BACKGROUND:Clear cell renal carcinoma is a common urological malignancy with poor prognosis and treatment outcomes. lncRNAs are important in metabolic reprogramming and the tumor immune microenvironment, but their role in clear cell renal carcinoma is unclear. METHODS:Renal clear cell carcinoma sample data from The Cancer Genome Atlas was used to establish a new risk profile by glycolysis-associated lncRNAs via machine learning. Risk profile-associated column-line plots were constructed to provide a quantitative tool for clinical practice. Patients with renal clear cell carcinoma were divided into high- and low-risk groups. Clinical features, tumor immune microenvironments, and immunotherapy responses were systematically analyzed. We experimentally confirmed the role of LINC01138 and LINC01605 in renal clear cell carcinoma. RESULTS:The risk profile, consisting of LUCAT1, LINC01138, LINC01605, and HOTAIR, reliably predicted survival in patients with renal clear cell carcinoma and was validated in multiple external datasets. The high-risk group presented higher levels of immune cell infiltration and better immunotherapy responses than the low-risk group. LINC01138 and LINC01605 knockdown inhibited the proliferation of renal clear cell carcinoma. CONCLUSIONS:The identified risk profiles can accurately assess the prognosis of patients with clear cell renal carcinoma and identify patient populations that would benefit from immunotherapy, providing valuable insights and therapeutic targets for the clinical management of clear cell renal carcinoma.

The association of programmed cell death-ligand 1 (PD-L1) expression with the activity of immune checkpoint inhibitor therapy setting has encouraged us to study the prevalence of PD-L1 in muscle invasive urothelial bladder cancer (MIUBC) patients. Since, the prevalence of PD-L1 expression and its prognostic role in MIUBC is unclear in China, we conducted a multi-center, prospective, epidemiological study to investigate prevalence of high PD-L1 expression and its correlation with other exploratory biomarkers including CD8+ T cells and tumor mutation burden (TMB) in Chinese MIUBC patients (NCT03433924). The study enrolled newly diagnosed, untreated MIUBC patients from 17 hospitals in China. High PD-L1 expression by Ventana PD-L1 [SP263] assay was defined as 1) ≥25% tumor cell (TC) + or 2) tumor associated immune cell (IC) area >1%: ≥25% IC+; 3) IC area=1%: 100% IC+. The primary outcome was prevalence of high PD-L1 expression; secondary outcome was PD-L1 expression profile in TC or IC in patients. An exploratory study was conducted to investigate the percentage of patients with high infiltration of CD8+ T cells and elevated TMB using immunohistochemistry and next generation sequencing, respectively. High CD8+ and TMB status was considered if it refers to ˃ median value (3%) and ˃10 mut/Mb, respectively. Association of PD-L1 expression with demographic and baseline characteristics was evaluated by logistic regression model while PD-L1 with tumor biomarkers were evaluated by spearman’s rank correlation coefficient. Overall, 248 MIUBC patients were enrolled and 229 patients with PD-L1 data were included. High PD-L1 expression was observed in 52.4% patients. High PD-L1 expression was positive in 59 (25.8%) patients of TC and 82 (35.8%) patients of IC. CD8+ T cell and TMB showed high expression in 44.5% and 54.1% patients, respectively, with mean TMB of 14.14 mut/Mb. The exploratory analysis showed no significant association of PD-L1 expression with age, gender, primary tumor site, metastatic disease, regional lymph node, tobacco use or AJCC stage. Further a weak and positive correlation was observed between percentage of TC with membrane PD-L1 positivity and CD8+ T cells (0.34, 95%Cl, 0.22 - 0.45, p˂0.001), IC with membrane PD-L1 positivity and CD8+ T cells (0.44, 95%Cl, 0.33 - 0.54, p˂0.001), TC with membrane PD-L1 positivity and TMB (0.05, 95%Cl, -0.08 - 0.19, p=0.441), IC with membrane PD-L1 positivity and TMB (0.16, 95%Cl, 0.02 - 0.29, p=0.020). In conclusion, our study showed high prevalence of PD-L1 expression in patients with MIUBC along with their weak, positive correlation with CD8+ T cells and TMB. These findings are consistent with other studies that show that PD-L1 and TMB stand as the most robust predictive biomarkers, but may be uncorrelated. With subsequent follow-up time, the effect of biomarkers on survival will be assessed in future study. Citation Format: Yu Fan, Tao Dai, Dahong Zhang, Hongqian Guo, Fangjian Zhou, Benkang Shi, Shaogang Wang, Zhigang Ji, Chunxi Wang, Xudong Yao, Qiang Wei, Nanhui Chen, Jinchun Xing, Jinjian Yang, Chuize Kong, Jian Huang, Dingwei Ye, Liqun Zhou. Prevalence of PD-L1 expression and its correlation with tumor biomarkers in the Chinese muscle invasive urothelial bladder carcinoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6658.

