray analysis in control vs. AR-knockdown bladder cancer sublines suggested that the expression of a GABA B receptor GABBR2 and AR was correlated. The present study aimed to determine the functional role of GABBR2 in modulating cisplatin sensitivity in bladder cancer. AR knockdown and dihydrotestosterone treatment considerably reduced and induced, respectively, GABBR2 expression, and the effect of dihydrotestosterone was at least partially restored by an antiandrogen hydroxyflutamide. A chromatin immunoprecipitation assay further revealed the binding of AR to the promoter region of GABBR2 in bladder cancer cells. Meanwhile, GABBR2 expression was significantly elevated in a cisplatin-resistant bladder cancer subline, compared with control cells. In AR-positive bladder cancer cells, knockdown of GABBR2 or treatment with a selective GABA B receptor antagonist, CGP46381, considerably enhanced the cytotoxic activity of cisplatin. However, no additional effect of CGP46381 on cisplatin-induced growth suppression was seen in GABBR2-knockdown cells. Moreover, in the absence of cisplatin, CGP46381 treatment and GABBR2 knockdown showed no significant changes in cell proliferation or migration. These findings suggest that GABBR2 represents a key downstream effector of AR signaling in inducing resistance to cisplatin treatment. Accordingly, inhibition of GABBR2 has the potential of being a means of chemosensitization, especially in patients with AR/GABBR2-positive bladder cancer.

ZNF500 abolishes breast cancer proliferation and sensitizes chemotherapy by stabilizing P53 via competing with MDM2.

Zinc finger protein 500 (ZNF500) has an unknown expression pattern and biological function in human tissues. Our study revealed that the ZNF500 mRNA and protein levels were higher in breast cancer tissues than those in their normal counterparts. However, ZNF500 expression was negatively correlated with advanced TNM stage (p = 0.018), positive lymph node metastasis (p = 0.014), and a poor prognosis (p < 0.001). ZNF500 overexpression abolished in vivo and in vitro breast cancer cell proliferation by activating the p53-p21-E2F4 signaling axis and directly interacting with p53 via its C2H2 domain. This may prevent ubiquitination of p53 in a manner that is competitive to MDM2, thus stabilizing p53. When ZNF500-∆C2H2 was overexpressed, the suppressed proliferation of breast cancer cells was neutralized in vitro and in vivo. In human breast cancer tissues, ZNF500 expression was positively correlated with p53 (p = 0.022) and E2F4 (p = 0.004) expression. ZNF500 expression was significantly lower in patients with Miller/Payne Grade 1-2 than in those with Miller/Payne Grade 3-5 (p = 0.012). ZNF500 suppresses breast cancer cell proliferation and sensitizes cells to chemotherapy.

Clinicopathologic features, tumor immune microenvironment and genomic landscape of EBV-related and EBV-unrelated poorly differentiated nonkeratinizing squamous cell carcinoma of the thymus.

OBJECTIVES: Knowledge regarding thymic EBV-related poorly differentiated nonkeratinizing squamous cell carcinoma (PDNKSCC), also known as lymphoepithelial carcinoma (LEC), is extremely limited due to its rarity. MATERIALS AND METHODS: This multi-institutional study enrolled 85 patients with thymic PDNKSCC. DNA in situ hybridization was performed to evaluate the EBV status of all 85 cases. Immunohistochemistry and next generation sequencing were performed to compare the differences in the clinicopathological and molecular features between EBV-related and EBV-unrelated PDNKSCC. Tumor-infiltrating lymphocytes (TILs) were also analyzed by these methods. RESULTS: The 85 cases were classified into 27 EBV-related PDNKSCCs (31.8 %) and 58 EBV-unrelated PDNKSCCs (68.2 %) according to the EBV status, and 35 Lymphoepithelioma pattern (LP) (41.2 %) and 50 desmoplastic pattern (DP) (58.8 %) according to the histological characteristics. Compared to the EBV-unrelated PDNKSCC, EBV-related PDNKSCC showed a younger patient predominance and more commonly displayed a LP subtype. Additionally, LP-type cases were divided into two groups: Group 1 (EBV-related, 20/85) and Group 2 (EBV-unrelated, 15/85); the DP-type cases were divided into Group 3 (EBV-unrelated, 43/85) and Group 4 (EBV-related, 7/85). The four Groups showed a significant association with patients' OS and PFS. EBV-related PDNKSCC had significantly higher PD-L1 + tumor cells (TCs) and PD-L1 + and CD8 + immune cells (ICs) than EBV-unrelated PDNKSCC. The tumor microenvironment immune type (TMIT) I (PDL1-Tumor+/CD8-High) was more common in EBV-related PDNKSCC, especially in Group 1(LP and EBV related) with more than 90 % cases belonged to TMIT I. Molecular analysis demonstrated that EBV-related PDNKSCC had a significantly higher tumour mutational burden and frequency of somatic mutations than EBV-unrelated cases. CONCLUSIONS: EBV-related PDNKSCC, especially the Group 1, could be a candidate for immunotherapy and EBV positivity may provide an indication for the selection of targeted therapy due to their high tumour mutational burden.

