Overall Survival In ccRCC Patients Research Articles

73 Fasting mediated gut microbiome modulation improves response to immune checkpoint therapy in Renal Cell Cancer

Abstract Background Gut microbiome and microbial metabolome has been shown to be a predictor and modulator of response to immune checkpoint blocker therapy (ICB) in clear cell renal cancer (ccRCC). Based on preliminary data from a prospective clinical study of 100 ccRCC patients, we showed differences in gut microbiome, microbial metabolome and progression free survival. Specifically higher abundance of indole acetic acid, indole acetaldehyde and indole pyruvic acid (IPyA), which represent microbial metabolism of tryptophan were significantly associated with immunotherapy resistance and shorter progression free survival. We hypothesized that time restricted eating would favorably modulate the gut microbiome, microbial metabolome to improve ICB response.We therefore evaluated the efficacy and mechanism of time restricted eating to improve response to immune checkpoint therapy in preclinical kidney cancer models. Methods Specific pathogen free (SPF) BL6 mice were injected subcutaneously with 0.5X106 LVRCC67(Renal adenocarcinoma) cell lines to develop kidney cancer preclinical models followed by randomization into two equal groups when average tumor size reached 100 mm3. Both groups were treated with anti-mouse PD1 twice weekly and daily tyrosine kinase inhibitor (TKI) while group 1 was additionally started on 14 hours of daytime fast(corresponding to period of inactivity), with feeding restricted to ten hours at night, 3 days prior to start of systemic treatment. Tumor volumes were measured twice weekly to evaluate progression free survival (defined as 30% increase in tumor volume), and mice were euthanized at end point, defined as completion of eight doses of antiPD1 treatment. To demonstrate that modulation of gut microbiome is the driving mechanism mediating favorable effect of TRE on ICB response, we conducted fecal microbiota transplant from fasting and non-fasting mice respectively into two groups of germ-free mice. Allowing two weeks for FMT to engraft, mice were injected with RenCa cell lines and subsequently treated with anti-mouse PD1 and TKI, without fasting, once tumors reached an average size of 100 mm 3.Tumors were measured twice weekly to assess PFS, until end point, defined similar to above. Results TRE was associated with significantly longer PFS via modulation of gut microbiome, which is necessary and sufficient to improve ICB response. Tumor growth was significantly slower and PFS significantly longer in SPF TRE mice as compared to non-TRE mice (median PFS 14 days vs 6 days, TRE vs non-TRE, p-0.02). Further GF mice engrafted with fasting microbiome developed significantly smaller tumors, which regressed after treatment, as opposed to those with non-fasting microbiota [(Mean tumor volume-45 mm3 vs 120 mm3, p-0.001 at day15 of injection), median PFS- not reached(NR) vs 7 days, P-0.007)]. Conclusions TRE improves response to ICB in renal cell cancer and can be developed into a cost effective, adjunctive therapy to improve PFS and overall survival of ccRCC patients treated with ICB. Deeper understanding of TRE mediated changes in gut microbiome and microbial metabolome can be leveraged to improve ICB response in patients who are unable to fast.

Prognostic Value and Clinical Significance of Vesicular Transport-Related Genes in Clear Cell Renal Cell Carcinoma

Introduction: Vesicular transport (VT) has a complex relationship with tumor progression and immunity. But prognostic significance of VT in clear cell renal cell carcinoma (ccRCC) is unclear. Thus, we aimed to establish a prognostic model according to VT to predict overall survival of ccRCC patients. Methods: We used patient data from TCGA database and built a prognostic model with 13 VT-related genes (VTRGs) by differential expression analysis, LASSO regression, and univariate/multivariate Cox analysis. The model was validated internally and externally, and survival analysis and ROC curves depicted excellent predictive ability. Furthermore, higher modeled riskscores corresponded to more advanced tumor progression. To further understand the potential reasons for different prognoses in patients, we did enrichment analysis on differentially expressed genes identified by the model in risk groups. The expression levels and roles of SAA1 and KIF18B in ccRCC were verified by qRT-PCR and cell function experiments. Results: Humoral immune response and cAMP signaling pathway may be the biological processes and pathways leading to poor prognosis. Further analysis of immune microenvironment presented that ccRCC patients with poor prognoses had highly immune-infiltrated characteristics. We compared the drug response data of samples from different prognostic patients in the GDSC database and identified drug sensitivity differences associated with prognosis. Finally, we demonstrated that SAA1 and KIF18B could increase the proliferation, migration, and invasion ability of ccRCC cells using cellular experiments. Conclusion: In summary, we further revealed the importance of VTRGs in ccRCC prognosis.

The ubiquitin ligase STUB1 suppresses tumorigenesis of renal cell carcinomas through regulating YTHDF1 stability.