Supplementary Figure 4 - PDF file 146K, mRNA expression of Cyclin D1 was evaluated in PC3 cells (A) and DU145 cells (B) that formed colonies in soft agar or those grew in the regular 2D culture condition by qRT-PCR. Values are mean s.d. (n = 4). (C), PC3 cells were cultured under 32˚C for 0 hr to 144 hr, and mRNA level of cyclin D1 was detected by qRT-PCR. Values are mean s.d. (n = 4). (D) Over-expression of CD44 variant and standard construct. LNCaP cells were transfected with pLenti-III-CD44s (CD44s), pLenti-III-CD44v8-v10 (CD44v8-10) or empty vector. After 48 hr of transfection, cells were collected and the expression of mRNA and protein of CD44 were detected by RT-PCR and Western blot, respectively. Data are mean s.d. (n = 3), *P < 0.05

Supplementary Figure 2 - PDF file 68K, (A), expression of cancer-related miRNAs was detected in the PC3-RBM3 and PC3-GFP clones using a cancer-specific miRNA PCR array. Left panel, selected miRNAs whose expression levels differed more than 1.5 fold in the clone R#1 compared to G#1. Red bars, differentiation-related miRNAs; black bars, other cancer-related miRNAs. Right panel, the up-regulation of miR-9 in PC3-RBM3 clones was validated by RT-PCR. Values are mean s.d. (n = 3), *P < 0.05. (B), left panel, β3-tubulin expression was detected by Western blot in PC3-GFP and PC3-RBM3 clones. Right panel, quantitative evaluation of protein levels of β3-tubulin. Values are mean s.d. (n = 3), *P < 0.05. (C), the immunohistochemical staining of RBM3 and β3-tubulin in the nerve fibers observed in human prostate tissue. Upper panels, low magnification; lower panels, high magnification. Scale bars, 100 m. In all panels: G#1, PC3-GFP-clone #1; G#2, PC3-GFP-clone #2; R#1, PC3-RBM3-clone #1; R#6, PC3-RBM3-clone #6.expression of CDH2 in PC3-GFP and PC3-RBM3 clones

Supplementary Figure 3 - PDF file 244K, (A), CD44 variant mRNAs were amplified by PCR using the general primers c13 and hs3'. The PCR products at size of 650 bp and 250 bp were cut and subjected to direct DNA sequencing. The numbers in the block are exons number of CD44. N, negative control; G#1, PC3-GFP-clone #1. (B), schematic diagram of designing siRNAs specific to CD 44 variant exons v8-v10 (siC1, siC2) and primers specific to CD44v8-v10 (CD44v) or standard CD44 (CD44s). Primers c13 and hs3' can amplify both CD44s and CD44v. Primers p1 and p2 can only amplify CD44v8-v10, while primers p3 and p4 are specific to standard CD44s. Primer hs5' is the outside primer of the nest PCR. (C), the specificity of siRNAs targeting for CD44v8-v10. Variant exon-specific PCR was performed using primer p1 and p2. CD44s was amplified using CD44 standard-specific primer p3 and p4. siN, non-target siRNA. Data are mean s.d. (n = 3), *P < 0.05. (D), the efficiency of siRNA pool targeting RBM3. Cells were treated with siRBM3 or non-targeting siRNA (siN) for 48 hr. The expression of mRNA and protein of CD44 were detected by RT-PCR and Western blot, respectively. R#1, PC3-RBM3-clone #1; R#6, PC3-RBM3-clone #6. Data are mean s.d. (n = 3), *P < 0.05

Supplementary Table 2 - PDF file 33K, Calculated size of PCR products for CD44 splicing variants

<p>Supplementary Table 1 - PDF file 43K, mRNA expression of stem cell-related gene using a Stem Cell PCR array</p>

Supplementary documents - PDF file 122K, Supplementary methods and supplementary figures legends

