GTSE1 promotes tumor growth and metastasis by attenuating of KLF4 expression in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is one of the most common malignant tumors and is characterized by a poor prognosis. Although G2- and S -phase expressed-1 (GTSE1) is known to be involved in the progression and metastasis of various cancers, its significance and mechanism in ccRCC remain unknown. In the present study, we found that GTSE1 was overexpressed in ccRCC tissues, especially in metastatic samples. Moreover, high GTSE1 expression was positively correlated with higher pT stage, tumor size, clinical stage, and WHO/ISUP grade and worse prognosis. And GTSE1 expression served as an independent prognostic factor for overall survival (OS). In addition, GTSE1 knockdown inhibited ccRCC cell proliferation, migration, and invasion, and enhanced cell apoptosis in vitro and in vivo. GTSE1 was crucial for epithelial-mesenchymal transition (EMT) in ccRCC. Mechanistically, GTSE1 depletion could upregulate the expression of Krüppel-like factor 4 (KLF4), which acts as a tumor suppressor in ccRCC. Downregulation of KLF4 effectively rescued the inhibitory effect induced by GTSE1 knockdown and reversed the EMT process. Overall, our results revealed that GTSE1 served as an oncogene regulating EMT through KLF4 in ccRCC, and that GTSE1 could also serve as a novel prognostic biomarker and may represent a promising therapeutic target for ccRCC.

Cumulative Sum Analysis of the Operator Learning Curve for Robot-Assisted Mayo Clinic Level I-IV Inferior Vena Cava Thrombectomy Associated with Renal Carcinoma: A Study of 120 Cases at a Single Center.

BACKGROUND This study aimed to use cumulative sum analysis of the operator learning curve for robot-assisted Mayo Clinic level I-IV inferior vena cava (IVC) thrombectomy associated with renal carcinoma, and describes the development of an optimized operative procedure at a single center. MATERIAL AND METHODS A retrospective study included 120 patients with Mayo Clinic level I-IV IVC thrombus who underwent robotic surgery between 2013 and 2018. Points in the learning curve were identified using cumulative sum analysis, and their impact was assessed by multiple regression analysis. Perioperative indicators analyzed included operative time, estimated blood loss, early complications, and the 90-day progression rate. RESULTS Cumulative sum analysis identified three phases in the learning curve of robot-assisted IVC thrombectomy. The median operative time decreased from 265 min (range, 212-401 min) to 207 min (range, 146-276 min) (p=0.003), the median estimated blood loss decreased from 775 ml (range, 413-1500 ml) to 300 ml (range, 163-813 ml) (p=0.006), and the early complication rate decreased from 52.5% to 15.0% (p<0.001). Multivariate analysis showed that for an initial 40 cases and a further 80 cases, the learning phase, the affected side, the Mayo Clinic level, and the surgical method were independent factors that affected operative time, estimated blood loss, and the rate of early complications. CONCLUSIONS Experience from an initial 40 cases and a further 80 cases of Mayo Clinic level I-IV IVC thrombectomy associated with renal carcinoma were found to provide acceptable surgical and clinical outcomes.

The values of neutrophil-lymphocyte ratio and/or prostate-specific antigen in discriminating real Gleason score ≥ 7 prostate cancer from group of biopsy-based Gleason score ≤ 6.