STIP1 homology and U-box protein 1 (STUB1), a crucial member of the RING family E3 ubiquitin ligase, serves dual roles as an oncogene and a tumor suppressor in various human cancers. However, the role and mechanism of STUB1 in clear cell renal cell carcinoma (ccRCC) remain poorly defined. Here, we identified YTHDF1 as a novel STUB1 interaction partner using affinity purification mass spectrometry (AP-MS). Furthermore, we revealed that STUB1 promotes the ubiquitination and degradation of YTHDF1. Consequently, STUB1 depletion leads to YTHDF1 up-regulation in renal cancer cells. Functionally, STUB1 depletion promoted migration and invasion of ccRCC cells in a YTHDF1-dependent manner. Additionally, depletion of STUB1 also increased the tumorigenic potential of ccRCC in a xenograft model. Importantly, STUB1 expression is down-regulated in ccRCC tissues, and its low expression level correlates with advanced tumor stage and poor overall survival in ccRCC patients. Taken together, these findings reveal that STUB1 inhibits the tumorigenicity of ccRCC by regulating YTHDF1 stability.

A Model to Predict Prognosis of Renal Cell Clear Cell Carcinoma Based on 3 Angiogenesis-related Long Non-coding RNAs.

Background: Tumor angiogenesis is closely related to the progression of clear cell renal cell carcinoma (ccRCC). Long non-coding RNAs (lncRNAs) regulating angiogenesis could be potential biomarkers for predicting ccRCC prognosis. With this study, we aimed to construct a prognostic model based on lncRNAs and explore its underlying mechanisms. Methods: RNA data and clinical information were obtained from The Cancer Genome Atlas (TCGA) database. Angiogenesis-related genes (ARGs) were extracted from the Molecular Signatures database. Pearson correlation and LASSO and COX regression analyses were performed to identify survival-related AR-lncRNAs (sAR-lncRNAs) and construct a prognostic model. The predictive power of the prognostic model was verified according to Kaplan‒Meier curve, receiver operating characteristic (ROC) curve and nomogram analyses. The correlation between the prognostic model and clinicopathological characteristics was assessed via univariate and multivariate analyses. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was subsequently performed to elucidate the mechanisms of the sAR-lncRNAs. In vitro qPCR, immunohistochemistry, migration and invasion assays were conducted to confirm the angiogenic function of sAR-lncRNAs. Results: Three sAR-lncRNAs were used to construct a prognostic model. The model was moderately accurate in predicting 1- , 3- and 5-year ccRCC prognosis, and the risk score according to this model was closely related to clinicopathological characteristics such as T grade and T stage. A nomogram was constructed to precisely estimate the overall survival of ccRCC patients. KEGG enrichment analysis indicated that the MAPK and Notch pathways were highly enriched in high-risk patients. Additionally, we found that the expression of the lncRNAs AC005324.4 and AC104964.4 in the prognostic model was lower in ccRCC cell lines and cancer tissues than in the HK-2 cell line and paracancerous tissues, while the expression of the lncRNA AC087482.1 showed the opposite trend. In a coculture model, knockdown of lncRNA AC005324.4 and lncRNA AC104964.4 significantly promoted the migration and invasion of human umbilical vein endothelial cells (HUVECs), but siR-AC087482.1 transfection alleviated these effects. Conclusions: We constructed a prognostic model based on 3 sAR-lncRNAs and validated its value in clinicopathological characteristics and prognostic prediction of ccRCC patients, providing a new perspective for ccRCC treatment decision making.

STAT3 phosphorylation at serine 727 activates specific genetic programs and promotes clear cell renal cell carcinoma (ccRCC) aggressiveness

The signal transducer and activator of transcription 3 (STAT3) is a transcription factor mainly activated by phosphorylation in either tyrosine 705 (Y705) or serine 727 (S727) residues that regulates essential processes such as cell differentiation, apoptosis inhibition, or cell survival. Aberrant activation of STAT3 has been related to development of nearly 50% of human cancers including clear cell renal cell carcinoma (ccRCC). In fact, phosho-S727 (pS727) levels correlate with overall survival of ccRCC patients. With the aim to elucidate the contribution of STAT3 phosphorylation in ccRCC development and progression, we have generated human-derived ccRCC cell lines carrying STAT3 Y705 and S727 phosphomutants. Our data show that the phosphomimetic substitution Ser727Asp facilitates a pro-tumoral phenotype in vitro, in a Y705-phosphorylation-independent manner. Moreover, we describe that STAT3 phosphorylation state determines the expression of different subsets of target genes associated with distinct biological processes, being pS727-dependent genes the most related to cellular hallmarks of cancer. In summary, the present study constitutes the first analysis on the role of overall STAT3 phosphorylation state in ccRCC and demonstrates that pS727 promotes the expression of a specific subset of target genes that might be clinically relevant as novel biomarkers and potential therapeutic targets for ccRCC.