Protective role of mineralocorticoid receptor signaling in urothelial tumorigenesis.

The expression status of mineralocorticoid receptor (MR) and its biological significance in human urothelial carcinoma remain unknown. The present study aimed to determine the functional role of MR in the development of urothelial cancer. In human normal urothelial SVHUC cells with exposure to a chemical carcinogen 3-methylcholanthrene (MCA), we assessed the effects of a natural MR ligand, aldosterone, and 3 MR antagonists, including spironolactone, eplerenone, and esaxerenone, as well as knockdown of MR via shRNA virus infection, on their neoplastic/malignant transformation. The in vitro system with carcinogen challenge showed that aldosterone and anti-mineralocorticoids significantly prevented and promoted, respectively, the neoplastic transformation of SVHUC cells. Similarly, MR knockdown in SVHUC cells considerably induced MCA-mediated neoplastic transformation, compared with a control subline. In addition, MR knockdown or antagonist treatment resulted in increases in the expression of ß-catenin, c-Fos, and N-cadherin, and a decrease in that of E-cadherin. Meanwhile, spironolactone, which is known to possess anti-androgenic activity, rather suppressed the neoplastic transformation of a SVHUC subline stably expressing wild-type androgen receptor, indicating its dominant effect via the androgen receptor pathway. Immunohistochemistry in surgical specimens detected MR signals in 77 (98.7%; 23.1% weak/1+, 42.3% moderate/2+, and 33.3% strong/3+) of 78 non-invasive bladder tumors, which was significantly (P<0.001) lower than in adjacent non-neoplastic urothelial tissues (100%; 20.5% 2+ and 79.5% 3+). Moreover, the risks for disease recurrence after transurethral surgery were marginally lower in female patients with MR-high (2+/3+) tumor (P=0.068) and significantly lower in all patients with MR-high/glucocorticoid receptor-high tumor (P=0.025), compared with respective controls. These findings suggest that MR signaling functions as a suppressor for urothelial tumorigenesis.

ARHGEF40 promotes non-small cell lung cancer proliferation and invasion via the AKT-Wnt axis by binding to RhoA.

Rho guanine nucleotide exchange factor 40 (ARHGEF40) is a member of the Dbl-family of guanine nucleotide factor proteins. However, its expression pattern and biological function in malignant tumors, notably in nonsmall cell lung cancer (NSCLC) are currently unknown. The present study demonstrated that ARHGEF40 was highly expressed in NSCLC specimens and that its expression was significantly associated with advanced TNM stage (p < 0.001), lymph node metastasis (p = 0.002), and poor prognosis (p = 0.0056). In addition, ARHGEF40 accelerated nuclear translocation of the key component ß-catenin and increased the expression levels of the Wnt signaling pathway targets c-myc, cyclin D1 and MMP7. Moreover, it promoted lung cancer cell proliferation and invasion in vitro and in vivo. To elucidate the underlying molecular mechanism, the current study demonstrated that ARHGEF40 could induce activation of the Wnt signaling pathway by increasing the phosphorylation levels of AKT and GSK3ß via interaction with RhoA. Moreover, the Dbl homology (DH)-pleckstrin homology (PH) domain of ARHGEF40 was responsible for this interaction. Its deletion abolished the binding, which blocked the activation of the Wnt signaling. Taken together, the data indicated that ARHGEF40 promoted the malignant phenotype of lung cancer cells by activating the AKT-Wnt axis. This was achieved by its interaction with RhoA via the DH-PH domain. ARHGEF40 may serve as a novel target for NSCLC treatment.

