Intraperitoneal laparoscopic single-site lymph node dissection in modified supine position during laparoscopic radical nephroureterectomy.

Technique modifications that aim to improve ergonomics of the surgical procedure without repositioning the upper tract urothelial carcinoma patients remain a challenge to urologists. We offer a novel technique to perform intraperitoneal laparoscopic single-site radical nephroureterectomy and pelvic lymph nodes dissection/retroperitoneal lymph nodes dissection in a supine position. Our novel technique is feasible and offers a significant improvement in operative efficiency, particularly in patients with locally advanced disease.

PABPN1 promotes clear cell renal cell carcinoma progression by suppressing the alternative polyadenylation of SGPL1 and CREG1.

Alternative polyadenylation (APA) is an important post-transcriptional regulatory mechanism in cancer development and progression. Poly(A) binding protein nuclear 1 (PABPN1) is a gene that encodes abundant nuclear protein, binds with high affinity to nascent poly(A) tails, and is crucial for 3'-UTR (3'-untranslated region) APA. Although PABPN1 has been recently reported as a dominant master APA regulator in clear cell renal cell carcinoma (ccRCC), the underlying functional mechanism remain unclear and the genes subject to PABPN1 regulation that contribute to ccRCC progression have not been identified. Here, we found that PABPN1 is upregulated in ccRCC, and its expression is highly associated with the clinical prognosis of ccRCC patients. PABPN1 promotes ccRCC cell proliferation, migration, invasion, and exerts an influence on sphingolipid metabolism and cell cycle. Moreover, PABPN1 depletion significantly suppressed cancer cell growth via induction of cell cycle arrest and apoptosis. In particular, we characterized PABPN1-regulated 3'-UTR APA of sphingosine-1-phosphate lyase 1 (SGPL1) and cellular repressor of E1A stimulated genes 1 (CREG1), which contribute to ccRCC progression. Collectively, our data revealed that PABPN1 promotes ccRCC progression at least in part, by suppressing SGPL1 and CREG1. Thus, PABPN1 may be a potential therapeutic target in ccRCC.

PABPN1 regulates mRNA alternative polyadenylation to inhibit bladder cancer progression.

BACKGROUND: About 10-20% of patients with bladder cancer (BC) progress to muscle-invasive diseases, of which the underlying key molecular events have yet to be addressed. RESULTS: Here, we identified poly(A) binding protein nuclear 1 (PABPN1), a general factor of alternative polyadenylation (APA), was downregulated in BC. Overexpression and knockdown of PABPN1 significantly decreased and increased BC aggressiveness, respectively. Mechanistically, we provide evidence that the preference of PABPN1-bound polyadenylation signals (PASs) depends on the relative location between canonical and non-canonical PASs. PABPN1 shapes inputs converging on Wnt signaling, cell cycle, and lipid biosynthesis. CONCLUSIONS: Together, these findings provide insights into how PABPN1-mediated APA regulation contributes to BC progression, and suggest that pharmacological targeting PABPN1 might have therapeutic potential in patients with BC.

Lengthening of 3' Untranslated Regions of mRNAs by Alternative Polyadenylation Is Associated With Tumor Progression and Poor Prognosis of Clear Cell Renal Cell Carcinoma.

Alternative polyadenylation (APA) is emerging as a major posttranscriptional mechanism for gene regulation in cancer. A prevailing hypothesis is that shortening of the 3' untranslated region (3'UTR) increases oncoprotein expression because of the loss of miRNA-binding sites (MBSs). We showed that the longer 3'UTR is associated with a more advanced tumor stage in patients with clear cell renal cell carcinoma (ccRCC). More surprisingly, 3'UTR shortening is correlated with better overall survival in patients with ccRCC. Furthermore, we identified a mechanism by which longer transcripts lead to increased oncogenic protein and decreased tumor-suppressive protein expression compared to the shorter transcripts. In our model, shortening of 3'UTRs by APA may increase the mRNA stability of the majority of the potential tumor-suppressor genes due to the loss of MBSs and AU-rich elements (AREs). Unlike potential tumor-suppressor genes, the potential oncogenes display much lower MBS and ARE density and globally much higher m6A density in distal 3'UTRs. As a result, 3'UTRs shortening decreases the mRNA stability of potential oncogenes and enhances the mRNA stability of potential tumor-suppressor genes. Our findings highlight the cancer-specific pattern of APA regulation and extend our understanding of the mechanism of APA-mediated 3'UTR length changes in cancer biology.