Abstract A011: Single-cell RNA sequencing reveals a potential renal tumor progenitor in conditional Vhlh knockout mouse model and ccRCC clinical samples

Abstract Introduction: The study aims to identify the progenitor of clear-cell renal cell carcinoma (ccRCC). Background: Inactivation of tumor suppressor gene VHL is the driver for the pathogenesis of ccRCC, the majority subtype of kidney tumors. However, it has also been observed that not all VHL-deficient cells develop into tumors. It is therefore likely that additional characteristics are required for ccRCC formation. Accumulating evidence suggests that tumor formation is initiated by a small pool of cells with tumor-initiating capacity, which are termed tumor progenitor cells (TPCs). Yet, the concept of TPCs in ccRCC has not been fully validated. Methods: LSL-tdTomato reporter was used to trace Vhlh mutant cells in the prior pre-malignant ccRCC mouse model, in which Vhlh was conditionally knocked out (VhlhKO) in renal epithelial cells under the control of Hoxb7-Cre. We also utilized single-cell RNA sequencing (scRNA-seq) to investigate the diversity of Vhlh-deficient renal tubule cells. Results: We show that the tubule phenotypes (traced by the tdTomato reporter) are associated with Vhlh inactivation; the abnormal tdTomato+ cells in Vhlh KO mice exhibit clear-cell phenotype, hyperplasia and multi-layered epithelial cell clusters. scRNA-seq was then used to analyze isolated tdTomato+ cells from Vhlh KO and wild-type (WT) kidneys. The results reveal that inactivation of Vhlh generates an expanded population of principal collecting duct cells characterized by the expression of keratin 19 (Krt19). These Krt19+ cells highly express stem cell-related genes (Wnt4, Sox4, Klf4, Ly6a). They also express multiple tubule segment-specific markers, suggesting the process of dedifferentiation. The expression of Krt19 is strikingly increased and colocalized with a subpopulation of tdTomato+ cells in Vhlh KO kidney, compared with WT where Krt19+ cells are a rare population scattered in tubule epithelia. Colocalization of KRT19 and CAIX in ccRCC samples confirms the ccRCC identity of these KRT19+ cells. High expression of KRT19 is significantly associated with poor overall survival of ccRCC patients. On kidney tissue microarray, KRT19 is strongly expressed in the early-stage peri-tumor tissue with the presence of immune cell infiltration and lipid droplets in the vicinity. In the high-grade ccRCC, KRT19 is expressed in the typical ccRCC cells and in multi-layered adenomas. Notably, Krt19+ progenitor-like cells are found in the fibrotic region with immune cell infiltration in Vhlh mutant kidney. The direct contact between macrophages and Krt19+ cells in mutant kidney, but not in WT, implies that macrophages may be a component in the progenitor-cell niche when Vhlh is conditionally knocked out. Conclusion: We suspect that VHL loss-of-function in the rare tissue progenitor cells may generate a tumor progenitor subset. Elucidation of the role of KRT19 in the context of VHL inactivation and characterization of the stem/progenitor cell niche are of importance as they will shed new lights on early diagnosis and therapeutic intervention. Citation Format: Dinh-Xuan Pham, Chen-Yun Chen, Hieu-Huy Nguyen-Tran, Tien Hsu. Single-cell RNA sequencing reveals a potential renal tumor progenitor in conditional Vhlh knockout mouse model and ccRCC clinical samples [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr A011.

LINC01480 as a Prognostic Biomarker Associated With Proliferation, Migration, and Invasion of Clear Cell Renal Cell Carcinoma.

The present study aimed to identify key long noncoding RNAs (lncRNAs) involved in survival and metastasis of clear cell renal cell carcinoma (ccRCC). A systemic screening for genes with differential expression in ccRCC was performed using publicly available databases. Cox regression analysis was used to identify lncRNAs associated with survival. A competing endogenous RNAs (ceRNA) regulation network of metastasis-related lncRNAs was constructed and hub lncRNAs were identified. Functional and pathway enrichment analyses were performed to investigate the role of lncRNA in ccRCC. Cell Counting Kit-8 and Transwell assays were used to determine the levels of cell proliferation, migration, and invasion. A total of 732 lncRNAs were found to be differentially expressed between ccRCC tumors and healthy samples. Among them, 139 lncRNAs were differentially expressed between metastasis and non-metastasis ccRCC samples and 75 lncRNAs were associated with overall survival and curated metastasis-related genes. Notably, LINC01480 was identified as the hub lncRNA involved in regulation of ccRCC metastasis. Clinically, LINC01480 may act as an independent factor for poor overall survival of ccRCC patients (log-rank p<0.05). Reverse transcription-quantitative PCR analysis validated that LINC01480 was significantly up-regulated in ccRCC compared to paired normal samples (n=20). Moreover, LINC01480 silencing inhibited the proliferation, migration, and invasion of ccRCC cells in vitro. Gene set enrichment analysis showed that high LINC01480 expression may promote ccRCC metastasis through enhancing immunodeficiency and amino acid metabolism. LINC01480 may act as a novel biomarker for overall prognosis in ccRCC and exhibit potential as a therapeutic target for the treatment of metastatic ccRCC.

