Study on Fu-Fang-Jin-Qian-Cao Inhibiting Autophagy in Calcium Oxalate-induced Renal Injury by UHPLC/Q-TOF-MS-based Metabonomics and Network Pharmacology Approaches.

INTRODUCTION: Fu-Fang-Jin-Qian-Cao is a Chinese herbal preparation used to treat urinary calculi. Fu-Fang-Jin-Qian-Cao can protect renal tubular epithelial cells from calcium oxalateinduced renal injury by inhibiting ROS-mediated autopathy. The mechanism still needs further exploration. Metabonomics is a new subject; the combination of metabolomics and network pharmacology can find pathways for drugs to act on targets more efficiently. METHODS: Comprehensive metabolomics and network pharmacology to study the mechanism of Fu-Fang-Jin-Qian-Cao inhibiting autophagy in calcium oxalate-induced renal injury. Based on UHPLC-Q-TOF-MS, combined with biochemical analysis, a mice model of Calcium oxalateinduced renal injury was established to study the therapeutic effect of Fu-Fang-Jin-Qian-Cao. Based on the network pharmacology, the target signaling pathway and the protective effect of Fu- Fang-Jin-Qian-Cao on Calcium oxalate-induced renal injury by inhibiting autophagy were explored. Autophagy-related proteins LC3-II, BECN1, ATG5, and ATG7 were studied by immunohistochemistry. RESULTS: Combining network pharmacology and metabolomics, 50 differential metabolites and 2482 targets related to these metabolites were found. Subsequently, the targets enriched in PI3KAkt, MAPK and Ras signaling pathways. LC3-II, BECN1, ATG5 and ATG7 were up-regulated in Calcium oxalate-induced renal injury. All of them could be reversed after the Fu-Fang-Jin-Qian- Cao treatment. CONCLUSIONS: Fu-Fang-Jin-Qian-Cao can reverse ROS-induced activation of the MAPK signaling pathway and inhibition of the PI3K-Akt signaling pathway, thereby reducing autophagy damage of renal tubular epithelial cells in Calcium oxalate-induced renal injury.

Two-Stage Deep-Learning Classifier for Diagnostics of Lung Cancer Using Metabolites.

We developed a machine-learning system for the selective diagnostics of adenocarcinoma (AD), squamous cell carcinoma (SQ), and small-cell carcinoma lung (SC) cancers based on their metabolomic profiles. The system is organized as two-stage binary classifiers. The best accuracy for classification is 92%. We used the biomarkers sets that contain mostly metabolites related to cancer development. Compared to traditional methods, which exclude hierarchical classification, our method splits a challenging multiclass task into smaller tasks. This allows a two-stage classifier, which is more accurate in the scenario of lung cancer classification. Compared to traditional methods, such a "divide and conquer strategy" gives much more accurate and explainable results. Such methods, including our algorithm, allow for the systematic tracking of each computational step.

HOXB3 drives WNT-activation associated progression in castration-resistant prostate cancer.

Enabled resistance or innate insensitiveness to antiandrogen are lethal for castration-resistant prostate cancer (CRPC). Unfortunately, there seems to be little can be done to overcome the antiandrogen resistance because of the largely unknown mechanisms. In prospective cohort study, we found that HOXB3 protein level was an independent risk factor of PSA progression and death in patients with metastatic CRPC. In vivo, upregulated HOXB3 contributed to CRPC xenografts progression and abiraterone resistance. To uncover the mechanism of HOXB3 driving tumor progression, we performed RNA-sequencing in HOXB3 negative (HOXB3-) and HOXB3 high (HOXB3 + ) staining CRPC tumors and determined that HOXB3 activation was associated with the expression of WNT3A and enriched WNT pathway genes. Furthermore, extra WNT3A and APC deficiency led HOXB3 to be isolated from destruction-complex, translocated to nuclei, and then transcriptionally regulated multiple WNT pathway genes. What's more, we also observed that the suppression of HOXB3 could reduce cell proliferation in APC-downregulated CRPC cells and sensitize APC-deficient CRPC xenografts to abiraterone again. Together, our data indicated that HOXB3 served as a downstream transcription factor of WNT pathway and defined a subgroup of CRPC resistant to antiandrogen which would benefit from HOXB3-targeted therapy.

