Synthetic lethality of combined ULK1 defection and p53 restoration induce pyroptosis by directly upregulating GSDME transcription and cleavage activation through ROS/NLRP3 signaling.

BACKGROUND: High expression of ubiquitin ligase MDM2 is a primary cause of p53 inactivation in many tumors, making it a promising therapeutic target. However, MDM2 inhibitors have failed in clinical trials due to p53-induced feedback that enhances MDM2 expression. This underscores the urgent need to find an effective adaptive genotype or combination of targets. METHODS: Kinome-wide CRISPR/Cas9 knockout screen was performed to identify genes that modulate the response to MDM2 inhibitor using TP53 wild type cancer cells and found ULK1 as a candidate. The MTT cell viability assay, flow cytometry and LDH assay were conducted to evaluate the activation of pyroptosis and the synthetic lethality effects of combining ULK1 depletion with p53 activation. Dual-luciferase reporter assay and ChIP-qPCR were performed to confirm that p53 directly mediates the transcription of GSDME and to identify the binding region of p53 in the promoter of GSDME. ULK1 knockout / overexpression cells were constructed to investigate the functional role of ULK1 both in vitro and in vivo. The mechanism of ULK1 depletion to activate GSMDE was mainly investigated by qPCR, western blot and ELISA. RESULTS: By using high-throughput screening, we identified ULK1 as a synthetic lethal gene for the MDM2 inhibitor APG115. It was determined that deletion of ULK1 significantly increased the sensitivity, with cells undergoing typical pyroptosis. Mechanistically, p53 promote pyroptosis initiation by directly mediating GSDME transcription that induce basal-level pyroptosis. Moreover, ULK1 depletion reduces mitophagy, resulting in the accumulation of damaged mitochondria and subsequent increasing of reactive oxygen species (ROS). This in turn cleaves and activates GSDME via the NLRP3-Caspase inflammatory signaling axis. The molecular cascade makes ULK1 act as a crucial regulator of pyroptosis initiation mediated by p53 activation cells. Besides, mitophagy is enhanced in platinum-resistant tumors, and ULK1 depletion/p53 activation has a synergistic lethal effect on these tumors, inducing pyroptosis through GSDME directly. CONCLUSION: Our research demonstrates that ULK1 deficiency can synergize with MDM2 inhibitors to induce pyroptosis. p53 plays a direct role in activating GSDME transcription, while ULK1 deficiency triggers upregulation of the ROS-NLRP3 signaling pathway, leading to GSDME cleavage and activation. These findings underscore the pivotal role of p53 in determining pyroptosis and provide new avenues for the clinical application of p53 restoration therapies, as well as suggesting potential combination strategies.

Inactivated cGAS-STING Signaling Facilitates Endocrine Resistance by Forming a Positive Feedback Loop with AKT Kinase in ER+HER2- Breast Cancer.

Endocrine-resistant ER+HER2- breast cancer (BC) is particularly aggressive and leads to poor clinical outcomes. Effective therapeutic strategies against endocrine-resistant BC remain elusive. Here, analysis of the RNA-sequencing data from ER+HER2- BC patients receiving neoadjuvant endocrine therapy and spatial transcriptomics analysis both show the downregulation of innate immune signaling sensing cytosolic DNA, which primarily occurs in endocrine-resistant BC cells, not immune cells. Indeed, compared with endocrine-sensitive BC cells, the activity of sensing cytosolic DNA through the cGAS-STING pathway is attenuated in endocrine-resistant BC cells. Screening of kinase inhibitor library show that this effect is mainly mediated by hyperactivation of AKT1 kinase, which binds to kinase domain of TBK1, preventing the formation of a trimeric complex TBK1/STING/IRF3. Notably, inactivation of cGAS-STING signaling forms a positive feedback loop with hyperactivated AKT1 to promote endocrine resistance, which is physiologically important and clinically relevant in patients with ER+HER2- BC. Blocking the positive feedback loop using the combination of an AKT1 inhibitor with a STING agonist results in the engagement of innate and adaptive immune signaling and impairs the growth of endocrine-resistant tumors in humanized mice models, providing a potential strategy for treating patients with endocrine-resistant BC.

