Knockdown of ADAM8 inhibits the proliferation, migration, invasion, and tumorigenesis of renal clear cell carcinoma cells to enhance the immunotherapy efficacy.

The current study performed bioinformatics and in vitro and in vivo experiments to explore the effects of ADAM8 on the malignant behaviors and immunotherapeutic efficacy of renal clear cell carcinoma (ccRCC) Cells. The modular genes most associated with immune cells were screened. Then, prognostic risk models were constructed by univariate COX analysis, LASSO regression analysis and multivariate COX analysis, and their diagnostic value was determined. The correlation between tumor mutation load (TMB) scores and the prognosis of ccRCC patients was clarified. Finally, six key genes (ABI3, ADAM8, APOL3, MX2, CCDC69, and STAC3) were analyzed for immunotherapy efficacy. Human and mouse ccRCC cell lines and human proximal tubular epithelial cell lines were used for in vitro cell experiments. The effect of ADAM8 overexpression or knockdown on tumor formation and survival in ccRCC cells was examined by constructing subcutaneous transplanted tumor model. Totally, 636 Black module genes were screened as being most associated with immune cell infiltration. Six genes were subsequently confirmed for the construction of prognostic risk models, of which ABI3, APOL3 and CCDC69 were low-risk factors, while ADAM8, MX2 and STAC3 were high-risk factors. The constructed risk model based on the identified six genes could accurately predict the prognosis of ccRCC patients. Besides, TMB was significantly associated with the prognosis of ccRCC patients. Furthermore, ABI3, ADAM8, APOL3, MX2, CCDC69 and STAC3 might play important roles in treatment concerning CTLA4 inhibitors or PD-1 inhibitors or combined inhibitors. Finally, we confirmed that ADAM8 could promote the proliferation, migration and invasion of ccRCC cells through in vitro experiments, and further found that in in vivo experiments, ADAM8 knockdown could inhibit tumor formation in ccRCC cells, improve the therapeutic effect of anti-PD1, and prolong the survival of mice. Our study highlighted the alleviative role of silencing ADAM8 in ccRCC patients.

A Network Meta-Analysis of the Differences in Effectiveness and Safety between Nivolumab and Targeted Drug Therapy in Metastatic Renal Cell Carcinoma.

Objective: Nivolumab plus other drugs have provided significant benefits in patients with metastatic renal cell carcinoma (mRCC), but most of the available comparisons were conducted with sunitinib, and differences in efficacy with targeted drugs were marginally reported. Thus, this study used a network meta-analysis to compare the difference in efficacy between nivolumab combination therapy and other targeted agents. Methods: In this systematic review and network meta-analysis, we searched PubMed, Embase, and Cochrane Library databases for randomized controlled trials (RCTs) with the time set from database establishment to December 10, 2021, using programmed death factor 1 (PD-1) inhibitors, nivolumab, and sunitinib in the treatment of mRCC. Progression-free survival (PFS), overall survival (OS), response rate (RR), and adverse events (AEs) were collated and analyzed using the gemtc package in the R language. Results: A total of ten studies satisfied the inclusion criteria, including 6568 RCC cases, 10 drugs, and 11 treatment protocols. The Ate_Axi protocol obtained similar PFS to the Niv_Cab protocol, which outperformed that of all other protocols. The Niv_Cab regimen showed better PFS benefits than the Niv_Ipi regimen (HR < 1, P < 0.05), and Niv_Ipi had superior PFS compared to the Ate, Eve, Paz, Sor, and Sun scheme. The regimens Cab, Niv_Cab, and Niv_Ipi were associated with the best PFS benefits, while Eve is the least favorable drug in terms of PFS. Niv_Cab showed better OS than Ate_Bev, Eve, Paz, Sor, and Sun. The patients given Ate_Bev, Eve, Paz, Sor, and Sun had inferior OS to those given Niv_Ipi. The Pem_Axi, Niv_Cab, and Niv_Ipi regimens had the best OS, and that of Eve is considered least promising. The Niv_Cab protocol showed significantly better RRs than the Eve, Paz, Sor, and Sun protocols, and the Ate_Bev, Eve, Paz, Sor, and Sun protocols presented superior RRs compared to the Niv_Ipi protocol. The Ate, Eve, and Niv_Ipi regimens had the lowest incidence of AEs, and the Sor regimen had the highest incidence of AEs. Conclusion: Among the targeted treatment options for mRCC, both Niv_Cab and Niv_Ipi yield better efficacy and safety in the treatment of mRCC, with Niv_Cab providing more survival benefit but with a less favorable safety profile.