Establishing a prognostic model based on five starvation-related long non-coding RNAs for clear cell renal cell carcinoma.

Starvation-induced tumor microenvironment significantly alters genetic profiles including long non-coding RNAs (lncRNAs), further regulating the malignant biological characteristics (invasion and migration) of clear cell renal cell carcinoma (ccRCC). Transcriptome RNA-sequencing data of 539 ccRCC tumors and 72 normal tissues were acquired from the TCGA and paired clinical samples of 50 ccRCC patients. In vitro experiments, such as qPCR, migration and invasion assays were applied to reveal the clinical relevance of LINC-PINT, AC108449.2 and AC007637.1. 170 lncRNAs were verified as starvation-related lncRNAs (SR-LncRs), of which 25 lncRNAs were associated with overall survival in ccRCC patients. Furthermore, a starvation-related risk score model (SRSM) was built based on the expression levels of LINC-PINT, AC108449.2, AC009120.2, AC008702.2 and AC007637.1. ccRCC patients with high level of LINC-PINT expression were divided into high-risk group and led to higher mortality, but AC108449.2 and AC007637.1 were contrary. Analogously, LINC-PINT was highly expressed in ccRCC cell lines and tumor tissues, especially in patients with advanced stage, T-stage and M-stage, while AC108449.2 and AC007637.1 showed the opposite results. In addition, the increased levels of AC108449.2 and AC007637.1 were significantly correlated with grade. Silencing LINC-PINT reduced the invasion and migration characteristics of ccRCC cells. SiR-AC108449.2 and siR-AC007637.1 enhanced the ability of invasion and migration in ccRCC cells. In this study, we find the clinical significance of LINC-PINT, AC108449.2 and AC007637.1 in predicting the prognosis of ccRCC patients and verify their correlation with various clinical parameters. These findings provide an advisable risk score model for ccRCC clinical decision-making.

Recent Advances in the Management of Clear Cell Renal Cell Carcinoma: Novel Biomarkers and Targeted Therapies.

Renal cell carcinoma (RCC) belongs to a heterogenous cancer group arising from renal tubular epithelial cells. Among RCC subtypes, clear cell renal cell carcinoma (ccRCC) is the most common variant, characterized by high aggressiveness, invasiveness and metastatic potential, features that lead to poor prognosis and high mortality rate. In addition, diagnosis of kidney cancer is incidental in the majority of cases, and this results in a late diagnosis, when the stage of the disease is advanced and the tumor has already metastasized. Furthermore, ccRCC treatment is complicated by its strong resistance to chemo- and radiotherapy. Therefore, there is active ongoing research focused on identifying novel biomarkers which could be useful for assessing a better prognosis, as well as new molecules which could be used for targeted therapy. In this light, several novel targeted therapies have been shown to be effective in prolonging the overall survival of ccRCC patients. Thus, the aim of this review is to analyze the actual state-of-the-art on ccRCC diagnosis, prognosis and therapeutic options, while also reporting the recent advances in novel biomarker discoveries, which could be exploited for a better prognosis or for targeted therapy.

Immunoglobulin as a predictive biomarker for the efficacy of immune checkpoint inhibitor combined with VEGF-TKI in metastatic renal cell carcinoma.