Metabolomic landscape of renal cell carcinoma in von Hippel-Lindau syndrome in a Chinese cohort.

Von Hippel-Lindau (VHL) syndrome is a rare autosomal dominant disorder, where renal cell carcinoma (RCC) serves as a significant cause of mortality. We collected peripheral blood from 61 VHL-RCC patients and 31 healthy individuals, along with 19 paired RCC tumor and adjacent non-malignant samples. Using liquid chromatography-mass spectrometry, we identified 238 plasma and 241 tissue differentially abundant metabolites (DAMs), highlighting key pathways such as arginine and proline metabolism. The top 10 of the 23 DAMs, common to both plasma and tissue, were instrumental in constructing a high-performance diagnostic model. These DAMs demonstrated significant correlations with VHL gene mutation types. Cox regression analysis revealed that plasma levels of N2,N2-dimethylguanosine were associated with the timing of RCC onset in VHL patients, acting as an independent predictive factor. This study enhances diagnostic accuracy for this rare condition and opens new avenues for exploring metabolic mechanisms of the disease and potential therapeutic directions.

TRPML1 triggers ferroptosis defense and is a potential therapeutic target in AKT-hyperactivated cancer.

Targeting ferroptosis for cancer therapy has slowed because of an incomplete understanding of ferroptosis mechanisms under specific pathological contexts such as tumorigenesis and cancer treatment. Here, we identify TRPML1-mediated lysosomal exocytosis as a potential anti-ferroptotic process through genome-wide CRISPR-Cas9 activation and kinase inhibitor library screening. AKT directly phosphorylated TRPML1 at Ser343 and inhibited K552 ubiquitination and proteasome degradation of TRPML1, thereby promoting TRPML1 binding to ARL8B to trigger lysosomal exocytosis. This boosted ferroptosis defense of AKT-hyperactivated cancer cells by reducing intracellular ferrous iron and enhancing membrane repair. Correlation analysis and functional analysis revealed that TRPML1-mediated ferroptosis resistance is a previously unrecognized feature of AKT-hyperactivated cancers and is necessary for AKT-driven tumorigenesis and cancer therapeutic resistance. TRPML1 inactivation or blockade of the interaction between TRPML1 and ARL8B inhibited AKT-driven tumorigenesis and cancer therapeutic resistance in vitro and in vivo by promoting ferroptosis. A synthetic peptide targeting TRPML1 inhibited AKT-driven tumorigenesis and enhanced the sensitivity of AKT-hyperactivated tumors to ferroptosis inducers, radiotherapy, and immunotherapy by boosting ferroptosis in vivo. Together, our findings identified TRPML1 as a therapeutic target in AKT-hyperactivated cancer.

Molecular classification and tumor microenvironment characteristics in pheochromocytomas.

Pheochromocytomas (PCCs) are rare neuroendocrine tumors that originate from chromaffin cells in the adrenal gland. However, the cellular molecular characteristics and immune microenvironment of PCCs are incompletely understood. Here, we performed single-cell RNA sequencing (scRNA-seq) on 16 tissues from 4 sporadic unclassified PCC patients and 1 hereditary PCC patient with Von Hippel-Lindau (VHL) syndrome. We found that intra-tumoral heterogeneity was less extensive than the inter-individual heterogeneity of PCCs. Further, the unclassified PCC patients were divided into two types, metabolism-type (marked by NDUFA4L2 and COX4I2) and kinase-type (marked by RET and PNMT), validated by immunohistochemical staining. Trajectory analysis of tumor evolution revealed that metabolism-type PCC cells display phenotype of consistently active metabolism and increased metastasis potential, while kinase-type PCC cells showed decreased epinephrine synthesis and neuron-like phenotypes. Cell-cell communication analysis showed activation of the annexin pathway and a strong inflammation reaction in metabolism-type PCCs and activation of FGF signaling in the kinase-type PCC. Although multispectral immunofluorescence staining showed a lack of CD8+ T cell infiltration in both metabolism-type and kinase-type PCCs, only the kinase-type PCC exhibited downregulation of HLA-I molecules that possibly regulated by RET, suggesting the potential of combined therapy with kinase inhibitors and immunotherapy for kinase-type PCCs; in contrast, the application of immunotherapy to metabolism-type PCCs (with antigen presentation ability) is likely unsuitable. Our study presents a single-cell transcriptomics-based molecular classification and microenvironment characterization of PCCs, providing clues for potential therapeutic strategies to treat PCCs.