BACKGROUND: The discrepant concordance between biopsy and radical prostatectomy (RP) specimen are well reported. To validate the clinical usefulness of neutrophil-lymphocyte ratio (NLR) in discriminating real GS ≥ 7 PCa from biopsy-based GS ≤ 6 PCa in comparison with serum total prostate-specific antigen (tPSA) and value of their combination. METHODS: One hundred one patients who underwent physical examinations incidentally found elevated tPSA and subsequently received biopsy with a conclusion of GS ≤ 6 and RP with an interval of 4-6 weeks after biopsy were enrolled. NLR and tPSA were obtained within 15 days prior to biopsy. Logistic regression model was applied appropriately; McNemar tests and AUC model were performed to evaluate differences among tPSA, NLR and their combination and corresponding diagnostic power respectively. RESULTS: The pathological results from RP specimen comprised 61 patients with GS ≤ 6 and 100 patients with GS ≥ 7. Higher tPSA and NLR were significantly associated with patients with actual GS ≥ 7 (All P < 0.05) concurrently. Multivariate logistic regression indicated that tPSA (OR = 1.088, 95% C.I. = 1.029-1.151, P = 0.003) and NLR (OR = 1.807, 95% C.I. = 1.021-3.200, P = 0.042) could be independent predictors for GS groupings. Under cutoff value of 14.09 ng/ml for tPSA and 2.25 for NLR, the sensitivity, specificity and accuracy were 60.0%, 80.3% and 67.7% for tPSA, 42%, 88.5% and 59.6% for NLR, and 71.0%, 75.4% and 72.7% for combination of tPSA and NLR (tPSA + NLR) respectively. The sensitivity of tPSA + NLR was significantly higher in comparison with tPSA (P = 0.001) and NLR (P < 0.001). Except for sensitivity, no significant difference was found between tPSA and NLR in specificity (P = 0.227) and accuracy (P = 0.132). tPSA got the largest AUC with 0.732 (p < 0.001, 95% C.I.: 0.651-0.813). CONCLUSIONS: Serum tPSA and NLR were significantly elevated among GS ≥ 7 PCa concurrently. The combination of tPSA and NLR might have additional benefit to biopsy on discriminating real GS ≥ 7 Pca from biopsy-based GS ≤ 6 PCa. More stratification models and prospectively multicenter studies are necessary.

Development and Validation of a Nomogram for Predicting Postoperative Recurrence-Free Survival in Patients With Nonmetastatic Pathological T3a Stage Renal Cell Carcinoma.

BACKGROUND: To establish a nomogram predicting postoperative recurrence-free survival (RFS) in patients with nonmetastatic renal cell carcinoma (RCC) of pathological T3a (pT3a) stage undergoing nephrectomy. MATERIALS AND METHODS: A retrospective review included 668 patients with pT3a RCC between 2008 and 2019, randomly divided into training and validation groups (7:3 ratio). Cox regression analysis established the RFS-predicting nomogram in the training group. Nomogram performance was assessed using Harrell's concordance index (C-index), time-dependent receiver operating characteristic curve, decision curve analysis, and Kaplan-Meier survival analysis. RESULTS: Of the 668 patients with pT3a RCC, 167 patients experienced local recurrence or distant metastasis. Using multivariable Cox regression analysis, tumor size, ISUP grade, necrosis, capsular invasion, pT3a invasion pattern were identified as the significant predictors for RFS to establish the nomogram. The C-index of the nomogram was 0.753 (95% CI, 0.710-0.796) and 0.762 (95% CI, 0.701-0.822) for the training and validating group, respectively. The areas under the 1-year, 3-year and 5-year RFS receiver operating characteristic curves were 0.814, 0.769 and 0.768, respectively. Decision curve analysis showed the optimal application of the model in clinical decision-making. Patients with low risk T3a RCC have better RFS than those with high risk T3a RCC. CONCLUSION: Tumor size, ISUP grade, necrosis, capsular invasion and T3a invasion patterns were independent risk factors for worse RFS in patients with nonmetastatic pT3a RCC. The current nomogram could effectively predict the RFS of patients with nonmetastatic pT3a RCC.

ATF4/NUPR1 axis promotes cancer cell survival and mediates immunosuppression in clear cell renal cell carcinoma.