Circular RNAs in Prostate Cancer: Is it Time to Further Explore Liquid Biopsies?

BACKGROUND: Although diagnosis and treatment of prostate cancer (PCa) have evolved rapidly in recent years, clinically significant molecular biomarkers are still needed to lower the mortality. Circular RNAs (circRNAs) are a poorly characterized component of PCa transcriptome. Recently, since the development of deep RNA sequencing and novel bioinformatic pipelines, emerging evidence suggests circRNAs to have diverse functions in the development and progression of PCa. Thus, we attempt to summarize the current situation and potential development prospects about the role of circRNAs in PCa liquid biopsies. METHODS: The role of circRNAs in PCa was summarized by searching the literature related to circRNAs in PubMed in recent years. RESULTS: Deregulation of circRNAs is associated with cell proliferation, apoptosis, cell invasion, migration, as well as metastasis in PCa. Because of the high stability and tissue specificity of circRNAs, with improved detection methodologies, circRNAs may be predictive biomarkers in liquid biopsies. CONCLUSION: From the perspective of recent research, with the development of high-throughput sequencing and novel bioinformatics tools, knowledge of circRNAs will be further expanded. Improved technologies will make personalized precision medicine less of a paper exercise. It is time to further explore circRNA in liquid biopsies.

Identification of LAT/ZAP70 characterized immune subtypes of prostate cancer.

BACKGROUND: While immunotherapy has shown potent efficacy in clinical practices, patient selection to receive checkpoint blockade is still challenging in prostate cancer (PCa). LAT and ZAP70 functions in lymphocyte activation and plays a critical role in T cell receptor (TCR) signal transduction. However, PCa genomic and clinical data regarding the role of LAT and ZAP70 are limited. We aim to identify and characterize LAT/ZAP70 defined subtypes of PCa. METHODS: We elaborated the TCGA PCa data and metastatic castration-resistant prostate cancer (mCRPC) RNA-seq data bioinformatic analysis and systematically elucidated the role of intra-tumoral expressed LAT and ZAP70 in the progression-free survival and immunotherapeutic-related signals. LAT/ZAP70-associated immune infiltration was evaluated using bioinformatic tools. Immunohistochemical staining of serial sections was used to confirm the expression and distribution of LAT, ZAP70 and androgen receptor (AR) in PCa tissues. RESULTS: Specifically, LAT and ZAP70 revealed increased expressions in PCa when compared to normal tissues and positively associated with intra-tumoral immune cells infiltration. LAT/ZAP70 defined immune-high early-stage PCa revealed higher TP53 mutation frequency and poor prognosis. Transcriptome analysis indicated immune-related signals and CTLA4 expression were highly enhanced in immune-high PCa parallel with higher protein level of MYC and lower AR expression. In mCRPC, LAT/ZAP70 defined immune-high patients also revealed upregulated immune related signals, higher CTLA4 expression and DNA repair deficiency. CONCLUSION: LAT/ZAP70 defined immune-high PCa linked to immune infiltration and predicts poor prognosis. Immune-high PCa may receive effective response from immune checkpoint inhibitor parallel with systemic treatment.

The Relationship Between Serum Folate Level and Residual Renal Function in CAPD Patients.

Objective: To investigate the relationship between serum folate (FA) levels and residual renal function (RRF) in continuous ambulatory peritoneal dialysis (CAPD) patients. Methods: Clinical data were collected from 180 hospitalized patients who received CAPD regularly. Patients were divided into the FA deficiency group and the FA non-deficiency group according to serum FA level. Data on age, sex, PD vintage, hemoglobin, mean corpuscular volume, serum FA, total Kt/V, residual kidney Kt/V, peritoneum Kt/V, creatinine clearance (Ccr), ultrafiltration volume, cystatin C (cytC), serum creatinine (Scr), urea nitrogen, retinol-binding protein and the primary disease were gathered from 2 groups. Statistical methods were used to analyze the relationship between serum FA level and RRF. Results: Peritoneal Kt/V, cytC, Scr were higher, and residual kidney Kt/V was lower in FA deficiency group than in non-deficiency group. Univariate correlation showed the peritoneal Kt/V, cytC, Scr negatively correlated with serum FA while residual kidney Kt/V positively correlated with serum FA, and there was a simple linear regression relationship between serum FA and residual kidney Kt/V. Differences were statistically significant (P<0.05). Conclusion: There is a relationship between serum FA and RRF in CAPD patients. Prospective studies or trials should be performed to clarify the importance of FA supplementation on RRF during peritoneal dialysis.