Suppressive stroma-immune prognostic signature impedes immunotherapy in ovarian cancer and can be reversed by PDGFRB inhibitors.

BACKGROUND: As the most lethal gynecologic cancer, ovarian cancer (OV) holds the potential of being immunotherapy-responsive. However, only modest therapeutic effects have been achieved by immunotherapies such as immune checkpoint blockade. This study aims to propose a generalized stroma-immune prognostic signature (SIPS) to identify OV patients who may benefit from immunotherapy. METHODS: The 2097 OV patients included in the study were significant with high-grade serous ovarian cancer in the III/IV stage. The 470 immune-related signatures were collected and analyzed by the Cox regression and Lasso algorithm to generalize a credible SIPS. Correlations between the SIPS signature and tumor microenvironment were further analyzed. The critical immunosuppressive role of stroma indicated by the SIPS was further validated by targeting the major suppressive stroma component (CAFs, Cancer-associated fibroblasts) in vitro and in vivo. With four machine-learning methods predicting tumor immune subtypes, the stroma-immune signature was upgraded to a 23-gene signature. RESULTS: The SIPS effectively discriminated the high-risk individuals in the training and validating cohorts, where the high SIPS succeeded in predicting worse survival in several immunotherapy cohorts. The SIPS signature was positively correlated with stroma components, especially CAFs and immunosuppressive cells in the tumor microenvironment, indicating the critical suppressive stroma-immune network. The combination of CAFs' marker PDGFRB inhibitors and frontline PARP inhibitors substantially inhibited tumor growth and promoted the survival of OV-bearing mice. The stroma-immune signature was upgraded to a 23-gene signature to improve clinical utility. Several drug types that suppress stroma-immune signatures, such as EGFR inhibitors, could be candidates for potential immunotherapeutic combinations in ovarian cancer. CONCLUSIONS: The stroma-immune signature could efficiently predict the immunotherapeutic sensitivity of OV patients. Immunotherapy and auxiliary drugs targeting stroma could enhance immunotherapeutic efficacy in ovarian cancer.

Preliminary Study of Whole-Genome Bisulfite Sequencing and Transcriptome Sequencing in VHL Disease-Associated ccRCC.

BACKGROUND: Von Hippel-Lindau (VHL) disease is an autosomal dominant hereditary tumor syndrome with an incidence of approximately 1/36,000. VHL disease-associated clear cell renal cell carcinoma (ccRCC) is the most common congenital RCC. Although recent advances in treating RCC have improved the long-term prognosis of patients with VHL disease, kidney cancer is still the leading cause of death in these patients. Therefore, finding new targets for diagnosing and treating VHL disease-associated ccRCC is still essential. METHODS: In this study, we collected matched tumor tissues and normal samples from 25 patients with VHL disease-associated ccRCC, diagnosed and surgically treated in the Department of Urology, Peking University First Hospital. After screening, we performed whole genome bisulfite sequencing (WGBS) on 23 pairs of tissues and RNA-seq on 6 pairs of tissues. And we also compared the VHL disease-associated ccRCC transcriptome data with the sporadic ccRCC transcriptome data from the The Cancer Genome Atlas (TCGA) public database RESULTS: We found that the methylation level of VHL disease-associated ccRCC tumor tissues was significantly lower than that of normal tissues. The tumor tissues showed a difference in the copy number of 3p loss and 5q and 7q gain compared with normal tissues. We integrated RNA-seq and WGBS data to reveal methylation candidate genes associated with VHL disease-associated ccRCC; our results showed 124 hypermethylated and downregulated genes, and 245 hypomethylated and upregulated genes. By comparing the VHL disease-associated ccRCC transcriptome data with the sporadic ccRCC transcriptome data from the TCGA public database, we found that the major pathways of differential gene enrichment differed between them. CONCLUSIONS: Our study mapped the multiomics of copy number variation, methylation and mRNA level changes in tumor and normal tissues of clear cell renal cell carcinoma with VHL syndrome, which provides a solid foundation for the mechanistic study, biomarker screening, and therapeutic target discovery of clear cell renal cell carcinoma.