Cancer cells encounter unavoidable stress during tumor growth. The stress-induced transcription factor, activating transcription factor 4 (ATF4), has been reported to upregulate various adaptive genes involved in salvage pathways to alleviate stress and promote tumor progression. However, this effect is unknown in clear cell renal cell carcinoma (ccRCC). In this study, we found that ATF4 expression was remarkably upregulated in tumor tissues and associated with poor ccRCC outcomes. ATF4 depletion significantly impaired ccRCC cell proliferation, migration, and invasion in vitro and in vivo by inhibiting the AKT/mTOR and epithelial-mesenchymal transition (EMT)-related signaling pathway. RNA sequencing and functional studies identified nuclear protein 1 (NUPR1) as a key downstream target of ATF4 for repressing ferroptosis and promoting ccRCC cell survival. In addition, targeting ATF4 or pharmacological inhibition using NUPR1 inhibitor ZZW115 promoted antitumor immunity in syngeneic graft mouse models, represented by increased infiltration of CD4+ and CD8+ T cells. Furthermore, ZZW115 could improve the response to the PD-1 immune checkpoint blockade. The results demonstrate that the ATF4/NUPR1 signaling axis promotes ccRCC survival and facilitates tumor-mediated immunosuppression, providing a set of potential targets and prognostic indicators for ccRCC patients.

Targeting STING elicits GSDMD-dependent pyroptosis and boosts anti-tumor immunity in renal cell carcinoma.

While Stimulator-of-interferon genes (STING) is an innate immune adapter cruicial for sensing cytosolic DNA and modulating immune microenvironment, its tumor-promoting role in tumor survival and immune evasion remains largely unknown. Here we reported that renal cancer cells are exceptionally dependent on STING for survival and evading immunosurveillance via suppressing ER stress-mediated pyroptosis. We found that STING is significantly amplified and upregulated in clear cell renal cell carcinoma (ccRCC), and its elevated expression is associated with worse clinical outcomes. Mechanically, STING depletion in RCC cells specifically triggers activation of the PERK/eIF2α/ATF4/CHOP pathway and activates cleavage of Caspase-8, thereby inducing GSDMD-mediated pyroptosis, which is independent of the innate immune pathway of STING. Moreover, animal study revealed that STING depletion promoted infiltration of CD4+ and CD8+ T cells, consequently boosting robust antitumor immunity via pyroptosis-induced inflammation. From the perspective of targeted therapy, we found that Compound SP23, a PROTAC STING degrader, demonstrated comparable efficacy to STING depletion both in vitro and in vivo for treatment of ccRCC. These findings collectively unveiled an unforeseen function of STING in regulating GSDMD-dependent pyroptosis, thus regulating immune response in RCC. Consequently, pharmacological degradation of STING by SP23 may become an attractive strategy for treatment of advanced RCC.

Effects of Retroperitoneal or Transperitoneal Pneumoperitoneum on Inferior Vena Cava Hemodynamics and Cardiopulmonary Function: A Prospective Real-Time Comparison.

Objective: To evaluate the effects of CO2 pneumoperitoneum on venous hemodynamics and cardiopulmonary function during transperitoneal or retroperitoneal laparoscopic surgery. Materials and Methods: A single-institution prospective study. Forty-three patients with renal-cell carcinoma undergoing retroperitoneal (22) or transperitoneal (21) laparoscopic partial nephrectomy were enrolled. Hemodynamic functions were monitored by minimally invasive FloTrac/Vigileo system. Transesophageal echocardiography was used to measure the diameter and blood flow of the inferior vena cava (IVC). Measured parameters were recorded at baseline, 10, 30, 60 minutes following insufflation to 14 mm Hg and 10 minutes following desufflation. Results: For hemodynamic changes in the transperitoneal laparoscopic surgery (TPL) and retroperitoneal laparoscopic surgery (RPL), transperitoneal CO2 insufflation resulted in a rapid parallel increase in central intravenous pressure (CVP), peak airway pressure (AWP), and IVC blood flow velocity after the first 30 minutes of pneumoperitoneum (p < 0.05). In contrast, CVP, AWP, and IVC blood flow velocity increased progressively in RPL. The variation of those parameters was significantly lower than that of TRL (p < 0.001; p = 0.002; p = 0.004). The mean maximum CVP in the two groups was 20 and 16 mm Hg, respectively. The IVC diameter at the cavoatrial junction was significantly reduced in TPL after 10 minutes of insufflation, but it remained unchanged in RPL throughout the surgery. For cardiopulmonary function changes, heart output decreased after a short period of pneumoperitoneum, but no statistical differences were observed between the two groups. The increments of partial pressure of arterial carbon dioxide and end-tidal carbon dioxide tension were significantly higher in RPL than TPL (p < 0.001; p < 0.001). Conclusions: Compared with retroperitoneal pneumoperitoneum, transperitoneal pneumoperitoneum has significant effects on IVC hemodynamics. Elevated intra-abdominal pressure (IAP) causes higher AWP and venous return resistance, which lead to the significant increase of CVP during transperitoneal approach. Adjusting the balance between IAP and CVP might be an effective way to control intravenous bleeding. Clinical Trial Registry: Registration number: ChiCTR2000038291.

