Castration resistance is a serious problem facing clinical treatment of prostate cancer (PCa). The underlying molecular mechanisms of acquired proliferation ability of tumor cells upon androgen deprivation are largely undetermined. In the present study, we identified that ubiquitin specific peptidase 39 (USP39) was significantly upregulated in PCa samples and cell lines. Elevated USP39 expression was positively correlated with Gleason score, predicted a poor outcome, and functioned as an independent risk factor for biochemical recurrence (BCR) especially in patients with a Gleason score ≤7. Our cell-based study showed that the expression level of USP39 was the highest in AR-negative PCa cell lines. Knockdown of USP39 in PCa cells inhibited cancer colony formation and tumor cell growth, and induced G2/M arrest and cell apoptosis. Microarray analysis suggested that knockdown of USP39 caused a reduced expression of EGFR. Silencing of USP39 inhibited the expression of EGFR 3'-end, and presented a remarkable block to the maturation of EGFR mRNA, suggesting that silencing of USP39 decreased the transcriptional elongation and maturation of EGFR mRNA. Oncomine datasets analysis showed that USP39 expression was positively correlated with EGFR level. The above findings suggest that USP39 plays a vital oncogenic role in the tumorigenesis of PCa and may prove to be a potential biomarker for predicting the prognosis of PCa patients.
Abstract Tumor growth, metastasis and therapeutic response are believed to be regulated by the tumor and its microenvironment (TME) in advanced renal cell carcinoma (RCC). However, the mechanisms underlying genomic, transcriptomic and epigenetic alternations in RCC progression have not been completely defined. In this study, single-cell RNA-sequencing (scRNA-seq) data were obtained from eight tissue samples of RCC patients, including two matched pairs of primary and metastatic sites (lymph nodes), along with Hi-C, transposable accessible chromatin by high-throughput (ATAC-seq) and RNA-sequencing (RNA-seq) between RCC (Caki-1) and human renal tubular epithelial cell line (HK-2). The identified target was verified in clinical tissue samples (microarray of 407 RCC patients, TMA-30 and TMA-2020), whose function was further validated by in vitro and in vivo experiments through knockdown or overexpression. We profiled transcriptomes of 30514 malignant cells, and 14762 non-malignant cells. Comprehensive multi-omics analysis revealed that malignant cells and TME played a key role in RCC. The expression programs of stromal cells and immune cells were consistent among the samples, whereas malignant cells expressed distinct programs associated with hypoxia, cell cycle, epithelial differentiation, and two different metastasis patterns. Comparison of the hierarchical structure showed that SERPINE2 was related to these NNMF expression programs, and at the same time targeted the switched compartment. SERPINE2 was highly expressed in RCC tissues and lowly expressed in para-tumor tissues or HK-2 cell line. SERPINE2 knockdown markedly suppressed RCC cell growth and invasion, while SERPINE2 overexpression dramatically promoted RCC cell metastasis both in vitro and in vivo. In addition, SERPINE2 could activate the epithelial-mesenchymal transition pathway. The above findings demonstrated that the role of distinct expression patterns of malignant cells and TME played a distinct role in RCC progression. SERPINE2 was identified as a potential therapeutic target for inhibiting metastasis in advanced RCC.
Genetic factors coordinate with environmental factors to drive the pathogenesis of prostate adenocarcinoma (PRAD). SPOP is one of the most mutated genes and LRP5 mediates lipid metabolism that is abnormally altered in PRAD. Here, we investigated the potential cross-talk between SPOP and LRP5 in PRAD. We find a negative correlation between SPOP and LRP5 proteins in PRAD. SPOP knockdown increased LRP5 protein while SPOP overexpression resulted in LRP5 reduction that was fully rescued by proteasome inhibitors. LRP5 intracellular tail has SPOP binding site and the direct interaction between LRP5 and SPOP was confirmed by Co-IP and GST-pulldown. Moreover, LRP5 competed with Daxx for SPOP-mediated degradation, establishing a dynamic balance among SPOP, LRP5 and Daxx. Overexpression of LRP5 tail could shift this balance to enhance Daxx-mediated transcriptional inhibition, and inhibit T cell activity in a co-culture system. Further, we generated human and mouse prostate cancer cell lines expressing SPOP variants (F133V, A227V, R368H). SPOP-F133V and SPOP-A227V have specific effects in up-regulating the protein levels of PD-1 and PD-L1. Consistently, SPOP-F133V and SPOP-A227V show robust inhibitory effects on T cells compared to WT SPOP in co-culture. This is further supported by the mouse syngeneic model showing that SPOP-F133V and SPOP-A227V enhance tumorigenesis of prostate cancer in in-vivo condition. Taken together, our study provides evidence that SPOP-LRP5 crosstalk plays an essential role, and the genetic variants of SPOP differentially modulate the expression and activity of immune checkpoints in prostate cancer.