Supplementary Figure 1 - PDF file 202K, (A), expression of RBM3 in different human tissues. Data are mean s.d. (n = 3). (B), expression of CIRBP in CD133+ and CD133- PrEC cells. mRNA levels of RBM3 and CIRBP was determined by RT-PCR. Data are mean s.d. (n = 4). (C), mRNA levels of RBM3 were detected in the PC3-GFP and PC3-RBM3 clones by RT-PCR. Data are mean s.d. (n = 3), *P < 0.05. (D), RBM3 protein was detected in both cytoplasmic (Cyto) and nuclear (Nu) components of PC3-GFP and PC3-RBM3 clones. Tubulin and Lammn A/C were used as loading controls for cytoplasmic and nuclear fractions, respectively. In all panels: G#1, PC3-GFP-clone #1; G#2, PC3-GFP-clone #2; R#1, PC3-RBM3-clone #1; R#6, PC3-RBM3-clone #6

Supplementary Figure 5 - PDF file 104K, (A), mRNA levels of CD44v8-v10 (CD44v) were determined in human normal and prostate cancer tissues by RT-PCR. Normal, normal prostate tissue (n = 19); OC-PCa, organ-confined prostate cancer (n = 79); Met-PCa, metastatic prostate cancer (n = 23). (B), mRNA levels of CD44v were compared between patients with recurrent PCa (Rec, n = 44) and those without recurrent disease (Non-rec, n = 30). (C) and (D), mRNA levels of standard CD44 (CD44s) (C) or CD44v (D) were compared between patients with low grade PCa (Gleason score 6, n = 21) and those with high grade disease (Gleason score > 6, n = 53). All experiments were repeated three times. Bars indicate median values

Abstract Background In the era of individualized therapy,there is a great need to incorporate lncRNAs into preclinical models to develop prognostic or therapeutic biomarkers.We sought to apply the tumor immune-associated lncRNA – SNHG16 to develop and validate a predictive model constructed from SNHG16 and associated immune gene signatures in BC patients obtained from independent public datasets and clinical internal cohorts, to assess the benefit of immune checkpoint inhibitors treatment and the prognosis in BC patients. Materials and methods A novel immune-related and independent predictive model was developed for prognosis and immunotherapeutic evaluation of bladder cancer, based on the identification and analysis of the immune-related SNHG16. Based on the training (TCGA-BLCA) and external validation datasets, the SNHG16-associated immune gene signature was applied to classify BC patients into low- and high-risk groups. Cell proliferation assay and Transwell assay were used to detect the function of related genes. Results There were significant differences in prognosis and response to immunotherapy among patients in the different risk groups. Univariate and multivariate analyses confirmed the SNHG16-associated immune gene signature to be an independent predictor of BC prognosis. In addition,in vitro functional assay data confirmed the cancer biological function of SNHG16 and its associated immune signature genes. Conclusions Immune-associated lncRNA-SNHG16 and its associated immune gene signatures are reliable tools for predicting BC prognosis and response to immunotherapy and may provide valuable insights for deciding the treatment for BC. Further, the model will provide useful guidance for clinical judgment and personalized regimen-selection for immunotherapy of bladder tumors.