The long-term outcome of nephron-sparing surgery versus radical nephroureterectomy for organ-localized upper urinary tract urothelial carcinoma: a population-based study of 1969 patients.

PURPOSE: To compare the long-term outcomes after nephron-sparing surgery (NSS) and radical nephroureterectomy (RNU) and investigate prognostic factors for organ-localized upper urinary tract urothelial carcinoma (UTUC) as the role of NSS for UTUC remains unclear. METHODS: Patients diagnosed with organ-localized UTUC between 2004 and 2020 were identified from the Surveillance, Epidemiology, and End Results (SEER) database. The propensity score overlap weighting (PSOW) process, Cox regression analysis, Kaplan‒Meier analysis, competing-risks models, and subgroup analysis were employed to compare the outcomes and identify prognostic factors. The overall survival (OS) and cancer-specific survival (CSS) nomograms were developed and evaluated using the concordance index (C-index) and calibration curve. RESULTS: A total of 1969 patients were included. After the process of PSOW, baseline data were well balanced. RNU was associated with similar OS and CSS than NSS in the overall cohort. Age, T stage, and histologic grade were independent prognostic factors for OS and CSS, while marital status was an independent prognostic factor only for OS. Four and three predictors were identified for developing the OS and CSS nomograms, respectively. C-index (OS 0.637, CSS 0.670), calibration curve, and Kaplan-Meier analysis proved excellent predictive accuracy of nomograms. CONCLUSION: Patients accepting RNU had a comparative or better outcome in each sample group. NSS achieved a similar oncologic control for selected patients with organ-localized UTUC.

Construction of a two-gene prognostic model related to ferroptosis in renal cell carcinoma.

Background: Renal cell carcinoma (RCC) is a common and aggressive tumor. A newly discovered form of programmed cell death, ferroptosis, plays an important role in tumor development and progression. However, a clear prognostic correlation between Ferroptosis-related genes (FRGs) and RCC has not yet been established. In this study, prognostic markers associated with FRGs were investigated to improve the therapeutic, diagnostic, and preventive strategies available to patients with renal cancer. Methods: The present study analyzed the predictive value of 23 FRGs in RCC through bioinformatics techniques, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) tools, Kaplan-Meier survival analysis, Cox regression modeling, tumor mutational burden (TMB), CIBERSORT, and half maximal inhibitory concentration (IC50) difference analysis. Results: We screened FRGs by differentially expressed genes (DEGs) and overall survival (OS). Four candidate genes were obtained by hybridization. Then, we constructed a two-gene prognostic signature (NCOA4 and CDKN1A) via univariate Cox regression and multivariate stepwise Cox regression, which classified RCC patients into high- and low-risk groups, and patients in the high-risk group were found to have worse OS and progression-free survival (PFS). We also found that patients with higher TNM stage, T stage, and M stage had higher risk scores than those with lower TNM stage, T stage, and M stage (P<0.05). Males had higher risk scores than females. This signature was identified as an independent prognostic indicator for RCC. These results were validated in both the test cohort and the entire cohort. In addition, we also constructed a nomogram that predicted the OS in RCC patients, the consistency index (C-index) of the nomogram was 0.731 [95% confidence interval (CI): 0.672-0.790], the areas under the receiver operating characteristic (ROC) curves (AUCs) were 0.728, 0.704, and 0.898 at 1-, 3-, and 5-year, respectively, which shows that nomogram has good prediction ability. and we also analyzed the immune status and drug sensitivity between the high- and low-risk groups. Conclusions: We constructed a prognostic model associated with ferroptosis, which may provide clinicians with a reliable predictive assessment tool and offer new perspectives for the future clinical management of RCC.