Germline Mutation Landscape and Associated Clinical Characteristics in Chinese Patients With Renal Cell Carcinoma.

BACKGROUND: Renal cell carcinoma (RCC) is a disease of genomic alterations, of which the complete panorama helps in facilitating molecular-guided therapy. Germline mutation profiles and associated somatic and clinical characteristics remains unexplored in Chinese RCC patients. METHODS: We retrospectively profiled the germline and somatic mutations of 322 unselected RCC patients using a panel consisting of 808 cancer-related genes. We categorized patients into three groups based on germline mutation status and compared the somatic mutation spectrum among different groups. RESULTS: Approximately one out of ten (9.9%) RCC patients were identified to carry pathogenic/likely pathogenic (P/LP) germline variants (PGVs), of which 3.7% were variants in syndromic RCC-associated genes and 6.2% were other cancer-predisposition genes. The most common PGV was found in VHL (2.2%), followed by FH, TSC2, ATM, BRCA1, NBN, and BLM (0.6% each). Young patients (≤46 years) were more likely to harbor PGVs. Variants in syndromic RCC-associated genes were predominant identified in young patients, while variants in other cancer-predisposition genes were found in patients >46 years more frequently. Furthermore, 39.3% (11/28) of patients carrying PGVs were detected to have somatic "second hit" events. Germline and somatic sequencing, including microsatellite instability (MSI) status analysis, provided potentially actionable therapeutic targets in 17.1% of patients in the whole cohort. CONCLUSIONS: Our results revealed that approximately 10% of RCC patients carried clinically significant germline mutations. Current guidelines recommendation for genetic testing seemed not sensitive enough to identify patients with hereditary RCC susceptibility. It is rational to promote genetic testing in RCC population.

Exosomes-mediated transfer of long noncoding RNA LINC01133 represses bladder cancer progression via regulating the Wnt signaling pathway.

Bladder cancer (BC), as one of the most common malignant cancers of the urinary system, has a high incidence and mortality rates. Recently, increasing studies have indicated that exosomes can mediate cellular communication in assorted cancers, including BC. Long noncoding RNAs (lncRNAs) have also been confirmed to take part in the regulation of many cancers. Long intergenic non-protein coding RNA 1133 (LINC01133) is an lncRNA and its roles in several cancers have been revealed. However, the functions of exosomes and LINC01133 in BC are still not elucidated. In our research, functional assays were conducted to evaluate the function of LINC01133, as well as the influence of exosomes and LINC01133 on BC cells. Western blot assay, immunofluorescence assay, electron microscope, and nanoparticle tracking analysis were applied for detecting the characteristics of exosomes. Bioinformatics tools and quantitative reverse-transcription polymerase chain reaction were performed to test the expression of LINC01133 in BC cells and exosomes of the immortalized human uroepithelial cell line (SV-HUC-1). Luciferase reporter assay was performed to measure the activity of the Wnt pathway. We discovered that LINC01133 expression was high in exosomes of SV-HUC-1 and low in that of BC cells. Additionally, exosomes restrained cell viability, proliferation, migration, and invasion. Similarly, LINC01133 exerted the same function on BC cells. In addition, the Wnt signaling pathway could be inactivated by LINC01133. Finally, in vivo experiments demonstrated that cell growth could be suppressed by overexpressed LINC01133. In short, exosomes-mediated transfer of lncRNA LINC01133 repressed BC progression via regulating the Wnt signaling pathway.

Prognostic Value of YTHDF2 in Clear Cell Renal Cell Carcinoma.

m6A, the main form of mRNA modification, participates in regulating multiple normal and pathological biological events, especially in tumorigenesis. However, there is little known about the association of m6A-related genes with prognosis of clear cell renal cell cancer (ccRCC). Therefore, the prognostic value of m6A-related genes was investigated using Kaplan-Meier curves of overall survival (OS) with the log-rank test and Cox regression analysis. The differential expression of YTHDF2 mRNA in ccRCC and tumor-adjacent normal tissues and associated with clinicopathological characteristics was also analyzed. The alteration of cancer signaling pathways was screened by Gene Set Enrichment Analysis (GSEA). Univariate analysis showed that 15 m6A-related genes (including YTHDF2) were closely related to prognosis. Multivariate analysis further confirmed that YTHDF2 could serve as an independent prognostic factor for the OS of ccRCC patients (P < 0.001). Low-level expression of YTHDF2 had poor prognosis in ccRCC patients with lower tumor-node-metastasis (TNM) stage, age > 61, non-distant metastasis, non-lymph node metastasis, female gender, and higher histological grade (P < 0.05). Moreover, YTHDF2 expression in ccRCC tissues (N = 529) is significantly lower than that of tumor-adjacent normal tissues (N = 72, P = 0.0086). Furthermore, GSEA demonstrated that AKT/mTOR/GSK3 pathway, EIF4 pathway, CHREBP2 pathway, MET pathway, NFAT pathway, FAS pathway, EDG1 pathway, and CTCF pathway are altered in tumors with high YTHDF2 expression. Taken together, our results demonstrated that YTHDF2 (an m6A-related gene) could serve as a potential prognostic biomarker of ccRCC, and targeting epigenetic modification may be a novel therapeutic strategy for the treatment of ccRCC.