hsa_circ_0092339 acts as a molecular sponge in castration-resistant prostate cancer via the hsa-mir-940/C-MYC axis.

Aims: We aimed to determine whether intronic circRNA acts as a molecular sponge in castration-resistant prostate cancer (CRPC). Materials & methods: A gene chip technique was used to conduct sequencing. A qPCR experiment was performed to verify the result. Radioimmunoprecipitation, RNA pull-down and dual-luciferase reporter assays were performed to particularly expound its function. Verification of downstream effects was carried out through qPCR and western blot, and a xenograft assay was performed in vivo for verification. Results: Intronic circRNA hsa_circ_0092339 in the nucleus was highly expressed in CRPC cell lines. hsa_circ_0092339 did not regulate the expression of its parental gene. hsa_circ_0092339 functions like a molecular sponge, preventing degradation of C-MYC mRNA by absorbing hsa-mir-940. Conclusion: hsa_circ_0092339 plays a critical role in CRPC through targeting C-MYC indirectly by absorbing hsa-mir-940.

Our research breaks the mold by investigating a novel function of RNA and a novel regulatory mechanism. Our research provides a new therapeutic target for prostate cancer treatment and broadens the understanding of prostate cancer.

Targeting KDM4A-AS1 represses AR/AR-Vs deubiquitination and enhances enzalutamide response in CRPC.

Castration-resistant prostate cancer (CRPC) is a highly malignant type of advanced cancer resistant to androgen deprivation therapy. One of the important mechanisms for the development of CRPC is the persistent imbalanced regulation of AR and AR splice variants (AR/AR-Vs). In this study, we reported KDM4A-AS1, a recently discovered lncRNA, as a tumor promoter that was significantly increased in CRPC cell lines and cancer tissues. Depletion of KDM4A-AS1 significantly reduced cell viability, proliferation, migration in vitro, and tumor growth in vivo. We found that by binding to the NTD domain, KDM4A-AS1 enhances the stability of USP14-AR/AR-Vs complex, and promoted AR/AR-Vs deubiquitination to protect it from MDM2-mediated ubiquitin-proteasome degradation. Moreover, KDM4A-AS1 was found to enhance CRPC drug resistance to enzalutamide by repressing AR/AR-Vs degradation; antisense oligonucleotide drugs targeting KDM4A-AS1 significantly reduced the growth of tumors with enzalutamide resistance. Taken together, our results indicated that KDM4A-AS1 played an important role in the progression of CRPC and enzalutamide resistance by regulating AR/AR-Vs deubiquitination; targeting KDM4A-AS1 has broad clinical application potential.

Oral Hydrogen-Rich Water Alleviates Oxalate-Induced Kidney Injury by Suppressing Oxidative Stress, Inflammation, and Fibrosis.