4543 Background: The combination of immune checkpoint inhibitor (ICI) and vascular endothelial growth factor tyrosine kinase inhibitor (VEGF-TKI) is recommended as the first-line treatment in patients with metastatic clear cell renal cell carcinoma (ccRCC). However, no effective serum biomarker has been used to predict the therapy response and potential benefits. Abnormal content and changes of immunoglobulin (IG) in serum or cancer tissue may indicate cancer progression and impact the prognosis. We examined the content of serum IG and analyzed the alteration after treatment to assess the efficacy of ICI and VEGF-TKI. Methods: Eighty-four patients with metastatic ccRCC were enrolled in this study. The total serum IG level was determined as the sum of IgA, IgG, and IgM. The data of baseline level and alteration of IG after 3-mo treatment was complete in 72 ccRCC patients. These patients were then divided into two groups based on alteration in their IG levels: the IG-increased group and the IG-decreased group. This study aimed to investigate the correlation between the baseline level of immunoglobulin (IG), changes in its content after 3-mo of treatment, and clinical outcomes. Results: Eighty-four patients were retrospectively enrolled with a median follow-up of 17.3 months (2.5-45.1). The objective response rate (ORR) was 41.67%, and the median overall survival (OS) was 31 months in our cohort. The baseline level of IG was not associated with ORR or OS. However, dynamic alteration in IG level after 3-mo treatment had a significant impact on ORR and OS in ccRCC patients. The ORR was 2.7% in the IG-increased group and 80.0% in the IG-decreased group. The OS was 21.0 months (95% CI 14.0–NA) in the IG-increased group and 48.0 months (28·0–NA) in the IG decreased-group (Hazard ratio [HR] 4.59, p=0·003). Conclusions: The change of serum IG level after 3-mo treatment served as an effective predictor of drug response and the prognosis for metastatic ccRCC patients. Value of serum IG needs to be validated in multi-centers and the mechanism of it should be further investigated.

A novel prognostic signature for clear cell renal cell carcinoma constructed using necroptosis-related miRNAs

BackgroundThis work aims to analyze the relationship between necroptosis-related microRNAs (miRNAs) and the prognosis of clear cell renal cell carcinoma (ccRCC).MethodsThe miRNAs expression profiles of ccRCC and normal renal tissues from The Cancer Genome Atlas (TCGA) database were used to construct a matrix of the 13 necroptosis-related miRNAs. Cox regression analysis was used to construct a signature to predict the overall survival of ccRCC patients. The genes targeted by the necroptosis-related miRNAs in the prognostic signature were predicted using miRNA databases. Gene Ontology (Go) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used to investigate the genes targeted by the necroptosis-related miRNAs. The expression levels of selected miRNAs in 15 paired samples (of ccRCC tissues and adjacent normal renal tissues) were investigated using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR).ResultsSix necroptosis-related miRNAs were found to differentially expressed between ccRCC and normal renal tissues. A prognostic signature consisting of miR-223-3p, miR-200a-5p, and miR-500a-3p was constructed using Cox regression analysis and risk scores were calculated. Multivariate Cox regression analysis showed that the hazard ratio was 2.0315 (1.2627–3.2685, P = 0.0035), indicating that the risk score of the signature was an independent risk factor. The receiver operating characteristic (ROC) curve showed that the signature has a favorable predictive capacity and the Kaplan-Meier survival analysis indicated that ccRCC patients with higher risk scores had worse prognoses (P < 0.001). The results of the RT-qPCR verified that all three miRNAs used in the signature were differentially expressed between ccRCC and normal tissues (P < 0.05).ConclusionThe three necroptosis-related-miRNAs used in this study could be a valuable signature for the prognosis of ccRCC patients. Necroptosis-related miRNAs should be further explored as prognostic indicators for ccRCC.

FAK-mediated phosphorylation at Y464 regulates p85β nuclear translocation to promote tumorigenesis of ccRCC by repressing RB1 expression

PI3K regulatory subunit p85s normally stabilizes and regulates catalytic subunit p110s in the cytoplasm. Recent studies show that p110-free p85s in the nucleus plays important roles in biological processes. However, the mechanisms by which p85s translocate into the nucleus remain elusive. Here, we describe the mechanism by which p85β translocates into the nucleus to promote ccRCC tumorigenesis. Phosphorylation of p85β at the Y464 by FAK facilitates its nuclear translocation in the kidney through enhancing the binding of p85β to KPNA1. PIK3R2/p85β is highly expressed in ccRCC samples and associated with overall survival of ccRCC patients. Nuclear but not cytoplasmic p85β performs oncogenic functions by repressing RB1 expression and regulating the G1/S cell cycle transition. Nuclear p85β represses RB1 expression by stabilizing histone methyltransferase EZH1/EZH2 proteins. Last, the FAK inhibitor defactinib significantly suppresses the tumor growth of ccRCC with high p85β Y464 levels.