TRAF2 promotes M2-polarized tumor-associated macrophage infiltration, angiogenesis and cancer progression by inhibiting autophagy in clear cell renal cell carcinoma.

BACKGROUND: The management of advanced clear cell renal cell carcinoma (ccRCC) remains a major challenge in clinical practice, and the construction of more reliable prognostic prediction models and the further elucidation of key molecular mechanisms of tumor progression are topics in urgent need of in-depth investigation. METHODS: We used CIBERSORT to estimate the proportion of 22 tumor-infiltrating immune cell types in the TCGA-KIRC cohort. Weighted gene co-expression network analysis, least absolute shrinkage and selection operator regression analysis were used to build risk prediction models. Expression patterns and clinical significance of TRAF2 were determined through bioinformatics analysis, real-time qPCR, Western Blot, immunohistochemistry. GSEA analysis, transmission electron microscopy, 2D/3D colony formation assay, cell migration and invasion assay, and tube-formation assay were used to investigate the underlying function and mechanism of the TRAF2/M2 macrophage/autophagy axis. RESULTS: We constructed a novel prognostic prediction model based on M2 macrophage-related genes, which was identified as an accurate, independent and specific prognostic risk model for ccRCC patients. A reliable nomogram was constructed to predict 1-, 3-, and 5-year overall survival for patients with ccRCC. As one of the constituent genes of the risk model, TRAF2 was determined to be upregulated in ccRCC and associated with poor clinical prognosis. We found that TRAF2 promotes malignant progression of ccRCC by regulating macrophage polarization, migration and angiogenesis. Mechanistically, we found that TRAF2 promotes the polarization of M2 macrophages, and this chemotaxis is achieved in an autophagy-dependent pathway. Orthotopic tumor growth assay results revealed that TRAF2 plays a key role as a promotor of ccRCC growth and metastasis. CONCLUSIONS: In conclusion, this risk model is highly predictive of prognostic in ccRCC patients, which is expected to promote improved treatment evaluation and comprehensive management of ccRCC. Moreover, our findings reveal that the TRAF2/M2 macrophage/autophagy axis plays a key regulatory role in the malignant progression of ccRCC, and suggest that TRAF2 is a potential novel therapeutic target for advanced ccRCC.

Clinical characteristics analysis and prognostic nomogram for predicting survival in patients with second primary prostate cancer: a population study based on SEER database.

BACKGROUND AND AIMS: Second primary prostate cancer (SPPCa) is a common type of secondary malignancy that negatively impacts patient prognosis. This study aimed to identify prognostic factors for SPPCa patients and develop nomograms to assess their prognosis. METHODS: Patients diagnosed with SPPCa between 2010 and 2015 were identified from the Surveillance, Epidemiology, and End Results (SEER) database. The study cohort was randomly divided into a training set and a validation set. Cox regression analysis, Kaplan‒Meier survival analysis, and least absolute shrinkage and selection operator regression analysis were used to identify independent prognostic factors and develop the nomogram. The nomograms were evaluated using the concordance index (C-index), calibration curve, area under the curve (AUC), and Kaplan-Meier analysis. RESULTS: A total of 5342 SPPCa patients were included in the study. Independent prognostic factors for overall survival (OS) and cancer-specific survival (CSS) were identified as age, interval between diagnoses, first primary tumor site, and AJCC stage, N stage, M stage, PSA, Gleason score, and SPPCa surgery. Nomograms were constructed based on these prognostic factors, and the performance was evaluated using the C-index (OS: 0.733, CSS: 0.838), AUC, calibration curve, and Kaplan-Meier analysis, which demonstrated excellent predictive accuracy. CONCLUSION: We successfully established and validated nomograms to predict OS and CSS in SPPCa patients using the SEER database. These nomograms provide an effective tool for risk stratification and prognosis assessment in SPPCa patients, which will aid clinicians in optimizing treatment strategies for this patient population.

Loss of HRD functional phenotype impedes immunotherapy and can be reversed by HDAC inhibitor in ovarian cancer.