Comprehensive analysis of alternative polyadenylation regulators concerning CD276 and immune infiltration in bladder cancer.

Alternative polyadenylation (APA) is emerging as a crucial regulatory mechanism in bladder cancer (BC), while it remains elusive whether APA influences the tumor immune microenvironment (TIME) in BC. We identified two distinct subtypes of BC by APA-related regulatory genes expression profiles. The two subtypes have different pathological grades, prognostic outcomes, tumor immune infiltration characteristics, and pathway enrichment. Subsequently, CPSF3 was identified as a potential immune infiltration-related gene in BC. Highly expressed CPSF3 was positively correlated with unfavorable prognosis and high CD276 expression in BC. Moreover, we verified the expression of CPSF3 in BC tissues and cell lines by qRT-PCR. In conclusion, the study indicates that APA regulatory factors play an important role in immune infiltration of BC, and that CPSF3 was a potentially prognostic marker and immunotherapy target for BC.

Comprehensive analysis of lower mitochondrial complex I expression is associated with cell metastasis of clear cell renal cell carcinoma.

Background: Clear cell renal cell carcinoma (ccRCC) is characterized by high metastasis potential. It is of great importance to explore the mechanisms underlying ccRCC metastasis and to enable development of potent therapeutics. The mitochondrial complex I (CI) had been considered to play an important role in the development of cancers, but less known in ccRCC. Methods: We utilized available public databases of ccRCC, including single-cell RNA sequencing (scRNA-seq) data GSE73121 and The Cancer Genome Atlas-kidney renal clear cell carcinoma (TCGA-KIRC). Principal component analysis (PCA) and t-Distributed Stochastic Neighbor Embedding (tSNE) analysis were evaluated the heterogeneity of metastatic renal cell carcinoma (mRCC) and primary renal cell carcinoma (pRCC). Protein-protein interaction (PPI) network identified critical gene. Gene set enrichment analysis (GSEA) and gene set variation analysis (GSVA) performed to explore the potential biologic pathways. Results: Our study revealed a significant gene expression heterogeneity between pRCC and mRCC. A PPI network based on differentially expressed genes (DEGs) identified electron transport chain (ETC), especially mitochondrial CI, as the key network hub. Further analysis revealed that the role of mitochondrial CI is associated with tumor metastasis and immune responds of ccRCC. Although CI had low frequency mutations in ccRCC, CI expression is associated with the high frequency mutated genes. A prognosis model included 7 CI genes, and these had a significant effect on overall survival (OS). The area under the curve at 1, 3, and 5 years was 0.717, 0.685, and 0.728, respectively. Transcription factor analysis predicted that PPARG possibly is a potential transcription activator of CI genes in ccRCC. Conclusions: Overall, we found that CI expression is associated with ccRCC progress. CI and PPARG may be potential biomarkers for metastatic ccRCC.

MiR-183-5p-PNPT1 Axis Enhances Cisplatin-induced Apoptosis in Bladder Cancer Cells.

OBJECTIVE: It has been reported that intrinsic apoptosis is associated with the progression of bladder cancer (BC). Recent evidence suggests that polyribonucleotide nucleotidyltransferase 1 (PNPT1) is a pivotal mediator involved in RNA decay and cell apoptosis. However, the regulation and roles of PNPT1 in bladder cancer remain largely unclear. METHODS: The upstream miRNA regulators were predicted by in silico analysis. The expression levels of PNPT1 were evaluated by real-time PCR, Western blotting, and immunohistochemistry (IHC), while miR-183-5p levels were evaluated by qPCR in BC cell lines and tissues. In vitro and in vivo assays were performed to investigate the function of miR-183-5p and PNPT1 in apoptotic RNA decay and the tumorigenic capability of bladder cancer cells. RESULTS: PNPT1 expression was decreased in BC tissues and cell lines. Overexpression of PNPT1 significantly promoted cisplatin-induced intrinsic apoptosis of BC cells, whereas depletion of PNPT1 potently alleviated these effects. Moreover, oncogenic miR-183-5p directly targeted the 3' UTR of PNPT1 and reversed the tumor suppressive role of PNPT1. Intriguingly, miR-183-5p modulated not only PNPT1 but also Bcl2 modifying factor (BMF) to inhibit the mitochondrial outer membrane permeabilization (MOMP) in BC cells. CONCLUSION: Our results provide new insight into the mechanisms underlying intrinsic apoptosis in BC, suggesting that the miR-183-5p-PNPT1 regulatory axis regulates the apoptosis of BC cells and might represent a potential therapeutic avenue for the treatment of BC.

circNUDT21 promotes bladder cancer progression by modulating the miR-16-1-3p/MDM2/p53 axis.