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology I (MP16)1 Apr 2019MP16-05 LOWERING MRNA EXPRESSION OF PLEKHO1 INHIBITS TUMOR GROWTH OF RCC CELLS IN VITRO AND IN VIVO, POTENTIALLY THROUGH MODULATING THE HIPPO AND MAPK/JNK SIGNALLING PATHWAYS Yu Zeng*, Zi Yu, Shui Fu, Gejun Zhang, Chengcheng Lv, Qingzhuo Dong, Chuize Kong, and Cheng Fu Yu Zeng*Yu Zeng* More articles by this author , Zi YuZi Yu More articles by this author , Shui FuShui Fu More articles by this author , Gejun ZhangGejun Zhang More articles by this author , Chengcheng LvChengcheng Lv More articles by this author , Qingzhuo DongQingzhuo Dong More articles by this author , Chuize KongChuize Kong More articles by this author , and Cheng FuCheng Fu More articles by this author View All Author Informationhttps://doi.org/10.1097/01.JU.0000555341.83310.20AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVES: Renal cell carcinoma (RCC) is the most common type of kidney cancer word widely. Although target therapy and checkpoint immunotherapy improve the survival of metastatic RCC, a large number of patients are still incurable. METHODS: The raw RNA-sequencing data of RCC were download from the Cancer Genome Atlas (TCGA) database and exported by using R-project. A high-content cell viability assay was used to screen genes those are potentially regulating cell proliferation after downregulating the mRNA expressions of each gene by shRNA. RT-PCR was conducted to detect the mRNA expression of pleckstrin homology domain containing O1 (PLEKHO1) gene in 30 paired localized tumor and para-tumor kidney tissue samples. The effects of lowering the expression of PLEKHO1 were assessed by CCK-8 assay, Celigo assay, flow cytometry and transwell assay, separately. Additionally, xenograft tumor models were established in nude mice to investigate the function of PLEKHO1 in vivo. Gene expression microarray and co-expression analysis followed by Western blot were further conducted to explore the involvement of PLEKHO1 in signalling pathways. RESULTS: Sixteen genes were identified to be consistently highly expressed in ccRCC tissue compared with the matched para-tumor tissue in TCGA data set, while downregulation of only two of those significantly inhibited the viability of RCC cells. Among two genes, PLEKHO1 was unknown in its relevance to RCC and its expression level was shown to be associated with prognosis of RCC in the TCGA data set. Next, upregulation of PLEKHO1 mRNA was confirmed in RCC tissue samples. Reducing the expression of PLEKHO1 by specific siRNAs significantly inhibited cell viability and cell migration, and facilitated apoptosis in 786-0 and Caki-1 cells. In addition, knocking-down the expression of PLEKHO1 greatly impeded xenografted tumor's growth in nude mice. Finally, signalling pathways exploring by microarray analysis indicated that PLEKHO1-knockdown decreased the expressions of TAZ and CTGF, which are two key factors in HIPPO signalling pathway, and simultaneously reduced the expressions of p-JNK and c-Jun, which are two key factors in the JNK signalling pathway, in 786-O cells. CONCLUSIONS: These results suggest that the aberrant expression of PLEKHO1 in RCC may contribute to tumor growth, and therefore the potential of PLEKHO1 as a therapeutic target needs to be further studied. Source of Funding: National Natural Science Foundation of China (grant nos. 81372766 and 81572532), and the Liaoning “Climbing” scholarship. Shenyang, China, People's Republic of© 2019 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 201Issue Supplement 4April 2019Page: e207-e207 Advertisement Copyright & Permissions© 2019 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yu Zeng* More articles by this author Zi Yu More articles by this author Shui Fu More articles by this author Gejun Zhang More articles by this author Chengcheng Lv More articles by this author Qingzhuo Dong More articles by this author Chuize Kong More articles by this author Cheng Fu More articles by this author Expand All Advertisement PDF downloadLoading ...

Xiaojun Man Tao Liu Yuanjun Jiang Zhe Zhang Yuyan Zhu Zhenhua Li Chuize Kong Jiani He 1Department of Urology, The First Hospital of China Medical University, Shenyang 110001, People’s Republic of China; 2Institute of Urology, Department of Urology, China Medical University, Shenyang 110001, People’s Republic of China; 3Breast Division, Department of Surgical Oncology, The First Hospital of China Medical University, Shenyang 110001, People’s Republic of China Background: Bladder cancer (BC) is the ninth most common cancer and the fourteenth leading death worldwide. CARD-containing MAGUK 3 (CARMA3) protein is a novel scaffold protein known to activate NF-κB pathway and is overexpressed in BC tissues. Purpose: The objective of this study was to identify how CARMA3 affects the metastasis of BC cells via the β-catenin signaling pathway. Materials and methods: In the present study, 5637 and T24 BC cells with stable low expression of CARMA3 were established, and their migratory and invasive capabilities were further evaluated by wound-healing and transwell assay. The activity and expression of βcatenin were determined by Luciferase assay and immunofluoresence staining. The mRNA and protein expression levels of CARMA3, matrix metallopeptidase (MMP) 9 and MMP2 were detected by quantitative real-time PCR (qRT-PCR) and Western blot analysis. The nude mouse tumor xenograft model was established for in vivo study. Results: By comparison to the control cells, CARMA3-silenced cells acquired a less aggressive phenotype: decreased migration and invasion. More importantly, we confirmed that CARM3 knockdown could inhibit β-catenin mRNA and protein expression and activity, and reduce the expression and/or activity of matrix metallopeptidase (MMP) 9, MMP2 and C-myc. Also, CARM3 silencing increased E-cadherin expression and attenuated the expression of βcatenin. Moreover, we demonstrated that β-catenin overexpression reversed the inhibiting effect of CARMA3 silencing on cell invasion and migration. Furthermore, our study illustrated that knockdown of CARMA3 suppressed BC cells xenograft tumor growth in nude mice. Conclusion: We demonstrated that CARMA3 contributes to the malignant phenotype of BC cells at least by activating β-catenin signaling pathway, and it may serve as a therapeutic target for clinic treatment in BC.