Progressive Obstruction of Inferior Vena Cava by Ameroid Constrictor: The Canine Model of Revealing Hemodynamic and Compensatory Patterns of Tumor Thrombus Formation at the Renal Hilus Level.

Background: To develop an efficient and stable canine model of inferior vena cava (IVC) progressive obstruction at the renal hilus level. Methods: The model was established in two beagles by encircling an ameroid constrictor (AC) on the IVC at the renal hilus level. Abdominal wall varicosity and animal weight variations were observed weekly after operation. Ultrasound examination was performed weekly after surgery to observe the AC position, the diameter, and the velocity in the IVC. Six weeks after surgery, IVC angiography and CT scan were performed to observe the collateral circulation establishment and internal organ variation. Blood samples were taken regularly to monitor for variation in critical biochemical parameters. Renal biopsy was performed at 0, 2, 4, and 6 weeks after surgery. Results: Superficial varicose veins were observed on the abdominal wall at 2 weeks after surgery. Four weeks after operation, the IVC diameter increased by ∼30%, whereas the IVC velocity decreased by more than 50%. Collateral circulation was observed by IVC angiography at 6 weeks through multiple dilated veins along with neovascularization. CT scan showed congestive alteration in the kidney. The body weight, kidney, and liver function were not significantly affected. Chronic congestive renal injury was detected in the renal tubular epithelium by kidney biopsy after surgery. Conclusions: A canine model of IVC progressive obstruction at the renal hilus level was stably and safely established for the first time by using an AC, which may be helpful for preserving pivotal collateral circulation and nontumor-side kidney function in the IVC thrombus surgery.

Loss of CMTM6 promotes DNA damage-induced cellular senescence and antitumor immunity.

Recent studies have revealed that chemokine-like factor-like MARVEL transmembrane domain-containing family member 6 (CMTM6) promotes tumor progression and modulates tumor immunity by regulating programmed death-ligand 1 stability; however, its intrinsic functions and regulatory mechanisms in clear cell renal cell carcinoma (ccRCC) remain poorly understood. Here, we show that CMTM6 is upregulated in ccRCC tissues and is strongly associated with advanced tumor grades, early metastases, and a worse prognosis. CMTM6 depletion significantly impaired the proliferation, migration, and invasion of ccRCC cells in vitro and in xenograft mouse models in vivo. In addition, targeting CMTM6 promotes anti-tumor immunity, represented by increased infiltration of CD4+ and CD8+ T cells in syngeneic graft mouse models. Further research revealed that loss of CMTM6 triggered aberrant activation of DNA damage response, resulting in micronucleus formation and G2/M checkpoint arrest, finally leading to cellular senescence with robust upregulation of numerous chemokines and cytokines. Our findings show for the first time the novel role of CMTM6 in maintaining cancer genome stability and facilitating tumor-mediated immunosuppression, linking DNA damage signaling to the secretion of inflammatory factors. Targeting CMTM6 may improve the treatment of patients with advanced ccRCC.

Loss of cell division cycle­associated 5 promotes cell apoptosis by activating DNA damage response in clear cell renal cell carcinoma.