MicroRNA-1298-5p inhibits cell proliferation and the invasiveness of bladder cancer cells via down-regulation of connexin 43.

MicroRNA (miR)-1298 is widely down-regulated in a variety of malignant tumors, which facilitates cell proliferation, invasiveness, and migration. However, the specific biological function of miR-1298 in bladder cancer (BC) is still unknown. Connexin 43 (Cx43) is often up-regulated in tumors. Identifying miRNAs that target Cx43 in the setting of BC will help to develop Cx43-based therapies for BC. In this study, the results demonstrated that the expression levels of miR-1298 and Cx43 were significantly down-regulated and up-regulated, respectively, in BC tissues. Overexpression of miR-1298 inhibited cell proliferation, migration, and invasiveness in two BC cell lines as determined using MTT assays, cell cycle assays, colony formation assays, Transwell assays, gelatin zymography, and Western blot. In addition, we found that miR-1298 decreased Cx43 expression by directly targeting the 3'-UTR. Further, we observed that the promotion of BC cell proliferation, migration, and invasiveness from Cx43 on could be partially attenuated by overexpressing miR-1298. Moreover, the protein expression of p-ERK was ameliorated after transfection with overexpressed-miR-1298. Knockdown of Cx43 reversed the promotion of cell migration and invasiveness due to decreased expression of miR-1298. All of the data from our study indicate that miR-1298 could be a diagnostic marker of BC and a potential therapeutic agent via inhibiting Cx43.

Silencing of lncRNA AFAP1-AS1 Inhibits Cell Growth and Metastasis in Clear Cell Renal Cell Carcinoma.

The lncRNA AFAP1-AS1, oriented from an antisense direction to the protein-coding gene AFAP1 in the opposite strand, was upregulated in a variety of tumors and associated with poor prognosis, including lung cancer, breast cancer, ovarian cancer, and so on. However, the biological role of AFAP1-AS1 in clear cell renal cell carcinoma (ccRCC) is still unknown. We observed that AFAP1-AS1 expression was significantly upregulated in ccRCC tissues and that patients with high-level expression of AFAP1-AS1 had a shorter overall survival. Knockdown of AFAP1-AS1 markedly suppressed the progression of proliferation, invasion, migration, and EMT in ccRCC cells. Downregulation of AFAP1-AS1 resulted in an increase in E-cadherin and a decrease in vimentin. Noticeably, we found that PTEN has a negative correlation with the lncRNA AFAP1-AS1 expression. Further studies verified that PTEN deficiency effectively attenuated the ability of AFAP1-AS1 in promoting ccRCC cell proliferation, invasion, migration, and EMT. Moreover, the similar biological response of silencing AFAP1-AS1 was observed in our ccRCC mice model. Knockdown of AFAP1-AS1 evidently suppressed tumor growth. Taken together, our results provide the evidences that silencing of AFAP1-AS1 inhibits cell proliferation, EMT, and metastasis through PTEN-dependent signaling, and our findings elucidate a novel potential therapeutic target or biomarker for the treatment of ccRCC.

Long non-coding RNA ZFAS1 promotes proliferation and metastasis of clear cell renal cell carcinoma via targeting miR-10a/SKA1 pathway.