Abstract Non-clear renal cell carcinomas (nccRCCs) are less frequent in kidney cancer with histopathological heterogeneity. A better understanding of the tumor biology of nccRCC can provide more effective treatment paradigms for different subtypes. To reveal the heterogeneity of tumor microenvironment (TME) in nccRCC, we performed 10x sing-cell genomics on tumor and normal tissues from patients with papillary renal cell carcinoma (pRCC), chromophobe RCC (chrRCC), collecting duct carcinoma (CDRCC) and sarcomatoid RCC (sarRCC). 15 tissue samples were finally included. 34561 cells were identified as 16 major cell clusters with 34 cell subtypes. Our study presented the sing-cell landscape for four types of nccRCC, and demonstrated that CD8+ T cells exhaustion, tumor-associated macrophages (TAMs) and sarcomatoid process were the pivotal factors in immunosuppression of nccRCC tissues and were closely correlated with poor prognosis. Abnormal metabolic patterns were present in both cancer cells and tumor-infiltrating stromal cells, such as fibroblasts and endothelial cells. Combined with CIBERSORTx tool, the expression data of bulk RNA-seq from TCGA were labeled with cell types of our sing-cell data. Calculation of the relative abundance of cell types revealed that greater proportion of exhausted CD8+ T cells, TAMs and sarRCC derived cells were correlated with poor prognosis in the cohort of 274 nccRCC patients. To the best of our knowledge, this is the first study that provides a more comprehensive sight about the heterogeneity and tumor biology of nccRCC, which may potentially facilitate the development of more effective therapies for nccRCC.
Renal cell carcinoma (RCC) is a hypermetabolic disease. Abnormal up-regulation of glycolytic signaling promotes tumor growth, and glycolytic metabolism is closely related to immunotherapy of renal cancer. The aim of the present study was to determine whether and how the glycolysis-related biomarker TCIRG1 affects aerobic glycolysis, the tumor microenvironment (TME) and malignant progression of clear cell renal cell carcinoma (ccRCC).Based on The Cancer Genome Atlas (TCGA, n = 533) and the glycolysis-related gene set from MSigDB, we identified the glycolysis-related gene TCIRG1 by bioinformatics analysis, analyzed its immunological properties in ccRCC and observed how it affected the biological function and glycolytic metabolism using online databases such as TIMER 2.0, UALCAN, LinkedOmics and in vitro experiments.It was found that the expression of TCIRG1, was significantly increased in ccRCC tissue, and that high TCIRG1 expression was associated with poor overall survival (OS) and short progression-free interval (PFI). In addition, TCIRG1 expression was highly correlated with the infiltration immune cells, especially CD4+T cell Th1, CD8+T cell, NK cell, and M1 macrophage, and positively correlated with PDCD1, CTLA4 and other immunoinhibitors, CCL5, CXCR3 and other chemokines and chemokine receptors. More importantly, TCIRG1 may regulate aerobic glycolysis in ccRCC via the AKT/mTOR signaling pathway, thereby affecting the malignant progression of ccRCC cell lines.Our results demonstrate that the glycolysis-related biomarker TCIRG1 is a tumor-promoting factor by affecting aerobic glycolysis and tumor immune microenvironment in ccRCC, and this finding may provide a new idea for the treatment of ccRCC by combination of metabolic intervention and immunotherapy.