Cell division cycle­associated 5 (CDCA5) protein, which is involved in cohesion, contributes to cell cycle regulation and chromosome segregation by maintaining genomic stability. Accumulating evidence indicates that CDCA5 expression is upregulated in a number of types of cancer associated with a poor prognosis. However, the biological function of CDCA5 in clear cell renal cell carcinoma (ccRCC) remains largely unknown. In the present study, The Cancer Genome Atlas data mining revealed that CDCA5 was more highly expressed in ccRCC than in adjacent normal tissues. Importantly, such a high expression was associated with a higher risk of distant metastasis and poorer clinical outcomes. Moreover, the clinical and prognostic value of CDCA5 expression was further investigated using immunohistochemistry on tissue microarrays containing paired tumor tissues and adjacent normal tissues from 137 patients with ccRCC. Functional analyses revealed that CDCA5 knockdown significantly inhibited the proliferation and migration of ccRCC cells, and suppressed the growth of xenografts in nude mice. Mechanistically, CDCA5 knockdown induced severe DNA damage with the persistent accumulation of γ­H2A histone family member X foci, resulting in G2/M cell cycle arrest and finally, in chromosomal instability and apoptosis. CDCA5 knockdown significantly decreased the phosphorylation levels of Stat3 and NF­κB, suggesting that CDCA5 plays a role in regulating the inflammatory response. Collectively, the findings of the present study indicate that ccRCC cells require CDCA5 for malignant progression, and that CDCA5 inhibition may enhance the outcomes of patients with high­risk ccRCC.

Robotic Retroperitoneal Versus Transperitoneal Inferior Vena Cava Thrombectomy: Right-Sided Cases with Level I-II Tumor Thrombus.

Objectives: To compare the perioperative hemodynamic consequences and oncology outcomes of robotic retroperitoneal vs transperitoneal inferior vena cava (IVC) thrombectomy (IVCT) for right renal cell carcinoma (RCC) with IVC tumor thrombus (IVCTT) that located below the first porta hepatis. Patients and Methods: Between January 2018 and June 2019, 35 patients of right RCC with IVCTT that located below the first porta hepatis underwent robotic retroperitoneal IVCT (16 patients) or transperitoneal IVCT (19 patients). We have described the procedures of transperitoneal IVCT earlier. The main procedure of robotic retroperitoneal IVCT include circumferential dissection of the IVC, sequentially clamping subhepatic IVC, the left renal vein and the caudal IVC with vessel loops, IVCT, IVC repair, and radical nephrectomy (RN). The following parameters were compared between the two groups: baselines characteristic, perioperative consequences, and hemodynamic changes. Results: Retroperitoneal and transperitoneal cohorts were comparable in terms of IVC thrombus length (3.2 vs 4.0 cm), IVC block time (18 vs 16 minutes, p = 0.64), postoperative hospital stay (6 vs 6 days, p = 0.67), postoperative complications (0 vs 0), and recurrence or metastasis rate (0 vs 0) for patients with similar baseline characteristic. The retroperitoneal cohort tended to less blood loss (160 vs 240 mL, p = 0.024), shorter operative time (130 vs 145 minutes, p = 0.003), lower central venous pressure (p < 0.05), and smaller diameter of IVC (p < 0.05). Conclusions: Robotic retroperitoneal RN and IVCT is feasible for patients of right RCC with IVCTT located below the first porta hepatis and is superior to transperitoneal IVCT in terms of bleeding control and operation time for skilled surgeons.