Objective: To explore the theraputic effects and potential mechanisms of hydrogen-rich water (HRW) against oxalate-induced kidney injury. Methods: The mouse model of Calcium oxalate (CaOx) crystallization was established by feeding a soluble oxalate diet. Crystal deposition, tubular injury, fibrosis and reactive oxygen species (ROS) production in kidneys were examined by histology. Serum indexes of renal injury, inflammation and oxidative stress were detected by commercial kits. RNA sequencing (RNA-seq) was performed to screen potential pathways and the expressions of key molecules in these pathways were determined by western blotting and immunohistochemistry. Results: Crystal deposition, tubular injury, fibrosis and increased ROS production in kidneys of mice induced by oxalate diet were improved with HRW administration. The indexes of renal injury, inflammation and oxidative stress in serum of mice were upregulated by oxalate diet, which were reduced by HRW. A total of 3,566 differential genes were screened by RNA-seq and these genes were analyzed by pathway enrichment and PI3K/AKT, NF-κB, and TGF-ß pathways were selected for further verification. The expressions of molecules related to PI3K-AKT pathway (PI3K, AKT, and p-AKT), NF-κB pathway (NF-κB p65, p- NF-κB p65, NLRP3, and IL-1ß) and TGF-ß pathway (TGF-ß, TGF-ßRI, TGF-ßRII, p-Smad2, and p-Smad3) in renal tissues were increased by oxalate diet, which were reduced by HRW administration. Conclusion: HRW may alleviate oxalate-induced kidney injury with its anti-oxidative, anti-inflammatory and anti-fibrotic effects via inhibiting PI3K/AKT, NF-κB, and TGF-ß pathways.

YAP1 plays a key role of the conversion of normal fibroblasts into cancer-associated fibroblasts that contribute to prostate cancer progression.

BACKGROUND: Cancer-associated fibroblasts (CAFs) are an important part of the tumour microenvironment, and their functions are of great concern. This series of experiments aimed to explore how Yes-associated protein 1 (YAP1) regulates the function of stromal cells and how the normal fibroblasts (NFs) convert into CAFs in prostate cancer (PCa). METHODS: The effects of conditioned media from different fibroblasts on the proliferation and invasion of epithelial cells TrampC1 were examined. We then analysed the interaction between the YAP1/TEAD1 protein complex and SRC, as well as the regulatory function of the downstream cytoskeletal proteins and actins. A transplanted tumour model was used to explore the function of YAP1 in regulating tumour growth through stromal cells. The relationship between the expression of YAP1 in tumour stromal cells and the clinical characteristics of PCa patients was analysed. RESULTS: The expression level of YAP1 was significantly upregulated in PCa stromal cells. After the expression level of YAP1 was increased, NF was transformed into CAF, enhancing the proliferation and invasion ability of epithelial cells. The YAP1/TEAD1 protein complex had the capability to influence downstream cytoskeletal proteins by regulating SRC transcription; therefore, it converts NF to CAF, and CAF can significantly promote tumour growth and metastasis. The high expression of YAP1 in the tumour stromal cells suggested a poor tumour stage and prognosis in PCa patients. CONCLUSION: YAP1 can convert NFs into CAFs in the tumour microenvironment of PCa, thus promoting the development and metastasis of PCa. Silencing YAP1 in tumour stromal cells can effectively inhibit tumour growth.

Impact of squamous differentiation on intravesical recurrence and prognosis of patients with upper tract urothelial carcinoma.

BACKGROUND: The prognostic role of squamous differentiation in upper urinary tract urothelial carcinoma (UTUC) is still unclear. This article describes the impact of squamous differentiation on prognosis and intravesical recurrence of patients with primary UTUC treated with radical nephroureterectomy (RNU). METHODS: Totally, we retrieved (I) 669 histologically confirmed UTUC patients without histologic variants; (II) 101 UTUC patients with squamous differentiation in our institution, dating from April 2003 to April 2016. The clinical pathological characteristics and survival outcomes were compared between these two cohorts. RESULTS: In our study, 13% UTUC patients were detected with squamous differentiation. The mean age of all the patients examined was 66, of whom 70% were males. Squamous differentiation significantly associated with tumor stage, tumor grade and lymphovascular invasion. The Kaplan-Meier and Cox regression analyses showed that presence of squamous differentiation was correlated with shorter cancer specific survival of UTUC patients. The 5-year cancer specific survival rates were 47% for squamous differentiation-present patients and 63% for squamous differentiation-absent patients. UTUC patients with squamous differentiation showed a higher frequency of high-grade disease in advanced stage (pT2/pT3/pT4), while the discrepancy was not shown in early stage (pTa/pT1). Intravesical recurrence was observed in 27% patients. We found that intravesical recurrence had little impact on the cancer specific survival of squamous differentiation-present patients, yet it tended to decrease cancer specific survival among squamous differentiation-absent patients. CONCLUSIONS: The presence of squamous differentiation in UTUC patients was a vital prognostic factor for cancer specific survival and correlated with intravesical recurrence after receiving RNU.