Bladder cancer (BC) is a common genitourinary malignancy. This study investigated the regulatory effects of an exonic circRNA, circNUDT21, in the progression of BC. The circNUDT21 level was overexpressed in BC tissues and cell lines as compared to normal controls. Overexpression and silencing of circNUDT21 promoted and inhibited, respectively, the proliferative and invasive abilities of BC cells. Mechanistical analysis showed that circNUDT21 acted as a miR-16-1-3p sponge and that MDM2 was a potential downstream target of miR-16-1-3p. We further verified that overexpression of circNUDT21 was associated with elevated MDM2 and reduced p53 expression. CircNUDT21 promoted BC progression by acting as a sponge of miR-16-1-3p to activate the miR-16-1-3p/MDM2/p53 axis. These findings suggest that circNUDT21 functions as an oncogenic circRNA and may be a potential therapy target for BC.

Downregulation of NUDT21 contributes to cervical cancer progression through alternative polyadenylation.

Nudix Hydrolase 21 (NUDT21), an alternative polyadenylation (APA)-regulatory protein, exhibits tumor-suppressive effects. However, its role in cervical cancer (CxCa) remains unknown. In the present study, we found that NUDT21 expression was reduced in CxCa tissues and cells, and NUDT21 levels were highly associated with the clinical prognosis of patients with CxCa. Knockdown of NUDT21 promoted CxCa cell proliferation, migration, and invasion in vitro, as well as tumorigenesis and lung metastasis in vivo. Overexpression of NUDT21 produces the opposite effects. Moreover, we performed polyadenylation site sequencing (PAS-Seq) and identified 457 transcripts with lengthened 3' untranslated regions (3' UTRs) upon NUDT21 overexpression. In particular, NUDT21 modulated the expression of several genes involved in fatty acid metabolism and the Wnt and NF-κB signaling pathways in CxCa development. Taken together, our study demonstrated that the APA regulatory effect of NUDT21 is an important mechanism for CxCa suppression.

NUDT21 inhibits bladder cancer progression through ANXA2 and LIMK2 by alternative polyadenylation.

Purpose: Nudix Hydrolase 21 (NUDT21) is a crucial mediator involved in alternative polyadenylation (APA), and this molecule has been reported to be a tumor suppressor in human cancers. However, neither the role NUDT21 plays in bladder cancer (BC) nor the mechanisms which are involved have been investigated. Methods: Expression levels of NUDT21 in BC were evaluated with real-time PCR, western blotting, and immunohistochemistry (IHC). In vitro and in vivo assays were performed to investigate the function of NUDT21 in tumorigenesis in bladder cancer cells. The TOP/FOP flash reporter assay, western blot, and global APA site profiling analysis were used to identify the pathway which mediates the biologic roles of NUDT21 in BC. Results: NUDT21 expression is reduced in BC tissue and cells, and BC patients with lower NUDT21 expression have shorter overall and recurrent-free survival than patients with higher NUDT21 expression. NUDT21 ectopic expression or knockdown respectively profoundly inhibited or promoted the capacity of BC cells for proliferation, migration and invasion. We also identified a number of genes with shortened 3'UTRs through modulation of NUDT21 expression, and further characterized the NUDT21-regulated genes ANXA2 and LIMK2. We found NUDT21 modulates the expression of ANXA2 and LIMK2 in the Wnt/ß-catenin and NF-κB signaling pathways. Conclusions: These findings show NUDT21 plays a crucial role in BC progression, at least in part through ANXA2 and LIMK2 which act by alternative polyadenylation. NUDT21 may thus have potential as a diagnostic and therapeutic target in treatment of BC.