Androgen Receptor Signaling Induces Cisplatin Resistance via Down-Regulating GULP1 Expression in Bladder Cancer.

The underlying molecular mechanisms of resistance to cisplatin-based systemic chemotherapy in bladder cancer patients remain to be elucidated, while the link between androgen receptor (AR) activity and chemosensitivity in urothelial cancer has been implicated. Our DNA microarray analysis in control vs. AR knockdown bladder cancer lines identified GULP1 as a potential target of AR signaling. We herein determined the relationship between AR activity and GULP1 expression in bladder cancer cells and then assessed the functional role of GULP1 in cisplatin sensitivity. Androgen treatment in AR-positive cells or AR overexpression in AR-negative cells considerably reduced the levels of GULP1 expression. Chromatin immunoprecipitation further showed direct interaction of AR with the promoter region of GULP1. Meanwhile, GULP1 knockdown sublines were significantly more resistant to cisplatin treatment compared with respective controls. GULP1 knockdown also resulted in a significant decrease in apoptosis, as well as a significant increase in G2/M phases, when treated with cisplatin. In addition, GULP1 was immunoreactive in 74% of muscle-invasive bladder cancers from patients who had subsequently undergone neoadjuvant chemotherapy, including 53% of responders showing moderate (2+)/strong (3+) expression vs. 23% of non-responders showing 2+/3+ expression (P = 0.044). These findings indicate that GULP1 represents a key downstream effector of AR signaling in enhancing sensitivity to cisplatin treatment.

Thymic adenocarcinoma accompanied by type A thymoma and pulmonary minimally invasive adenocarcinoma and harboring distinct gene alterations: A case report.

RATIONALE: Thymic adenocarcinoma is an extremely rare thymic carcinoma. The exact genetic alteration associated with thymic adenocarcinoma is unclear. Here, we report a case of thymic adenocarcinoma accompanied by type A thymoma and pulmonary minimally invasive adenocarcinoma (MIA). PATIENT CONCERNS: A 53-year-old woman presented with multiple nodules in the mediastinum and lung. Thoracic computed tomography revealed nodules in the anterior superior mediastinum and anterior mediastinum near the right pericardium and ground-glass opacity (GGO) in the right superior lobe of the lung. DIAGNOSIS: The tumor in the anterior superior mediastinum was diagnosed as primary thymic papillary adenocarcinoma. The tumor in the anterior mediastinum near the right pericardium was diagnosed as type A thymoma. The GGO of the right superior lobe of the lung was diagnosed as a MIA. INTERVENTION: The patient underwent thoracoscopic mediastinal tumor resection and partial lobectomy in our hospital. OUTCOMES: The postoperative course was uneventful. The patient is alive and free of the disease for 22 months after diagnosis. LESSONS: Thyroid transcription factor 1 (TTF-1) was positive in this case of thymic adenocarcinoma, which indicated that a thymic adenocarcinoma with TTF-1-positive may not necessarily be a metastasis of lung or thyroid adenocarcinoma. The positive staining of CD5 and CD117 can help us to confirm the thymic origin. Molecular genetic analysis indicated that these tumors harbored different mutations. The thymic adenocarcinoma and type A thymoma both had the mutation of KMT2A, but the mutation sites were different. KMT2A mutation may be a common genetic change in thymic tumorigenesis. The genetic alterations disclosed in this study will help expand the understanding of thymic tumors.