In recent years, homologous recombination deficiency (HRD) has not achieved the expected substantial promotion of immunotherapeutic efficacy in ovarian cancer. This study aims to explore the role of HRD functional phenotype as a powerful biomarker in identifying HRD patients who may benefit from immunotherapy. HRD functional phenotype, namely HRD-EXCUTE, was defined as the average level of the 15 hub genes upregulated in HRD ovarian cancer. A decision tree was plotted to evaluate the critical role of HRD-EXCUTE in HRD patients. Agents inducing HRD-EXCUTE were identified by CMAP web (Connectivity Map). The mechanisms and immunotherapeutic effect of PARPi and HDACi in promoting HRD-EXCUTE was examined in vitro and in vivo. The decision tree plotted on the basis of HRD and HRD-EXCUTE indicated the HRD patients without the HRD functional phenotype were largely unresponsive to immunotherapy, which was validated by the immunotherapeutic cohorts. Furthermore, loss of HRD-EXCUTE in the HRD patients attenuated immunogenicity and inhibited immune cells in tumor microenvironment. Moreover, Niraparib combined with Entinostat induced HRD-EXCUTE by activating the cGAS-STING pathway and increasing the histone acetylation. The combination therapy could enhance the cytotoxicity of immune cells, and promote pro-immune cells infiltrating into ascites, resulting in inhibited ovarian cancer growth. The HRD functional phenotype HRD-EXCUTE was set up as a potent biomarker to identify whether HRD patients can benefit from immunotherapy. Loss of HRD-EXCUTE in HRD patients were largely insensitive to immunotherapy. The combination of PARPi with HDACi could improve the efficacy of the PARPi-based immunotherapy in ovarian cancer by augmenting the HRD functional phenotype.

Genotype-phenotype correlations and clinical outcomes of patients with von Hippel-Lindau disease with large deletions.

BACKGROUND: Approximately 20%-40% of patients with von Hippel-Lindau (VHL) disease, an autosomal dominant hereditary disease, exhibit large deletions (LDs). Few studies have focused on this population. Hence, we aimed to elucidate the genotype-phenotype correlations and clinical outcomes in VHL patients with LDs. METHODS: In this retrospective study, we included 119 patients with VHL disease from 50 unrelated families in whom LDs were detected using traditional and next-generation sequencing methods. Other germline mutations were confirmed by Sanger sequencing. Genotype-phenotype correlations and survival were analysed in different groups using Kaplan-Meier and Cox regression. We also evaluated therapeutic response to tyrosine kinase inhibitor (TKI) therapy. RESULTS: The overall penetrance of patients aged <60 was 95.2%. Two VHL patients with LDs also carried CHEK2 and FLCN germline mutations. An earlier age of onset of retinal haemangioblastoma was observed in the next generation. Patients with exon 2 deletion of VHL had an earlier onset age of renal cell carcinoma and pancreatic lesions. The risk of renal cell carcinoma was lower in VHL patients with LDs and a BRK1 deletion. The group with earlier age of onset received poorer prognosis. Four of eight (50%) patients showed partial response to TKI therapy. CONCLUSION: The number of generations and the status of exon 2 could affect age of onset of VHL-related manifestations. Onset age was an independent risk factor for overall survival. TKI therapy was effective in VHL patients with LDs. Our findings would further support clinical surveillance and decision-making processes.

Tubastatin A potently inhibits GPX4 activity to potentiate cancer radiotherapy through boosting ferroptosis.

Ferroptosis, an iron-dependent lipid peroxidation-driven programmed cell death, is closely related to cancer therapy. The development of druggable ferroptosis inducers and their rational application in cancer therapy are critical. Here, we identified Tubastatin A, an HDAC6 inhibitor as a novel druggable ferroptosis inducer through large-scale drug screening. Tubastatin A directly bonded to GPX4 and inhibited GPX4 enzymatic activity through biotin-linked Tubastatin A putdown and LC/MS analysis, which is independent of its inhibition of HDAC6. In addition, our results showed that radiotherapy not only activated Nrf2-mediated GPX4 transcription but also inhibited lysosome-mediated GPX4 degradation, subsequently inducing ferroptosis tolerance and radioresistance in cancer cells. Tubastatin A overcame ferroptosis resistance and radioresistance of cancer cells by inhibiting GPX4 enzymatic activity. More importantly, Tubastatin A has excellent bioavailability, as demonstrated by its ability to significantly promote radiotherapy-induced lipid peroxidation and tumour suppression in a mouse xenograft model. Our findings identify a novel druggable ferroptosis inducer, Tubastatin A, which enhances radiotherapy-mediated antitumor effects. This work provides a compelling rationale for the clinical evaluation of Tubastatin A, especially in combination with radiotherapy.