e15553^ Background: Systemic adjuvant therapy in RCC patients may eliminate residual micrometastases and reduce risk of relapse. Sorafenib is a candidate for investigation as adjuvant therapy. Methods: 69 patients at multiple centers between Nov 2008 and Jul 2010 with locally advanced clear cell RCC at high risk for disease recurrence postoperatively were enrolled. The criteria for high risk were defined according to Leibovich Score (≥6), which was based on tumor stage, regional lymph node status, tumor size, nuclear grade. The patients received sorafenib 400 mg bid in 4-12 weeks post radical nephrectomy for a total 9 cycles (6weeks/cycle). The primary endpoint was median disease free survival (DFS). Overall survival (OS) and other safety profiles were investigated, and the efficacy of Udderly Smooth Udder Cream in management of hand and foot skin reaction (HFSR) induced by sorafenib was determined by stratification into two groups (consistent usage or only start use the scream since the onset of HFSR). Results: Median age was 54 years, and the surgical features for the patients are listed in the Table. Leibovich Score 6,7,8,9,10 is respectively 34 (49.28%), 17 (24.64%), 11 (15.94%), 5 (7.24%), 2 (2.90%). 35 patients had already either progression or died till the time of analysis, the median DFS is 41.4 months. Median OS has not yet been reached, and 1, 2, 3, 4 years survival rate was 92.4%, 86.0%, 76.2%, 67.5%.The most common AEs were HFSR (81.2%), alopecia (42.0%), and diarrhea (36.2%), and most AEs (82.6%) were grade 1 or 2 severity. The AE profile was consistent with reported data in sorafenib Asian population. Udderly Smooth udder cream had not been proven to decrease the incidence, grade, onset or duration of HFSR. Conclusions: In high risk of relapse RCC patients who underwent radical nephrectomy, sorafenib seemed to provide longer DFS while comparing with previously reported literatures, and side effects were acceptable. Due to the single arm study design and limited sample size, the effect of sorafenib adjuvant therapy will require further validation in randomized controlled trials. Clinical trial information: NCT01041482. TNM stage N (%) T2N0 13 (18.84) T2N1-2 5 (7.25) T3aN0 24 (34.78) T3aN1-2 4 (5.80) T3bN0-1 13 (18.84) T3cN0 3 (4.35) T4N0 7 (10.14)

Aim: The present study was conducted to investigate whether alpha-lipoic acid (α-LA) is able to reverse impaired bladder function induced by diabetes in a rat model and to explore the possible mechanism mediating the effect. Methods: Male Sprague-Dawley rats were divided randomly into 3 age-matched groups: control, diabetes mellitus (DM) treated with vehicle, and DM with α-LA treatment. The diabetic rats were induced by a single intraperitoneal (ip) injection of streptozotocin (60 mg/kg). Six weeks after the induction of DM, the two groups received another 6 weeks of treatment with vehicle or α-LA (100 mg/kg, ip). Body weight and blood glucose levels were measured weekly. The bladder function was evaluated by in vitro cystometry. The oxidative stress status was determined by biochemical methods, and the level of nerve growth factor was investigated by enzyme-linked immunosorbent assay. Results: The streptozotocin-induced diabetic rats showed impaired bladder function characterized by increased bladder capacity, decreased bladder contractility (voiding efficiency), and an increase in residual urine. Treatment with α-LA significantly normalized the increased bladder capacity for inducing voiding, single-voided volume, and post-void residual volume. α-LA treatment significantly reversed the increased level of malondialdehyde and reduced the activities of both superoxide dismutase and catalase. DM caused a decrease in the bladder nerve growth factor (NGF) level, and α-LA upregulated the level of NGF in the diabetic rat bladder. Conclusion: These results indicate that α-LA has a beneficial effect on diabetes-induced cystopathy by ameliorating oxidative stress and normalizing the NGF level in the bladder.