BACKGROUND: LncRNA ZFAS1 (ZNFX1 antisense RNA1) plays key roles in the occurrence and progression of various cancers, including colorectal cancer, glioma, lung cancer, gastric cancer, and so on. To date, relatively little is known about its potential role in development and/or progression of clear cell renal cell carcinoma (ccRCC). METHODS: Expression level of ZFAS1 and miR-10a in 60 ccRCC and 20 adjacent non-tumor tissues were determined by using qRT-PCR. The effect of knockdown of ZFAS1 on cell proliferation, migration and invasion were measured by CCK-8 assay, transwell migration and invasion assay, respectively. The correlation of ZFAS1 and miR-10a was analyzed by bioinformatics DIANA TOOLS. Protein and mRNA expression of spindle and kinetochore-associated protein 1(SKA1) were determined by western blot and qRT-PCR analysis, respectively. Interactions between ZFAS1 and miR-10a were verified by luciferase reporter assay and RNA immunoprecipitation (RIP) assay, and interactions between miR-10a and SKA1 was verified by a luciferase reporter assay. RESULTS: We observed that high-level expression of ZFAS1 is positively correlated with poor prognosis and shorter overall survival (OS) in patients with ccRCC. Knockdown of ZFAS1 significantly suppressed proliferation, migration and invasion of ccRCC cells. Furthermore, miR-10a was identified as a target of ZFAS1. Silencing miR-10a could attenuate the ability of ZFAS1 in promoting proliferation and metastasis of ccRCC. Subsequently, our studies validated that SKA1, as a key downstream target protein for miR-10a, is responsible for the biological role of ZFAS1. ZFAS1 positively regulated SKA1 expression via sponging miR-10a. CONCLUSIONS: Taken together, our findings suggest that ZFAS1 promotes growth and metastasis of ccRCC via targeting miR-10a/SKA1 pathway, which may represent a novel therapeutic target or biomarker for ccRCC.

Efficacy and Safety of Chemotherapy Regimens in Advanced or Metastatic Bladder and Urothelial Carcinomas: An Updated Network Meta-Analysis.

Background: Gemcitabine plus cisplatin (GC) and methotrexate, vinblastine, adriamycin, and cisplatin (MVAC) have been the first-line treatments for advanced or metastatic urothelial carcinoma (AMUC). However, their effects are unsatisfactory, and more drugs and regimens still need to be explored. Objective: We aimed to comprehensively compare all possible regimens with GC or MVAC in randomized controlled trials (RCTs) by network meta-analysis. Methods: We searched the PubMed, Embase, and Cochrane databases for RCTs that evaluated regimens compared to GC or MVAC on AMUC patients. The major outcomes were progression-free survival (PFS), overall survival (OS), and objective response rate (ORR). A network meta-analysis was used to assess the effectiveness and safety of the included treatment regimens, and the regimens were then clustered by the average linkage method. Results: A total of 19 trials that assessed 3,363 AMUC patients were included. For PFS, paclitaxel plus GC (PGC) was significantly superior to GC (log hazard ratio (HR): -0.16; 95% confidence interval (CI): -0.32, 0.00) with a moderate level of reliability. However, there was no significant difference between PGC and MVAC (log HR: -0.03; 95% CI: -0.27, 0.20). For OS, PGC was significantly superior to GC (log HR:-0.17; 95% CI: -0.33, -0.00) with a moderate reliability level but not significantly different from MVAC (log HR: -0.10; 95% CI: -0.35, 0.15). Analysis of ORR showed that PGC was superior to MVAC (log odds ratio (OR): 0.59; 95% CI: 0.02, 1.16) with a low reliability level and GC (log OR: 0.41; 95% CI: 0.12, 0.71) with a moderate reliability level. In the cluster results, PGC and sorafenib plus GC (GCS) exhibited relative advantages in efficiency, followed by MVAC and apatorsen plus GC (GCA); however, PGC, gemcitabine plus carboplatin (GP), and MVAC had more serious side effects. Conclusions: In our analysis, PGC was superior to MVAC and GC in only the ORR results and superior to GC in the OS and PFS results but was not significantly different from MVAC. More individualized therapies with targeted drugs need to be studied.

ZFAS1: a novel tumor-related long non-coding RNA.

Long non-coding RNAs (lncRNA) are classified as a kind of RNA, which are longer than 200 nucleotides in length and cannot be translated into proteins. Multiple studies have demonstrated that lncRNAs are involved in various cellular processes, including proliferation, differentiation, cell death, and metastasis. In addition, aberrant expression of lncRNAs has been discovered in human tumors, where they function as either oncogenes or tumor suppressor genes. Among numerous lncRNAs, we focus on ZNFX1 antisense RNA 1 (ZFAS1), a well-known lncRNA that is aberrant overexpression in various tumors, including melanoma, esophageal squamous cell carcinoma, non-small cell lung cancer, gastric cancer, colon cancer, and Hepatocellular carcinoma, in which it functions as oncogene. In contrast, ZFAS1 is downregulated in breast cancer, which may function as tumor suppressor gene. In this review, we provide an overview of current evidence concerning the role and potential clinical utilities of ZFAS1 in human cancers.

Collagen Type VI Alpha 3 Chain Promotes Epithelial-Mesenchymal Transition in Bladder Cancer Cells via Transforming Growth Factor ß (TGF-ß)/Smad Pathway.