Identification of BXDC2 as a Key Downstream Effector of the Androgen Receptor in Modulating Cisplatin Sensitivity in Bladder Cancer.

Underlying mechanisms for resistance to cisplatin-based chemotherapy in bladder cancer patients are largely unknown, although androgen receptor (AR) activity, as well as extracellular signal-regulated kinase (ERK) signaling, has been indicated to correlate with chemosensitivity. We also previously showed ERK activation by androgen treatment in AR-positive bladder cancer cells. Because our DNA microarray analysis in control vs. AR-knockdown bladder cancer lines identified BXDC2 as a potential downstream target of AR, we herein assessed its functional role in cisplatin sensitivity, using bladder cancer lines and surgical specimens. BXDC2 protein expression was considerably downregulated in AR-positive or cisplatin-resistant cells. BXDC2-knockdown sublines were significantly more resistant to cisplatin, compared with respective controls. Without cisplatin treatment, BXDC2-knockdown resulted in significant increases/decreases in cell proliferation/apoptosis, respectively. An ERK activator was also found to reduce BXDC2 expression. Immunohistochemistry showed downregulation of BXDC2 expression in tumor (vs. non-neoplastic urothelium), higher grade/stage tumor (vs. lower grade/stage), and AR-positive tumor (vs. AR-negative). Patients with BXDC2-positive/AR-negative muscle-invasive bladder cancer had a significantly lower risk of disease-specific mortality, compared to those with a BXDC2-negative/AR-positive tumor. Additionally, in those undergoing cisplatin-based chemotherapy, BXDC2 positivity alone (p = 0.083) or together with AR negativity (p = 0.047) was associated with favorable response. We identified BXDC2 as a key molecule in enhancing cisplatin sensitivity. AR-ERK activation may thus be associated with chemoresistance via downregulating BXDC2 expression in bladder cancer.

Estrogen receptor-ß signaling induces cisplatin resistance in bladder cancer.

The efficacy of cisplatin-based chemotherapy in patients with bladder cancer is often limited due to the development of therapeutic resistance. Our recent findings in bladder cancer suggested that activation of prostaglandin receptors (e.g. EP2, EP4) or cyclooxygenase (COX)-2 induced cisplatin resistance. Meanwhile, emerging evidence indicates the involvement of estrogen receptor-ß (ERß) signals in urothelial cancer progression. In this study, we aimed to investigate whether ERß activity was associated with cisplatin sensitivity in bladder cancer. Immunohistochemistry in muscle-invasive bladder cancer specimens from 55 patients who had subsequently received at least 3 cycles of cisplatin + gemcitabine neoadjuvant chemotherapy showed that ERß was positive in 38% of responders vs. 71% of non-responders (P = 0.016), including 42% of male responders vs. 65% of male non-responders (P = 0.142) and 20% of female responders vs. 100% of female non-responders (P = 0.048). Then, cisplatin cytotoxicity was compared in human bladder cancer cell lines. Control sublines endogenously expressing ERß were significantly more resistant to cisplatin treatment at its pharmacological concentrations, compared with ERß knockdown sublines via short hairpin RNA virus infection. An ER modulator tamoxifen increased sensitivity to cisplatin in ERα-negative/ERß-positive cell lines, while, in an estrogen-depleted condition, 17ß-estradiol reduced it. Additionally, western blot showed considerable elevation in ERß expression in cisplatin-resistant bladder cancer sublines, compared with respective controls. Moreover, treatment with tamoxifen or a COX-2 inhibitor celecoxib increased cisplatin sensitivity even in resistant cells, while COX-2/EP2/EP4 inhibitor treatment resulted in reduced expression of ERß. The expression and activity of ß-catenin known to involve cisplatin resistance was also up-regulated in cisplatin-resistant cells, which was further induced by 17ß-estradiol treatment. The present results suggest that estrogen-mediated ERß signaling plays an important role in modulating cisplatin sensitivity in bladder cancer cells. Targeting ERß during chemotherapy may thus be a useful strategy to overcome cisplatin resistance especially in female patients with ERß-positive bladder cancer.