ZNF582 overexpression restrains the progression of clear cell renal cell carcinoma by enhancing the binding of TJP2 and ERK2 and inhibiting ERK2 phosphorylation.

Recent evidences have suggested that Zinc finger protein 582 (ZNF582) plays different important roles in various tumors, but its clinical role, biological function and regulatory mechanism in clear cell renal cell carcinoma (ccRCC) are still vague. Through analyzing GEO and TCGA-KIRC data and validation with local samples, we identified the low expression pattern of ZNF582 in ccRCC. Decreased ZNF582 expression is correlated with higher tumor stage and grade, distant metastasis and poor prognosis. By analyzing the DNA methylation data of ccRCC in TCGA-KIRC and using Massarray DNA methylation and demethylation analysis, we confirmed the hypermethylation status of ZNF582 in ccRCC and its negative regulation on ZNF582 expression. Using cell phenotype experiments and orthotopic kidney tumor growth models, we determined the inhibitory effect of ZNF582 overexpression on ccRCC growth and metastasis in vivo and in vitro. Mechanistically, using TMT (Tandem mass tags) quantitative proteomics test, Co-IP (Co-immunoprecipitation) and Western Blot experiments, we clarified that ZNF582 binds to TJP2 and up-regulates TJP2 protein expression. Increased TJP2 protein combines with ERK2 to promote ERK2 protein expression and suppresses the phosphorylation of ERK2, thereby inhibiting the growth and metastasis of ccRCC. In general, our findings provide the first solid theoretical rationale for targeting ZNF582/TJP2/ERK2 axis to improve ccRCC treatment.

A Novel Cuproptosis-Related Prognostic Model and the Hub Gene FDX1 Predict the Prognosis and Correlate with Immune Infiltration in Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) is a common malignancy of the urological system with poor prognosis. Cuproptosis is a recently discovered novel manner of cell death, and the hub gene FDX1 could promote cuproptosis. However, the potential roles of cuproptosis-related genes (CRGs) and FDX1 for predicting prognosis, the immune microenvironment, and therapeutic response have been poorly studied in ccRCC. In the present study, The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) data were downloaded. CRGs were subjected to prognosis analysis, and three of them were used to construct the prognostic model by least absolute shrinkage and selection operator (LASSO) regression. The CRGs prognostic model showed excellent performance. Moreover, based on the risk score of the model, the nomogram was developed to predict 1-year, 3-year, and 5-year survival. Furthermore, the hub gene of cuproptosis, FDX1, was an independent prognostic biomarker in multivariate Cox regression analysis. The pan-cancer analysis showed that FDX1 was significantly downregulated and closely related to prognosis in ccRCC among 33 cancer types. Lower FDX1 was also correlated with worse clinicopathologic features. The lower expression of FDX1 in ccRCC was verified in the external database and our own database, which may be caused by DNA methylation. We further demonstrated that the tumor mutational burden (TMB) and immune cell infiltration were related to the expression of FDX1. Immune response and drug sensitivity analysis revealed that immunotherapy or elesclomol may have a favorable treatment effect in the high FDX1 expression group and sunitinib or axitinib may work better in the low FDX1 expression group. In conclusion, we constructed a CRGs prognostic model and revealed that FDX1 could serve as a prognostic biomarker and predict therapeutic response in ccRCC. The study will provide a novel, precise, and individual treatment strategy for ccRCC patients.

FTO-mediated autophagy promotes progression of clear cell renal cell carcinoma via regulating SIK2 mRNA stability.