Correction: CLCA4 inhibits bladder cancer cell proliferation, migration, and invasion by suppressing the PI3K/AKT pathway.

[This corrects the article DOI: 10.18632/oncotarget.21724.].

RSPO3 promotes the aggressiveness of bladder cancer via Wnt/ß-catenin and Hedgehog signaling pathways.

R-spondin 3 (RSPO3) is a secreted protein that associates directly with Wnt/ß-catenin signaling. However, its functional contribution and prognostic value in human bladder cancer remain unclear. Here, we showed that RSPO3 is upregulated in bladder cancer tissues and cells, and high expression of RSPO3 correlates with advanced clinicopathological features, poor prognosis and disease progression in bladder cancer patients. Furthermore, we observed that ectopic expression or knockdown of RSPO3 profoundly promoted or inhibited, respectively, the invasive ability of bladder cancer cells. Mechanistically, RSPO3 promoted bladder cancer progression via mediating the Wnt/ß-catenin and Hedgehog signaling pathways. These findings demonstrate, for the first time, that RSPO3 exhibited a tumor-promoting effect in bladder cancer cells through activation of Wnt/ß-catenin and Hedgehog signaling pathways. Thus, RSPO3 may be served as a potential therapeutic target for bladder cancer treatment.

Prognostic Implication of Urothelial Stem Cell Markers Differs According to Primary Tumour Location in Non-Muscle-Invasive Bladder Cancer.

BACKGROUND/AIMS: This study aimed to validate the value of urothelial stem cell (USC) markers ΔNp63, integrin ß4, CD47, and CD44v6 in predicting the prognosis of non-muscle invasive bladder cancer (NMIBC) located in different anatomic regions of bladder. METHODS: The study reviewed the clinicopathologic data of 169 patients with NMIBC. Using real-time PCR and immunohistochemistry, the expression of ΔNp63, integrin ß4, CD47, and CD44v6 in archived specimens of patients with NMIBC were validated. Kaplan-Meier analysis and Cox proportional hazards model were used to assess the prognostic impact of USC markers for recurrent-free survival (RFS). RESULTS: The Real-time PCR data showed that the expression of USC markers were higher in tumors located in the trigone and posterior wall than that in other regions of bladder (P< 0.05). Statistical analysis showed that high expression of ΔNp63 was correlated with tumor stage (P=0.023) and tumor size (P=0.001), that high expression of integrin ß4 was correlated with tumor stage (P=0.026), tumor grade (P=0.005) and tumor size (P=0.003), and that high integrin ß4, CD47, and CD44v6 expression were significantly associated with tumor recurrence (P=0.032, P=0.010, and P=0.043, respectively). Moreover, high expression of ΔNp63 and integrin ß4 was correlated with poor RFS in patients with tumors located in the trigone (P=0.025 and P=0.023, respectively). High expression of integrin ß4, CD47, and CD44v6 was correlated with poor RFS in patients with tumors in the posterior wall (P=0.017, P=0.033 and P=0.047, respectively). High expression of integrin ß4 and CD47 was correlated with poor RFS in patients with tumors in the trigone/posterior wall area (P=0.002 and P=0.005, respectively). CONCLUSION: Our results suggest that USC markers are linked with poor prognosis of NMIBC patients, especially in patients with tumors in the trigone and posterior wall.

Leupaxin Promotes Bladder Cancer Proliferation, Metastasis, and Angiogenesis Through the PI3K/AKT Pathway.

BACKGROUND/AIMS: Leupaxin (LPXN) is a member of the paxillin protein family. Several studies have reported that LPXN regulates cancer development; however, the role of LPXN in bladder cancer remains unknown. METHODS: The expression of LPXN in bladder cancer cells and tissues was determined by real-time PCR, western blotting, and immunohistochemistry, respectively. The biological role of LPXN in bladder cancer cell proliferation, invasion, and angiogenesis was explored both in vitro and in vivo. RESULTS: LPXN expression was elevated in bladder cancer tissues and cell lines compared to adjacent non-tumor tissues and normal urothelial cells. High LPXN expression was correlated with large tumor size, advanced tumor stage, and poor survival in bladder cancer patients. Overexpression of LPXN significantly promoted the proliferation, invasion, and angiogenesis of bladder cancer cells, while suppressing LPXN had the opposite effects. The impact on tumor progression was abolished by inhibiting PI3K/ AKT signaling pathway. We further demonstrated that LPXN probably up-regulated S100P via the PI3K/AKT pathway. CONCLUSIONS: LPXN may facilitate bladder cancer progression by upregulating the expression of S100P via PI3K/AKT pathway. These results provide a novel insight into the role of LPXN in tumorigenesis and progression of bladder cancer and potential therapeutic target of bladder cancer.