BACKGROUND Collagen type VI alpha 3 chain (COL6A3) has been proven to be a biomarker in the occurrence and development of bladder cancer, which is the most common malignant tumor in the urinary system. This study aimed to explore the effect and molecular mechanism of COL6A3 on EMT in vitro induced by TGF-ß/Smad in bladder carcinoma. MATERIAL AND METHODS There were 42 patients included in the Kaplan-Meier survival analysis. A cell counting kit-8 (CCK-8) assay and an angiogenesis assay were used to measure cell proliferation and tube formation, respectively. Western blot analysis and quantitative reverse transcription-polymerase chain reaction (qPCR) were conducted for the proteins and mRNAs expression. RESULTS COL6A3 was highly expressed in tissues and cells of bladder cancer. COL6A3 silencing could inhibit the cell proliferation and angiopoiesis. In addition, COL6A3 silencing obviously suppressed the levels of matrix metalloproteinase-2 (MMP2), Matrix metalloproteinase-9 (MMP9), and vimentin. On the contrary, the levels of epithelium-specific cell-cell adhesion molecule (E-cadherin) and tumor inhibitor of metalloproteinase-1 (TIMP-1) were significantly increased. Furthermore, we found that COL6A3 silencing reduced the activity of p-Smad2, p-Smad3, and transforming growth factor ß (TGF-ß). CONCLUSIONS COL6A3 could influence the viability and angiogenesis of bladder cancer cells. COL6A3 may have a certain relationship with the TGF-ß/Smad-induced EMT process.

Prognostic value of long noncoding RNA ZFAS1 in various carcinomas: a meta-analysis.

A number of studies have revealed that zinc finger antisense 1 (ZFAS1), a long noncoding RNA (lncRNA), is aberrantly regulated in various cancers, and high ZFAS1 expression is associated with poor prognosis and increased risk of lymph node metastasis (LNM). This meta-analysis was conducted to identify the potential value of ZFAS1 as a biomarker for cancer prognosis. We searched electronic database PubMed, Web of Science, and China Wanfang Data (up to June 1, 2017) to collect all relevant studies and explore the association of ZFAS1 expression with overall survival (OS) and LNM. The results showed that cancer patients with high ZFAS1 expression had a worse OS than those with low ZFAS1 expression (HR: 1.94, 95% confidence interval [CI]: 1.41-2.47, P < 0.001), and high ZFAS1 expression was significantly associated with LNM (OR: 2.60, 95% CI: 1.54-4.42, P < 0.001). Subgroup analysis revealed that high ZFAS1 expression was significantly related to high incidence of LNM in subgroups of sample size more than 88 (OR: 3.16, 95% CI: 2.06-4.86, P < 0.001), non-digestive system malignancies (OR: 4.05, 95% CI: 2.49-6.60, P < 0.001), and studies reported in 2017 (OR: 4.86, 95% CI: 2.67-8.84, P < 0.001) without significant heterogeneity. Further meta-regression by the covariates showed that tumor type, sample size, quality score, cut off value and publication year did not result in the inter-study heterogeneity. In conclusion, the present meta-analysis demonstrates that high ZFAS1 expression may potentially serve as a reliable biomarker for poor clinical outcome in various cancers.

Ectopic WWOX Expression Inhibits Growth of 5637 Bladder Cancer Cell In Vitro and In Vivo.

WW domain-containing oxidoreductase (WWOX) gene located in the common fragile site FRA16D region exhibits loss or reduction of expression in multiple types of carcinomas including bladder cancer. However, the role of WWOX in the tumorigenesis and development of bladder cancer remains elusive. In this study, WWOX overexpression construct was transfected into 5637 bladder cancer cell line in which WWOX expression was compromised. Constitutive expression of ectopic WWOX in 5637 cells suppressed cell proliferation and cell cycle progression, which was associated with downregulation of Cyclin B, D1, and E. Moreover, WWOX overexpression promoted apoptosis in 5637 cells and resulted in upregulation of Bax, downregulation of Bcl-2, and elevated levels of cleaved caspase-3 and cleaved PARP, indicating activation of the intrinsic apoptosis pathway. Furthermore, WWOX overexpression suppressed tumorigenicity of 5637 cells and promoted apoptosis in the xenograft tumors as demonstrated in a xenograft mouse model. In summary, our data indicate that WWOX plays a critical role in the regulation of proliferation, cell cycle, apoptosis, and tumorigenesis of bladder cancer cells, suggesting that WWOX may have potential clinical implications in bladder cancer therapy.