Primary thymic carcinoma with adenoid cystic carcinoma-like features: A case report and literature review.

RATIONALE: Thymic carcinoma with adenoid cystic carcinoma-like features is a special subtype of thymic adenocarcinoma, and the occurrence of this condition is extremely rare. Herein, we report a case of primary thymic carcinoma with adenoid cystic carcinoma-like features in a young man. PATIENT CONCERNS: A 38-year-old man had an incidental finding of space-occupying lesion in the anterior mediastinum during a routine health examination. The patient complained of occasional mild chest tightness during hot weather but had no obvious cough, sputum, chest pain, or fever. Contrast-enhanced computed tomography scan of the chest revealed a space-occupying lesion in the anterior mediastinum, which is likely benign. DIAGNOSIS: The lesion was diagnosed as a primary thymic carcinoma with adenoid cystic carcinoma-like features. INTERVENTION: The patient underwent thoracoscopic resection of left anterior mediastinal mass and enlarged resection of thymectomy and mediastinal fat in our hospital. OUTCOMES: The postoperative course was uneventful. LESSONS: The tissue characteristic of this tumor was extremely similar to that of adenoid cystic carcinoma. A precise pathological examination is extremely important to prevent misdiagnoses of the lesion as adenoid cystic carcinoma or other thymic tumors. Immunohistochemical staining is extremely useful for the pathological and differential diagnoses of this tumor.

Androgen Receptor Signaling Reduces the Efficacy of Bacillus Calmette-Guérin Therapy for Bladder Cancer via Modulating Rab27b-Induced Exocytosis.

Although intravesical bacillus Calmette-Guérin (BCG) immunotherapy has been the gold standard for nonsurgical management of non-muscle-invasive bladder cancer, a considerable number of patients exhibit resistance to the adjuvant treatment with unexplained mechanisms. This study aimed to investigate whether and how androgen receptor (AR) signals modulate BCG cytotoxicity in bladder cancer. AR knockdown or overexpression in bladder cancer lines resulted in induction or reduction, respectively, in intracellular BCG quantity and its cytotoxic activity. Microarray screening identified Rab27b, a small GTPase known to mediate bacterial exocytosis, which was upregulated in BCG-resistant cells and downregulated in AR-shRNA cells. Knockdown of Rab27b, or its effector SYTL3, or overexpression of Rab27b also induced or reduced, respectively, BCG quantity and cytotoxicity. In addition, treatment with GW4869, which was previously shown to inhibit Rab27b-dependent secretion, induced them and reduced Rab27b expression in bladder cancer cells. Meanwhile, AR expression was upregulated in BCG-resistant lines, compared with respective controls. In a mouse orthotopic xenograft model, Rab27b/SYTL3 knockdown or GW4869 treatment enhanced the amount of BCG within tumors and its suppressive effect on tumor growth. Moreover, in non-muscle-invasive bladder cancer specimens from patients subsequently undergoing BCG therapy, positivity of AR/Rab27b expression was associated with significantly higher risks of tumor recurrence. AR activation thus correlates with resistance to BCG treatment, presumably via upregulating Rab27b expression. Mechanistically, it is suggested that BCG elimination from urothelial cells is induced by Rab27b/SYTL3-mediated exocytosis. Accordingly, Rab27b inactivation, potentially via antiandrogenic drugs and/or exocytosis inhibition are anticipated to sensitize the efficacy of BCG therapy, especially in patients with BCG-refractory AR/Rab27b-positive bladder cancer.

FOXO1 inactivation induces cisplatin resistance in bladder cancer.

We found that FOXO1-shRNA sublines or FOXO1-positive cells co-treated with a FOXO1 inhibitor were significantly more resistant to cisplatin treatment at pharmacological concentrations, compared with respective control sublines or those with mock treatment. Western blot demonstrated considerable increases in the expression levels of a phosphorylated inactive form of FOXO1 (p-FOXO1) in cisplatin-resistant sublines established by long-term culture with low/increasing doses of cisplatin, compared with respective controls. Immunohistochemistry in surgical specimens from patients with muscle-invasive bladder cancer undergoing cisplatin-based neoadjuvant therapy further showed a strong trend to associate between p-FOXO1 positivity and unfavorable response to chemotherapy.