SF3B4 promotes Twist1 expression and clear cell renal cell carcinoma progression by facilitating the export of KLF 16 mRNA from the nucleus to the cytoplasm

Splicing factor 3B subunit 4 (SF3B4) plays important functional roles not only in pre-mRNA splicing, but also in the regulation of transcription, translation, and cell signaling, and its dysregulation contributes to various diseases including Nager syndrome and tumorigenesis. However, the role of SF3B4 and underlying mechanisms in clear cell renal cell carcinoma (ccRCC) remain obscure. In the present study, we found that the expression of SF3B4 was significantly elevated in ccRCC tissues and negatively correlated with the overall survival of ccRCC patients. Upregulation of SF3B4 promotes migration and invasion of ccRCC cells in vitro and in vivo. The promoting effect of SF3B4 on cell migration and invasion is mediated by Twist1, a key transcription factor to mediate EMT. Interestingly, SF3B4, a component of the pre-mRNA spliceosome, is able to promote KLF16 expression by facilitating the transport of KLF16 mRNA into the cytoplasm. Mechanistically, SF3B4 promotes the export of KLF16 mRNA from the nucleus to the cytoplasm and thus enhances KLF16 expression, and in turn elevated KLF16 directly binds to the Twist1 promoter to activate its transcription, leading to EMT and ccRCC progression. Our findings provide evidence that the SF3B4-KLF16-Twist1 axis plays important functional roles in the development and progression of ccRCC, and manipulating this pathway may be a novel therapeutic target for the treatment of ccRCC.

Identification and validation of the molecular subtype and prognostic signature for clear cell renal cell carcinoma based on neutrophil extracellular traps.

Background: Renal cell carcinoma (RCC) is one of the most common cancers, with an annual incidence of nearly 400,000 cases worldwide. Increasing evidence has also demonstrated the vital role of neutrophil extracellular traps (NETs) in cancer progression and metastatic dissemination. Methods: Consensus cluster analysis was performed to determine the number of ccRCC subtypes. The Kruskal-Wallis test or Student t-test was performed to evaluate the difference of infiltrating immune cell and gene expression in different groups. The Kaplan-Meier (KM) method was used to draw the survival curve. LASSO cox regression analysis was conducted to construct a NET-related prognostic signature. We also constructed a lncRNA-miRNA-mRNA regulatory axis by several miRNA and lncRNA target databases. Results: A total of 23 differentially expressed NET-related genes were obtained in ccRCC. Three clusters of ccRCC cases with significant difference in prognosis, immune infiltration, and chemotherapy and targeted therapy were identified. LASSO Cox regression analysis identified a NET-related prognostic signature including six genes (G0S2, DYSF, MMP9, SLC22A4, SELP, and KCNJ15), and this signature had a good performance in predicting the overall survival of ccRCC patients. The expression of prognostic signature genes was significantly correlated with the pTMN stage, immune infiltration, tumor mutational burdens, microsatellite instability, and drug sensitivity of ccRCC patients. MMP9 was identified as the hub gene. We also identified the lncRNA UBA6-AS1/miR-149-5p/MMP9 regulatory axis for the progression of ccRCC. Conclusion: Collectively, the current study identified three molecular clusters and a prognostic signature for ccRCC based on neutrophil extracellular traps. Integrative transcriptome analyses plus clinical sample validation may facilitate the biomarker discovery and clinical transformation.

High expression of TTC21A predicts unfavorable prognosis and immune infiltrates in clear cell renal cell carcinoma.

Background: Clear cell renal cell carcinoma (ccRCC) is the most common pathological type of renal cell carcinoma. Tetratricopeptide repeat domain 21A (TTC21A), known as a component of intraflagellar transport complex A which is essential for the function of cilia, However, the role of TTC21A remains unclear in ccRCC. For the first time, we explore the role and potential mechanism of TTC21A in ccRCC based on multiple databases. Methods: TTC21A expression across all TCGA tumor was analyzed via Tumor Immune Estimation Resource (TIMER) site. The correlation between TTC21A and clinicopathologic characteristics of ccRCC was analyzed with TCGA database. The diagnostic and prognostic value of TTC21A was evaluated by receiver operation characteristic curve, Kaplan-Meier plotter and Cox regression respectively. Moreover, functional enrichment analysis of TTC21A and the co-expression genes were performed by Gene Set Enrichment Analysis. The correlation of TTC21A and immune infiltration were evaluated by single sample Gene Set Enrichment Analysis. Results: Pan-cancer analysis indicated that TTC21A was highly expressed in ccRCC and other cancer. In addition, elevated expression of TTC21A was associated with worse overall survival in ccRCC patients. Functional enrichment analysis showed that TTC21A and the co-expressed genes enriched in glucose metabolism and energy metabolism. Moreover, TTC21A expression was associated with infiltrating levels of dendritic cell, nature killer cell and other immune marker sets. Conclusion: The results of analysis indicate that expression of TTC21A is associated with poor prognosis and immune infiltrating in ccRCC, which suggested TTC21A might be used as a potential predictor and target of treatment in ccRCC.

Cuproptosis-related gene FDX1 expression correlates with the prognosis and tumor immune microenvironment in clear cell renal cell carcinoma.