KIF20A Affects the Prognosis of Bladder Cancer by Promoting the Proliferation and Metastasis of Bladder Cancer Cells.

OBJECTIVE: To investigate the expression of kinesin family member 20A (KIF20A) in bladder cancer, the effect of KIF20A on the proliferation and metastasis of bladder cancer cells, and the effect of KIF20A expression on the prognosis of bladder cancer patients. METHODS: Bladder cancer tissue and its adjacent tissues were collected from tumour patients. The mRNA and protein expression levels of KIF20A in the tissue samples were detected by qRT-PCR and western blot. Immunohistochemical (IHC) staining was used to identify the expression and distribution of KIF20A proteins in the tissue samples. The relationship between the KIF20A expression and the clinical pathology of bladder cancer was analysed. The effect of the differential expression of KIF20A on the prognosis of patients with bladder cancer was analysed by the TCGA database. The plasmid was transfected into the bladder cell lines T24 and 5637 to construct two stable cell lines with knocked down KIF20A. The effect of KIF20A expression on the proliferation and invasion of T24 and 5637 bladder cells was explored in vitro using the abovementioned stable cell lines. The effect of the KIF20A expression on the proliferation of bladder cancer cells was evaluated by a mouse xenograft model. RESULTS: The expression of KIF20A was significantly higher in the bladder cancer tissues than in the adjacent control tissues. The expression of KIF20A was significantly associated with the degree of pathological differentiation of bladder cancer. Patients with a higher expression of KIF20A had a higher tumour grade and a more advanced stage. The mean survival of patients with a high KIF20A expression was significantly lower than the mean survival of patients with a low KIF20A expression. The in vitro experiments demonstrated that the knockdown of KIF20A significantly inhibited T24 and 5637 cell proliferation and invasion. The in vivo experiments showed that the knockdown of KIF20A significantly inhibited the proliferation of the bladder tumours. CONCLUSION: KIF20A promotes the proliferation and metastasis of bladder cancer cells. Bladder cancer patients with a high KIF20A expression have a worse tumour differentiation and a poor prognosis. KIF20A may become an independent factor that affects the prognosis of bladder cancer patients and a therapeutic target for bladder cancer.

LncRNA PCAT1 activates AKT and NF-κB signaling in castration-resistant prostate cancer by regulating the PHLPP/FKBP51/IKKα complex.

In PTEN-deficient prostate cancers, AKT signaling may be activated upon suppression of androgen receptor signaling. Activation of AKT as well as NF-κB signaling involves a key regulatory protein complex containing PHLPP, FKBP51 and IKKα. Here, we report a critical role of lncRNA PCAT1 in regulating the PHLPP/FKBP51/IKKα complex and progression of castration-resistant prostate cancer (CRPC). Using database queries, bioinformatic analyses, as well as RIP and RNA pull-down assays, we discovered and validated that the lncRNA-PCAT1 perturbs the PHLPP/FKBP51/IKKα complex and activates AKT and NF-κB signaling. Expression of lncRNA-PCAT1 is positively linked to CRPC progression. PCAT1 binds directly to FKBP51, displacing PHLPP from the PHLPP/FKBP51/IKKα complex, leading to activation of AKT and NF-κB signaling. Targeting PCAT1 restores PHLPP binding to FKBP1 leading to suppression of AKT signaling. Preclinical study in a mouse model of CRPC suggests therapeutic potential by targeting lncRNA PCAT1 to suppress CRPC progression. Together, the newly identified PCAT1/FKBP51/IKKα complex provides mechanistic insight in the interplay between AKT, NF-κB and AR signaling in CRPC, and the preclinical studies suggest that a novel role for PCAT1 as a therapeutic target.

A retrospective analysis of the correlation between AXL expression and clinical outcomes of patients with urothelial bladder carcinoma.