Loss of Fezf2 promotes malignant progression of bladder cancer by regulating the NF-κB signaling pathway.

Forebrain embryonic zinc finger 2 (Fezf2) is an evolutionarily conserved zinc finger transcription repressor. It has been reported to be a tumor suppressor; however, neither the role that Fezf2 plays in bladder cancer nor the mechanisms involved have been investigated. In this study, we showed that Fezf2 expression is downregulated in bladder cancer tissues and cell lines compared to adjacent non-tumor tissues and normal urothelial cells. We also retrospectively analyzed the association between Fezf2 and various clinicopathologic characteristics in 196 bladder cancer patients, and showed that low expression of Fezf2 is correlated with larger tumor size, advanced tumor stage, and poor clinical prognosis. Moreover, we found that overexpression of Fezf2 significantly inhibited the proliferation, growth, migration, and invasion of bladder cancer cells, and attenuated angiogenesis, while knockdown of Fezf2 had the opposite effect. Fezf2 suppressed bladder cancer aggressiveness by activating the NF-κB signaling pathway. These findings suggest that Fezf2 holds promise as a prognostic biomarker, and provide a putative mechanism for bladder cancer progression.

HBO1 promotes cell proliferation in bladder cancer via activation of Wnt/ß-catenin signaling.

Histone acetyltransferase binding to ORC1 (HBO1), a histone acetyltransferase, was recently identified as an oncoprotein; however, its role in bladder cancer remains unknown. In this study, we showed that HBO1 was highly expressed at both the mRNA and the protein levels in bladder cancer. HBO1 expression was associated with the clinical features of human bladder cancer, including tumor size (P = 0.018) and T (P = 0.007) classifications. Patients with higher HBO1 expression had shorter recurrence-free survival time, whereas patients with lower HBO1 expression had better survival time. Moreover, we found that ectopic overexpression of HBO1 promoted, whereas HBO1 silencing inhibited tumor growth in bladder cancer cells both in vitro and in vivo. We further demonstrated that upregulation of HBO1 activated the Wnt/ß-catenin signaling pathway and led to nuclear localization of ß-catenin and upregulation of downstream targets of of Wnt/ß-catenin signaling. These findings suggest that HBO1 plays a key role in the progression of bladder cancer via the Wnt/ß-catenin pathway, and may serve as a potential therapeutic target for the treatment of bladder cancer.

CLCA4 inhibits bladder cancer cell proliferation, migration, and invasion by suppressing the PI3K/AKT pathway.

Calcium activated chloride channel A4 (CLCA4), a tumor suppressor, was shown to contribute to the progression of several human cancers, while its role in bladder carcinoma remains unclear. In this study, we showed CLCA4 expression was down-regulated in bladder carcinoma tissues and cells compared to adjacent non-tumor tissues and normal urothelial cells. Low CLCA4 expression was correlated with larger tumor size, advanced tumor stage, and poor prognosis in bladder carcinoma patients. Overexpression of CLCA4 profoundly attenuated the proliferation, growth, migratory and invasive capabilities of bladder cancer cells, whereas CLCA4 knockdown had the opposite effect. Mechanistically, CLCA4 is involved in PI3K/AKT signaling and its downstream molecules can promote bladder cancer cell proliferation. Additionally, CLCA4 could mediate the migration and invasion of bladder cancer cells by regulating epithelial-mesenchymal transition and PI3K/Akt activation. This study suggests that CLCA4 may represent a promising prognostic biomarker for bladder cancer and provides a possible mechanism for bladder cancer growth and invasion.

Expression of COL6A1 predicts prognosis in cervical cancer patients.