BackgroundCuproptosis, a newly discovered form of cell death, is regulated by protein lipoylation and is related to mitochondrial metabolism. However, further research is needed to determine how the cuproptosis-related gene ferredoxin 1 (FDX1) affects the tumor immune response and its prognostic significance in clear cell renal cell carcinoma (ccRCC).MethodsThe Cancer Genome Atlas was used to screen for FDX1 gene expression in ccRCC and healthy tissue samples. The results were validated using the Gene Expression Omnibus and the Human Protein Atlas. Multivariable analysis and Kaplan-Meier survival curves were used to examine the relationship between FDX1 gene expression, clinicopathological parameters, and overall survival (OS). The protein network containing FDX1 gene interaction was constructed using the online Search Tool for the Retrieval of Interacting Genes/Proteins. The relationship between FDX1 gene expression and immune cell infiltration in ccRCC was examined using Gene Ontology, gene set enrichment analysis (GSEA), and a single-sample GSEA. Using the Gene Expression Profiling Interactive Analysis and Tumor Immune Estimation Resource databases, we investigated the relationship between FDX1 gene expression, the degree of immune cell infiltration, and the corresponding gene marker sets.ResultsccRCC samples had significantly (p < 0.05) lower FDX1 gene expression levels than normal tissue samples. Lower FDX1 gene expression levels were strongly associated with higher cancer grades and more advanced tumor–node–metastasis stages. The findings of multivariate and univariate analyses illustrated that the OS in ccRCC patients with low FDX1 expression is shorter than in patients with high FDX1 expression (p < 0.05). Ferredoxin reductase and CYP11A1 are key proteins interacting with the FDX1 gene, and ccRCC with an FDX1 enzyme defect was associated with a low number of invading immune cells and their corresponding marker.ConclusionIn ccRCC, decreased FDX1 expression was linked to disease progression, an unfavorable prognosis, and dysregulated immune cell infiltration.

Abstract 1274: Prognostic relevance of ZnF 649 in clear cell carcinoma

Abstract Clear cell renal carcinoma (ccRCC) is the most common form of neoplastic disease affecting the kidneys that accounts for at least 70% of all kidney cancers. Given its resistance to medications and high mortality at later stages, there is a need to identify biomarkers that can detect ccRCC. KRAB Zinc finger proteins (KRAB-ZFPs) are the largest family of mammalian transcription regulators. They are differentially expressed in various tissues during cellular development and phenotypic differentiation. Though poorly understood, these transcription factors, are also differentially expressed in several cancers though very few have been reported to be implicated in ccRCC. Using bioinformatics techniques, we have demonstrated that ZNF649 and its paralog ZNF613 are suppressed in clear cell renal carcinomas (p&amp;lt;0.001) relative to normal tissues. Moreover, The ZNF649 and ZNF613 transcripts were downregulated across all histological grades and pathological stages (p&amp;lt;0.001). In addition, both ZNF649 and ZNF613 downregulation was associated with metastasis and unsatisfactory patient survival (p&amp;lt;0.001). Univariate and multivariante Cox regression analysis of age, gene expression, gender, pathological stage, histological grade, and pathological N in relationship to overall survival in ccRCC patients revealed that ZNF649 expression was predictive of overall survival. Gene Set Enrichment Analysis of the Gene Ontology Biological Processes database indicate that the top pathways enriched with genes correlated to ZNF649 expression were associated with angiogenesis, cellular motility and mitochondrial regulation (p&amp;lt;0.001 and FDR &amp;lt;0.05). Taken collectively, our observations indicate that ZNF649 is a prognostic marker and is a putative tumor suppressor gene in clear cell renal carcinoma. Citation Format: Renee Reams, Simone O. Heyliger, marilyn Saulsbury, Karam F. Soliman. Prognostic relevance of ZnF 649 in clear cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1274.

Nanoparticles targeting at methylases with high correlation to N6-methyladenosine-related lncRNA signatures as potential therapy of kidney clear cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is a heterogeneous malignancy with poor prognosis. Methylation of the N6 position of adenosine (m6A), the most common epigenetic modification in both messenger RNAs and noncoding RNAs, has been reported to regulate the initiation and progression of ccRCC. However, whether and how m6A-related long noncoding RNAs (m6ArlncRNAs) signify the progression of ccRCC remain unclear. We found m6ArlncRNAs are effective signatures illustrating immune landscape and risk stratification in ccRCC. We identified two differently expressed m6ArlncRNAs (DEm6ArlncRNAs), AC008870.2 and EMX2OS, as independent risk factors for overall survival of ccRCC patients, by applying stringent variable selection procedure to data from the Cancer Genome Atlas Kidney Renal Clear Cell Carcinoma project. The risk score generated from the DEm6ArlncRNA expression categorizes patients into either high or low-risk groups, between which, enrichment analysis indicated an enrichment in immune-related pathways. Under different DEm6ArlncRNA transcription pattern, the two risk groups differ in immune cell population composition and expression levels of therapy targeting genes. Nanoparticle is satisfactory strategy to delivering therapeutic drugs. For further clinical translation, we designed a novel nanoparticle delivery system packaged STM2457 (STM@8P4 NPs), which selectively inhibits AC008870.2-correlated m6A writer. STM@8P4 NPs loaded drug successfully with uniform particle size, long-term stability and high release efficiency. STM@8P4 NPs can easily enter ccRCC cells and showed a highly efficient ccRCC killing activity in vitro. Our results therefore indicate that m6ArlncRNAs expression can depict tumor microenvironment, predict prognosis for ccRCC patient and give hint to therapeutic strategies in ccRCC.