Background: Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a protein expressed primarily in the liver, formerly known to maintain plasma lipid homeostasis by regulating low-density lipoprotein receptor levels, and its exact role in the radioresistance of prostate cancer (PCa) remains unclear. We aim to investigate the function of PCSK9 in the radioresistance of PCa cells. Methods: PCSK9 small interfering RNA (siRNA) was introduced into the PCa cells by transient transfection. Then, cells were exposed to ionizing radiation (IR) at indicated dose rates. Cell damage was detected using cell counting kit-8 (CCK-8) and Hoechest 33342/propidium iodide (PI) staining. Rhodamine-123 (Rho-123) dye was used to assay mitochondrial membrane potential alteration. Western blot was used to detect the apoptosis-related protein expression. Results: PCSK9 siRNA treatment significantly protected PCa cells from IR-induced cell damage, including enhancing cell viability, reducing apoptosis, and inhibiting MMPs. Moreover, PCSK9 siRNA repressed the increase of cytochrome C (cyto C), caspase-3, and B-cell leukemia/lymphoma 2 (Bcl-2)-associated X (Bax) expressions induced by IR and promoted Bcl-2 expression, which might partially interpret the radioprotective role of PCSK9 siRNA in PCa cells. Conclusion: PCSK9 might impact on radiosensitivity through mitochondrial pathways and serve as a novel therapeutic target for PCa patients. Keywords: PCSK9, prostate cancer, radioresistance, mitochondrial pathway
Objective
To analyze the value of early sequential unclamping method in laparoscopic partial nephrectomy.
Methods
From April 2017 to October 2017, a total of 8 cases of renal tumor patients by early sequential unclamping method of laparoscopic partial nephrectomy (LPN) were reviewed, with 5 males and 3 females and average age of 56.4 years (43-70 years). Three cases of renal tumor were located on the left side, 5 cases on the right side. The mean tumor diameter was 5.6 (4.6-6.4) cm. The preoperative R. E.N.A.L. score was 8.8 (7-10), and the mean ASA score was 1.4 (1-2). Preoperative serum creatinine level was 89.5(72.1-104.2) μmol/L, and the GFR level of the kidney with tumor before operation was 55. 5 (40.4-62.3) ml/min. The early sequential unclamping method was used for retroperitoneal laparoscopic partial nephrectomy: according to the preoperative CTA results, the main branches and branches of the renal artery were routinely separated. Before the tumor resection, the branches of renal artery and the main renal artery were sequentially blocked. After removal of the tumor, the first layer of bare kidney wound blood vessels and collection system were sutured and repaired. Then released the main renal artery occlusion clamp, restored most of the blood supply to the kidney, but kept the tumor-specific segmental renal artery blocked. Continuous suture of the kidney created a rough combination of the renal wound. After second layers of suture completed, unclamped the segmental renal artery and sutured the renal wound again, made the third layers of suture intersecting with the second seam suture to strengthen the hemostatic effect.
Results
All the 8 patients were performed LPN with early sequential unclamping method successfully. The average operative time was 132.5 (90-180) min, the intraoperative blood loss was 142.5 (100-200) ml, the completely warm ischemia time was 15.5 (12.0-20.0) min, and no blood transfusion was performed intraoperatively and postoperatively. The operative margin was negative. The postoperative pathology showed that 7 cases were clear cell carcinoma and 1 cases of papillary cell carcinoma. Postoperative complications such as urinary leakage, incision infection and fever were not found. Drainage tube removal time was 3.5 (3-5) days and the time of postoperative hospitalization was 4.8(4-6) days. At 1 months after operation, the serum creatinine level was 94.0 (83.6-101.2) μmol/L and the GFR level of one side kidney with tumor was 52.3(43.2-59.6) ml/min. After 2-9 months of follow-up, there was no recurrence of the tumor.
Conclusions
Early sequential unclamping method could shorten the warm ischemia time and reduce the risk of bleeding during the operation. It also maintains a clear operative field, which could reduce the difficulty of laparoscopic partial nephrectomy and make a more accurate tumor resection in the complex renal tumor patients.
Key words:
Kidney neoplasms; Partial nephrectomy; Segmental renal artery clamping; Early unclamping; Warm ischemia time
Background: Cancer stem cells (CSCs) are biologically characterized by self-renewal, multi-directional differentiation and infinite proliferation, inducing anti-tumor drug resistance and metastasis.In the present study, we attempted to depict the baseline landscape of CSC-mediated biological properties, knowing that it is vital for tumor evolution, anti-tumor drug selection and drug resistance against fatal malignancy.Methods: We performed single-cell RNA sequencing (scRNA-seq) analysis in 15208 cells from a pair of primary and metastatic sites of collecting duct renal cell carcinoma (CDRCC).Cell subpopulations were identified and characterized by t-SNE, RNA velocity, monocle and other computational methods.Statistical analysis of all single-cell sequencing data was performed in R and Python.Results: A CSC population of 1068 cells was identified and characterized, showing excellent differentiation and self-renewal properties.These CSCs positioned as a center of the differentiation process and transformed into CDRCC primary and metastatic cells in spatial and temporal order, and played a pivotal role in promoting the bone destruction process with a positive feedback loop in the bone metastasis microenvironment.In addition, CSC-specific marker genes BIRC5, PTTG1, CENPF and CDKN3 were observed to be correlated with poor prognosis of CDRCC.Finally, we pinpointed that PARP, PIGF, HDAC2, and FGFR inhibitors for effectively targeting CSCs may be the potential therapeutic strategies for CDRCC. Conclusion:The results of the present study may shed new light on the identification of CSCs, and help further understand the mechanism underlying drug resistance, differentiation and metastasis in human CDRCC.