BACKGROUND: Immunohistochemistry (IHC) analysis of primary tumors revealed that AXL expression is associated with survival in patients with different cancers. The objective of our study is to investigate the relationship between the expression of AXL and clinical outcomes of patients with bladder carcinoma (BC). METHODS: A total of 407 samples from The Cancer Genome Atlas (TCGA) database and 203 patients with clinical and pathological diagnosis of BC in our hospital were used to assess the association of AXL and clinical outcomes of BC patients. IHC was performed to evaluate the expression of AXL in tumor tissue collected after surgical treatment. RESULTS: Data from TCGA showed that AXL mRNA expression was significantly associated with poor clinical-pathological characters and short overall survival (OS) of BC patients. In our study, AXL was significantly related to worse pathological T-stage (pT), lymph node metastasis (pN), and tumor grade. In univariate analysis, abundant AXL was significantly associated with the worse OS. In multivariate analysis, AXL, as well as pT, pN, and Ki67, was an independent prognostic factor in predicting the survival of patients. CONCLUSIONS: AXL was an unfavorable prognostic factor, which was associated with poor clinical outcomes of BC patients.

Restoration of FKBP51 protein promotes the progression of castration resistant prostate cancer.

BACKGROUND: As deregulation of androgen receptor (AR) signaling target genes is associated with tumorigenesis and the development of prostate cancer (PCa), AR signaling is the primary therapeutic target for PCa. Although patients initially responses to first-line androgen deprivation therapies (ADTs), most of them with advanced PCa progress to lethal castration-resistant prostate cancer (CRPC). Recent studies have suggested the molecular mechanisms by which AR elicit the robust up-regulation of the FKBP51 gene. We suggest that restored expression of FKBP51 gene, modulated by androgen receptor splicing variant 7 (AR-V7) which replaces full length androgen receptor (AR-FL) in androgen ablation status, promotes CRPC progression through activating NF-κB signaling. METHODS: Immunohistochemistry assays were used to detect the expression of AR-V7, FKBP51 and NF-κB signaling correlated proteins in CRPC tissues. An androgen ablation resistant PCa cell line model established by Long-term culturing in androgen depleted medium, named androgen-independent LNCaP (LNCaP-AI) cells, were used to dynamically monitor FKBP51 expression during the process of androgen dependent PCa cells transforming into androgen-independent cells, as well as its association with NF-κB signal pathway. LNCaP-AI cell line was determined to express AR-V7 protein continuously. Luciferase reporter assays and DNA pull down were used to determine the association between AR-V7 and FKBP51. RESULTS: Our results suggested that CRPC patients with AR-V7 high expression tend to have higher expression of FKBP51 and enhanced NF-κB signaling compared with AR-V7 negative patients. Knockdown of AR-V7 or FKBP51 in LNCaP-AI cells attenuated the level of p-NF-κB (Ser536) and androgen-resistant cells growth. Luciferase reporter assays and DNA pull down results indicated that FKBP51 was transcriptionally promoted by AR-V7 in absence of androgen, which enhanced NF-κB signaling. CONCLUSIONS: Because of upregulation of AR-V7 in androgen-independent PCa cells, increasing of FKBP51 induced NF-κB signaling, leading to progression of CRPC.

High Expression of KIF22/Kinesin-Like DNA Binding Protein (Kid) as a Poor Prognostic Factor in Prostate Cancer Patients.

BACKGROUND Kid (kinesin-like DNA binding protein), a member of microtubule-dependent molecular motor proteins, also known as KIF22, is reported to be associated with carcinogenesis and cancer progression in different types of malignant tumor, but the biologic behavior and clinical outcome of KIF22 in prostate cancer (PCa) has not been well studied. This study aimed to analyze the association between KIF22 and clinical outcome in PCa patients. MATERIAL AND METHODS The expression of KIF22 in tumor specimens compared with paired paracancerous tissue from 114 patients undergoing radical prostatectomy was detected by immunohistochemistry; results were verified using The Cancer Genome Atlas (TCGA) database. Subsequently, the relationship between KIF22 expression and clinical prognosis of PCa patients was then statistically analyzed. RESULTS Both immunohistochemistry and database analysis showed that KIF22 was obviously overexpressed in PCa tissues compared with paracancerous tissue. The overexpression of KIF22 at the protein level was significantly related to higher clinical stage (P=0.025), Gleason score (P=0.002), seminal vesicle invasion (P=0.007), and lymph node metastasis (P=0.009). Furthermore, with the overexpression of KIF22 mRNA level in PCa patients, the oncological prognosis of PCa patients was much poorer. CONCLUSIONS High-level expression of KIF22 was related to both tumor progression and adverse clinical outcome. For this reason, KIF22 may become a potential prognostic factor for PCa.