Identification of a Methylation-Regulating Genes Prognostic Signature to Predict the Prognosis and Aid Immunotherapy of Clear Cell Renal Cell Carcinoma.

Methylation is one of the most extensive modifications of biological macromolecules and affects cell-fate determination, development, aging, and cancer. Several methylation modifications, including 5-methylcytosine and N6-methyladenosine, play an essential role in many cancers. However, little is known about the relationship between methylation and the prognosis of clear cell renal cell carcinoma (ccRCC). Here, we established a methylation-regulating genes prognostic signature (MRGPS) to predict the prognoses of ccRCC patients. We obtained ccRCC samples from The Cancer Genome Atlas and identified methylation-regulatingd genes (MRGs) from the Gene Set Enrichment Analysis database. We also determined differentially expressed genes (DEGs) and performed cluster analysis to identify candidate genes. Subsequently, we established and validated an MRGPS to predict the overall survival of ccRCC patients. This was also verified in 15 ccRCC samples collected from the Fujian Provincial Hospital via quantitative real-time transcription (qRT-PCR). While 95 MRGs were differentially expressed (DEGs1) between tumor and normal tissues, 17 MRGs were differentially expressed (DEGs2) between cluster 1 and 2. Notably, 13 genes common among DEGs1 and DEGs2 were identified as hub genes. In fact, we established three genes (NOP2, NSUN6, and TET2) to be an MRGPS based on their multivariate Cox regression analysis coefficients (p < 0.05). A receiver operating characteristic curve analysis confirmed this MRGPS to have a good prognostic performance. Moreover, the MRGPS was associated with characteristics of the tumor immune microenvironment and responses to inhibitor checkpoint inhibitors. Data from “IMvigor 210” demonstrated that patients with a low MRGPS would benefit more from atelozumab (p < 0.05). Furthermore, a multivariate analysis revealed that MRGPS was an independent risk factor associated with ccRCC prognosis (p < 0.05). Notably, a nomogram constructed by combining with clinical characteristics (age, grade, stage, and MRGPS risk score) to predict the overall survival of a ccRCC patient had a favorable predictive value. Eventually, our qRT-PCR results showed that tumor tissues had higher NOP2 and NSUN6 expression levels and lower TET2 expression than normal tissues of ccRCC samples. While the proposed MRGPS comprising NOP2, NSUN6, and TET2 can be an alternative prognostic biomarker for ccRCC patients, it is a promising index for personalized ICI treatments against ccRCC.

CGN Correlates With the Prognosis and Tumor Immune Microenvironment in Clear Cell Renal Cell Carcinoma.

This study aimed to screen and verify the important prognostic genes related to clear cell renal cell carcinoma (ccRCC) and further analyze their relationship with the immune microenvironment. Gene expression profiles from the TCGA-KIRC, GSE46699, GSE36895, and GSE16449 datasets were utilized to explore differentially co-expressed genes in ccRCC. We screened 124 differentially co-expressed genes using a weighted gene co-expression network and differential gene expression analyses. Univariate and multivariate Cox survival analyses revealed that the expressions of genes CGN, FECH, UCHL1, and WT1 were independently related to the overall survival of ccRCC patients. Kaplan–Meier survival analysis was performed, and CGN was found to have the strongest correlation with the prognosis of ccRCC patients and was consequently selected for further analyses and experimental verification. The results showed that NK cell activation, resting dendritic cells, resting monocytes, and resting mast cells were positively correlated with CGN expression; CD4+ memory activated T cells, regulatory T cells, and M0 macrophages were negatively correlated with CGN expression. Finally, using western blotting and reverse transcription polymerase chain reaction, we verified that the CGN protein level was down-regulated in ccRCC samples, which was consistent with the mRNA levels. CGN was thus identified as diagnosis and prognosis biomarker for ccRCC and is related to the immune microenvironment.