Compound A inhibits urothelial tumorigenesis via both the androgen receptor and glucocorticoid receptor signaling pathways.

Recent preclinical evidence has indicated that both androgen receptor (AR) inactivation and glucocorticoid receptor (GR) transrepression are associated with suppression of urothelial carcinogenesis. We therefore assessed the effect of a unique compound, 2-(4-acetoxyphenyl)-2-chloro-N-methylethylammonium chloride (Compound A; CpdA), which could function as an AR antagonist as well as a GR ligand, on urothelial tumorigenesis. Using the in vitro system with GR-positive non-neoplastic urothelial SVHUC cells stably expressing AR (SVHUC-AR), neoplastic transformation induced by a chemical carcinogen 3-methylcholanthrene (MCA) was inhibited similarly by an anti-androgen hydroxyflutamide and a glucocorticoid prednisone, and more strongly by CpdA. CpdA also prevented the neoplastic transformation of AR-negative MCA-SVHUC cells, which was diminished by a GR antagonist RU486, but failed to prevent that of GR knockdown MCA-SVHUC cells. In MCA-SVHUC-AR cells, CpdA significantly reduced the expression levels of oncogenes (c-Fos/c-Jun/c-Myc) and induced those of tumor suppressors (UGT1A/p21/p27/p53/PTEN). Additionally, a potent carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine induced bladder cancer in all of 8 mock-treated mice versus 4 (50%) of flutamide-treated (P = 0.021), 4 (50%) of prednisone-treated (P = 0.021), or 2 (25%) of CpdA-treated (P = 0.002) animals. Finally, CpdA was found to reduce AR transactivation and selectively induce GR transrepression (i.e. suppression of NF-κB transactivation and expression of its regulated genes), but not GR transactivation (i.e. activation of glucocorticoid-response element-mediated transcription and expression of its targets) in SVHUC cells. These findings suggest that CpdA suppresses urothelial tumorigenesis via both the AR and GR pathways, which may consequently provide an effective option of chemoprevention for bladder cancer, especially in patients with superficial disease following transurethral surgery.

FOXO1 as a tumor suppressor inactivated via AR/ERß signals in urothelial cells.

Androgen receptor (AR) and estrogen receptor-ß (ERß) have been implicated in urothelial tumor outgrowth as promoters, while underlying mechanisms remain poorly understood. Our transcription factor profiling previously performed identified FOXO1 as a potential downstream target of AR in bladder cancer cells. We here investigated the functional role of FOXO1 in the development and progression of urothelial cancer in relation to AR and ERß signals. In non-neoplastic urothelial SVHUC cells or bladder cancer lines, AR/ERß expression or dihydrotestosterone/estradiol treatment reduced the expression levels of FOXO1 gene and induced those of a phosphorylated inactive form of FOXO1 (p-FOXO1). In chemical carcinogen-induced models, FOXO1 knockdown via shRNA or inhibitor treatment resulted in considerable induction of the neoplastic transformation of urothelial cells or bladder cancer development in mice. Similarly, FOXO1 inhibition considerably induced the viability, migration, and invasion of bladder cancer cells. Importantly, in FOXO1 knockdown sublines, an anti-androgen hydroxyflutamide or an anti-estrogen tamoxifen did not significantly inhibit the neoplastic transformation of urothelial cells, while dihydrotestosterone or estradiol did not significantly promote the proliferation or migration of urothelial cancer cells. In addition, immunohistochemistry in surgical specimens showed that FOXO1 and p-FOXO1 expression was down-regulated and up-regulated, respectively, in bladder tumor tissues, which was further associated with worse patient outcomes. AR or ERß activation is thus found to correlate with inactivation of FOXO1 which appears to be their key downstream effector. Moreover, FOXO1, as a tumor suppressor, is likely inactivated in bladder cancer, which contributes in turn to inducing urothelial carcinogenesis and cancer growth.