Sox5 contributes to prostate cancer metastasis and is a master regulator of TGF-ß-induced epithelial mesenchymal transition through controlling Twist1 expression.

BACKGROUND: Metastatic castration-resistant prostate cancer (mCRPC) is one of the main contributors to the death of prostate cancer patients. To date, the detailed molecular mechanisms underlying mCRPC are unclear. Given the crucial role of epithelial-mesenchymal transition (EMT) in cancer metastasis, we aimed to analyse the expression and function of Transforming growth factor-beta (TGF-ß) signal-associated protein named Sox5 in mCRPC. METHODS: The protein expression levels were analysed by western blot, immunohistochemistry and immunofluorescence. Luciferase reporter assays and chromatin immunoprecipitation were employed to validate the target of Sox5. The effect of Smad3/Sox5/Twist1 on PCa progression was investigated in vitro and in vivo. RESULTS: Here, we found that TGF-ß-induced EMT was accompanied by increased Sox5 expression. Interestingly, knockdown of Sox5 expression attenuated EMT induced by TGF-ß signalling. Furthermore, we demonstrated that Smad3 could bind to the promoter of Sox5 and regulate its expression. Mechanistically, Sox5 could bind to Twist1 promoter and active Twist1, which initiated EMT. Importantly, knockdown of Sox5 in prostate cancer cells resulted in less of the mesenchymal phenotype and cell migration ability. Furthermore, targeting Sox5 could inhibit prostate cancer progression in a xenograft mouse model. In clinic, patients with high Sox5 expression were more likely to suffer from metastases, and high Sox5 expression also has a lower progression-free survival and cancer specific-survival in clinic database. CONCLUSIONS: Therefore, we propose a new mechanism in which Smad3/Sox5/Twist1 promotes EMT and contributes to PCa progression.

Squamous differentiation in patients with superficial bladder urothelial carcinoma is associated with high risk of recurrence and poor survival.

BACKGROUND: The independent prognostic role of squamous differentiation in pT1 bladder urothelial carcinoma has not been reported in previous studies. This article describes the impact of squamous differentiation on tumor recurrence and survival, and whether this histologic variant could indeed alter definitive treatment, based on single center-based retrospective data. METHODS: Totally, we retrieved (1)1449 histologically confirmed pT1 bladder urothelial carcinoma patients without histologic variants; (2)227 pT1 bladder urothelial carcinoma patients with squamous differentiation in our institution, from May 2004 to Oct 2015. The total amount of high/low grade urothelial carcinoma patients was 991/685 respectively. Transurethral resection of bladder tumor (TURBT) and intravesical chemotherapy were performed as initial treatments for all the patients. The clinical and pathological characteristics, treatment and survival outcomes were compared between squamous differentiation-positive and squamous differentiation-negative patients. RESULTS: In our study, 14% urothelial carcinoma patients were detected with squamous differentiation. The mean age of all the patients examined was 66.4, of whom 82% were males. The 5-year cancer specific survival rates were 69% for squamous differentiation-positive patients and 91% for squamous differentiation-negative patients (p < 0.001). Recurrence proved to be more common in squamous differentiation-positive patients than in negative patients. In the results of the univariate and multivariate Cox proportional hazard analysis, tumor size, lymphovascular invasion, recurrence and squamous differentiation were confirmed to be the prognostic factors associated with patients' survival. CONCLUSIONS: Squamous differentiation in pT1 bladder urothelial carcinoma is correlated to high risk of recurrence and poor prognosis as an independent prognostic factor. Radical cystectomy is essential for recurred high grade pT1 bladder urothelial carcinoma with squamous differentiation accompanied by lymphovascular invasion.