The progression of clear cell renal cell carcinoma (ccRCC) remains a major challenge in clinical practice, and elucidation of the molecular drivers of malignancy progression is critical for the development of effective therapeutic targets. Recent studies have demonstrated that N6-methyladenosine (m6A) is the most abundant modification of eukaryotic mRNA and plays a key role in tumorigenesis and progression. However, the biological roles and underlying mechanisms of m6A-mediated autophagy in cancers especially in ccRCC remain poorly elucidated. m6A dot blot assay, m6A RNA methylation assay kit and immunofluorescence analysis were used to profile m6A levels in tissue samples and their correlation with autophagic flux. Expression patterns and clinical significance of fat mass and obesity-associated protein (FTO) were determined through bioinformatics analysis, real-time PCR, western blotting, immunohistochemistry. RNA-seq, MeRIP-seq, MeRIP-qRT-PCR, RIP-qRT-PCR, transmission electron microscopy, immunofluorescence analysis and luciferase reporter assay were used to investigate the underlying mechanism of the FTO-autophagy axis. The role of FTO and autophagy in ccRCC progression was evaluated both in vitro and in vivo. Here we found that m6A modification was suppressed and closely related to autophagic flux in ccRCC. Elevated FTO was inhibited by rapamycin, whereas silencing FTO enhanced autophagic flux and impaired ccRCC growth and metastasis. SIK2 was identified as a functional target of m6A-mediated autophagy, thereby prompting FTO to play a conserved and important role in inhibiting autophagy and promoting tumorigenesis through an m6A-IGF2BP2 dependent mechanism. Moreover, the small molecule inhibitor FB23-2 targeting FTO inhibited tumor growth and prolonged survival in the patient-derived xenograft (PDX) model mice, suggesting that FTO is a potential effective therapeutic target for ccRCC. Our findings uncovered the crucial role of FTO/autophagy/SIK2 axis in modulating the progression of ccRCC, suggesting that FTO may serve as a valuable prognostic biomarker and promising therapeutic target in ccRCC.

Downregulation of FXYD2 Is Associated with Poor Prognosis and Increased Regulatory T Cell Infiltration in Clear Cell Renal Cell Carcinoma.

Background: FXYD2, a gene coding for the γ subunit of Na+/K+-ATPase, was demonstrated to involve in carcinogenesis recently. However, the specific role of FXYD2 in clear cell renal cell carcinoma (ccRCC) remains unknown. The current study was conducted to investigate the expression, biological function, and potentially immune-related mechanisms of FXYD2 in ccRCC. Materials and methods. The data from TCGA-KIRC, ICGC, GEO, Oncomine, ArrayExpress, TIMER, HPA datasets, and our clinical samples were used to determine and validate the expression level, prognostic roles, and potentially immune-related mechanisms in ccRCC. Cell function assays were performed to investigate the biological role of FXYD2 in vitro. Results: FXYD2 was identified to be downregulated in ccRCC tissue compared to normal tissue, which was confirmed by our RT-PCR, WB, and IHC analyses. Kaplan-Meier survival analysis and Cox regression analysis suggested that downregulated FXYD2 could independently predict poor survival of ccRCC patients. Through the ESTIMATE algorithm, ssGSEA algorithm, CIBERSORT algorithm, TIMER database, and our laboratory experiment, FXYD2 was found to correlate with the immune landscape, especially regulatory T cells (Treg), in ccRCC. Gain-of-function experiment revealed that FXYD2 could restrain cell proliferation, migration, and invasion in vitro. Functional enrichment analysis illustrated that TGF-ß-SMAD2/3, Notch, and PI3K-Akt-mTOR signaling pathways may be potential signaling pathways of FXYD2 in ccRCC. Conclusions: Downregulation of FXYD2 is associated with ccRCC tumorigenesis, poor prognosis, and increased Treg infiltration in ccRCC, which may be related to TGF-ß-SMAD2/3, Notch, and PI3K-Akt-mTOR signaling pathways. This will probably provide a novel prognostic marker and potential therapeutic target for ccRCC.