COL6A1 has been shown to play an important role in tumor initiation and progression. The present study is to investigate the clinical significance of COL6A1 in cervical cancer. In this study, the COL6A1 expression levels in 10 paired cervical cancer tissues and the adjacent non-tumor tissues were examined by real-time PCR. The expression of COL6A1 protein was examined in 162 cervical cancer samples by immunohistochemistry, and the correlation of COL6A1 expression with clinicopathologic factors was analyzed. The overall and recurrent-free survival rates were estimated using Kaplan-Meier method and compared with the log-rank test. The prognostic analysis was carried out with multivariate Cox regressions model. The result showed that COL6A1 expression was up-regulated in cervical cancer tissues in compared with that in non-tumor tissues. High expression of COL6A1 was significantly correlated with FIGO stage (P<0.001), tumor size (P=0.025) and lymph node metastasis (P=0.028) of the disease. Moreover, survival analysis showed that high expression of COL6A1 was significantly associated with poorer overall (OS) and recurrent free (RFS) survival (p=0.004 and =0.001, respectively) of cervical cancer patients. Multivariate analysis suggested that COL6A1 expression was an independent prognostic marker of cervical cancer (P=0.029). Thus, COL6A1 may serve as an oncogene in the initiation and progression of cervical cancer, and as a predictor of poor prognosis in cervical cancer patients.

Musashi-2 promotes migration and invasion in bladder cancer via activation of the JAK2/STAT3 pathway.

Musashi-2 (Msi2) is considered to have a crucial role in regulating various key cellular functions. However, the clinical significance and biological role of Msi2 in bladder cancer remains unknown. We examined the expression of Msi2 in bladder cancer cell lines in 167 clinical samples and the biological role of Msi2 in bladder cancer cells. Western blotting was used to investigate the possible mechanism of Msi2-induced migration and invasion in bladder cancer. Msi2 was significantly upregulated in bladder cancer cells and tissues compared with normal bladder urothelial cells and tissues. Immunohistochemical analysis revealed high expression of Msi2 in 57 of 167 (34.1%) bladder cancer specimens. Statistical analysis showed a significant correlation of Msi2 expression with advanced clinical stage, lymph node metastasis, and poor prognosis. Overexpression and ablation of Msi2 promoted and inhibited, respectively, the migration and invasion of bladder cancer cells. Furthermore, we found that Msi2 activated the JAK2/STAT3 pathway and promoted expression of genes downstream of JAK2/STAT3 in bladder cancer. This study demonstrates that Msi2 can induce bladder cancer cell migration and invasion by activating the JAK2/STAT3 pathway, and that Msi2 may be a valuable prognostic biomarker for bladder cancer patients.

Putative stem cell markers in cervical squamous cell carcinoma are correlated with poor clinical outcome.

BACKGROUND: The aim of this study was to elucidate the value of putative cancer stem cell markers Musashi-1, ALDH1, Sox2, and CD49f in predicting the prognosis in cervical squamous cell carcinoma (CSCC). METHODS: Real-time PCR and immunohistochemistry staining was performed to examine Musashi-1, ALDH1, Sox2, and CD49f expression in archived specimens of CSCC patients with postoperative chemotherapy. Kaplan-Meier analysis and Cox proportional hazards model were used to assess the prognostic impact of CSC markers for overall survival (OS) and recurrent-free survival (RFS). RESULTS: The Real-time PCR data showed that the expression of all markers were increased in CSCC tissues compared with in paired normal cervical tissues (P < 0.05). The IHC result showed that high expression of Msi1, ALDH1, Sox2, and CD49f was found in 25.7%, 43.0%, 62.0% and 29.0% CSCC samples, respectively. Moreover, high expression of Msi1 (P = 0.033 and P = 0.003, respectively), ALDH1 (P = 0.015 and P = 0.002, respectively), and Sox2 (P = 0.005 and P = 0.003, respectively), and low expression of CD49f (P = 0.027 and P = 0.025, respectively) were correlated with poor OS and PFS in CSCC patients. Interestingly, tumors with Msi1(high)/CD49f(low) expression had the poorest prognosis according to Msi1/CD49f stratification. In multivariate Cox regression analysis, Sox2 expression (P = 0.047 and P = 0.018, respectively), ALDH1 expression (P = 0.013 and P = 0.003, respectively), and CD49f expression (P = 0.008 and P = 0.003, respectively) were independent prognostic markers for both OS and RFS. CONCLUSIONS: Our results suggest that cancer stem cell markers are linked with poor prognosis of CSCC patients.