Background: Increasing evidence, including ours, reveals that the interaction between tumor cells and tumor-associated macrophages (TAMs) facilitates progression of prostate cancer (PCa), but the related mechanisms have not yet been elucidated. This study determined how gankyrin regulates progression and androgen deprivation therapy (ADT) resistance in PCa through tumor cell–TAM interactions. Methods: In vitro functional experiments and in vivo subcutaneous tumor models were used to explore the biological role and molecular mechanisms of gankyrin in PCa cell–TAM interactions. Two hundred thirty-four PCa patients were randomly divided into training and validation cohorts to examine the prognostic value of gankyrin through immunohistochemistry (IHC) and statistical analyses. Findings: High gankyrin expression was observed in PCa specimens, which was positively correlated with poor prognosis of PCa patients. In addition, gankyrin facilitated PCa progression and ADT resistance. Mechanistically, gankyrin recruited and upregulated NONO expression, resulting in increased AR expression. AR bound to the HMGB1 promoter to trigger HMGB1 transcription, expression, and secretion. Moreover, HMGB1 was found to promote recruitment and activation of TAMs, which secreted IL-6 to reciprocally promote PCa progression, ADT resistance and gankyrin expression via STAT3, thus forming a gankyrin/NONO/AR/HMGB1/IL-6/STAT3 positive feedback loop. Furthermore, targeting the interaction between tumor cells and TAMs by blocking this loop inhibited ADT resistance in a tumor xenograft model. Interpretation: Gankyrin serves as a reliable prognostic indicator and oncogene for PCa. Combined inhibition of gankyrin and HMGB1, which blocks the regulatory loop between PCa and TAMs, enhances the therapeutic effect of ADT on advanced PCa.Funding: National Natural Science Foundation of China (No. 81773154, 82173357)， Shanghai Natural Science Foundation (No. 20ZR1449600), Pudong New Area Science and technology development fund special fund for people's livelihood Research (medical and health) (PKJ2019-Y19), Shanghai Sailing Program (19YF1447000), the Science and Technology Foundation of the Health Commission of Guizhou province （gzwkj2022-102）.Declaration of Interest: The authors declare no conflict of interest.Ethical Approval: All experiments were approved by the Ethics Committee of Changhai Hospital,and written informed consent was obtained from all prostate cancer patients.
There are no effective therapies for advanced renal cell carcinoma (RCC), except for VEGFR inhibitors with only ~50% response rate. To identify novel targets and biomarkers for RCC is of great importance in treating RCC. In this study, we observed that eukaryotic initiation factor 3d (EIF3D) expression was significantly increased in RCC compared with paracarcinoma tissue using immunohistochemistry staining and western blot analysis. Furthermore, bioinformatics meta-analysis using ONCOMINE microarray datasets showed that EIF3D mRNA expressions in CCRCC tissue specimens were significantly higher than that in normal tissue specimens. In addition, RCC tissue microarray demonstrated that elevated EIF3D expression was positively correlated with TNM stage and tumor size. EIF3D silencing in human 786-O and ACHN CCRCC cell lines by RNA interference demonstrated that EIF3D knockdown obviously inhibited cell proliferation and colony formation, caused G2/M arrest through downregulation of Cyclin B1 and Cdk1 and upregulation of p21, and induced apoptosis shown by sub-G1 accumulation and RARP cleavage. Moreover, correlation analysis using ONCOMINE microarray datasets indicated that increased EIF3D mRNA expression was positively correlated to PCNA, Cyclin B1 and CDK1 mRNA expression in RCC. Collectively, these results provide reasonable evidences that EIF3D may function as a potential proto-oncogene that participates in the occurrence and progression of RCC.
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