Genomic Landscape of Chinese Clear Cell Renal Cell Carcinoma Patients With Venous Tumor Thrombus Identifies Chromosome 9 and 14 Deletions and Related Immunosuppressive Microenvironment.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) with venous tumor thrombus (VTT) is associated with a poor clinical outcome. Although several studies have examined the genomic features of ccRCC, the genetic profile of VTT along with its matched primary tumor has not been fully elucidated. MATERIALS AND METHODS: Samples of VTT tissues and matched primary tumor tissues from ccRCC patients (n = 25), as well as primary tumor tissues from patients without VTT (n = 25) were collected and analyzed using whole-exome sequencing. Four additional ccRCC patients who were unfit for surgery were treated with an anti-programmed death receptor-1 (PD-1) monoclonal antibody (Toripalimab, 240 mg, Q3W, IV). RESULTS: By comparing the primary kidney tumors from ccRCC patients with or without VTT, a relatively higher prevalence of BAP1 and KDM5C alterations were found in ccRCC patients with VTT, and these alterations were associated with worse overall survival in the kidney renal clear cell carcinoma (KIRC) database. Based on subclone analysis, VTT was predicted to primarily originate directly from the primary renal mass. A significantly higher prevalence of CELSR2 and TET2 alterations were identified in the VTTs compared with the matched primary tumors. An increased prevalence of DNA damage repair genes, especially those involved in homologous recombination repair and non-homologous end joining, was found in ccRCC patients with VTT. Notably, VTT was characterized by the increase incidence of copy number loss in the whole exome (p < 0.05), particularly in the chromosome 9 and 14 regions. Deletion of chromosome 9 and 14 was associated with worse survival, unfavorable clinical features, and the presence of an immunosuppressive microenvironment, which was characterized by higher infiltration of regulatory T cells, follicular helper T cells, and resting mast cells, but lower counts of resting CD4 memory T cells and CD8 positive T cells. A significantly lower count of CD4+ and CD8+ tumor-infiltrated lymphocytes was identified in the VTT samples comparing with matched primary tumor. Of note, three out of the four ccRCC patients with VTT in our cohort who were treated with the anti-PD-1 therapy exhibited remarkable remission in the renal mass but no notable shrinkage in the VTT mass. CONCLUSION: Our study revealed the genetic profile of Chinese ccRCC patients with VTT, and identified multiple features associated with known poor outcomes, including gene alterations and copy number loss. The deletions in chromosomes 9 and 14, and the associated immunosuppressive microenvironment may indicate limited sensitivity to anti-PD-1/PD-L1 monotherapy in VTT.

Prognostic role of bland thrombus in patients treated with resection of renal cell carcinoma with inferior vena cava tumor thrombus.

OBJECTIVES: To assess the impact of the presence of bland thrombus (BT) on prognosis of patients treated with resection of renal cell carcinoma (RCC) with inferior vena cava tumor thrombus (IVCTT). MATERIALS AND METHODS: The medical records of a total of 145 consecutive postsurgical RCC patients with level I-IV IVCTT were reviewed from January 2008 to August 2018. Associations of BT with clinicopathological variables were estimated by chi-square test or Student's t-test. Kaplan-Meier method and multivariate Cox proportional hazard model were used. The eighth TNM staging system, "Spiess PE" model, University of California at Los Angeles Integrated Staging System and Stage, Size, Grade, and Necrosis (SSIGN) score were selected to assess whether BT could improve their predictive abilities. RESULTS: BT was observed in 34 (23.4%) patients and was significantly associated with increased levels of IVCTT (P = 0.004) and invasion of IVC wall (P = 0.030). Multivariable Cox analyses revealed that tumor grade, T stage, M stage, tumor thrombus consistency and BT were independent risk factors for both progression-free survival and overall survival. The concordance indexes ranged from a low of 0.652 in TNM to a high of 0.731 in SSIGN, and integrating BT into each base model led to an increased predictive accuracies of 6.2% for TNM (P = 0.025), 4.0% for "Spiess PE" model (P = 0.069), 2.1% for University of California at Los Angeles Integrated Staging System (P = 0.149) and 1.2% for SSIGN (P = 0.290), respectively. CONCLUSIONS: Presence of BT was independently associated with survival in postsurgical patients with RCC-IVCTT. Routine consideration of BT as an adjunct to TNM staging system may be suggested.

E2F1 promotes proliferation and metastasis of clear cell renal cell carcinoma via activation of SREBP1-dependent fatty acid biosynthesis.

Enhanced synthesis or uptake of lipids contributes to rapid cancer cell proliferation and tumor progression. In recent years, cell cycle regulators have been shown to be involved in the control of lipid synthesis, in addition to their classical function of controlling the cell cycle. Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer and is characterized by lipid-rich cytoplasmic deposition. However, the relationship between altered lipid metabolism and tumor progression in ccRCC is poorly understood. Here, we demonstrated that E2F transcription factor 1 (E2F1), in addition to its key role in regulating the cell cycle, induces extensive lipid accumulation and elevated levels of lipogenic enzymes in ccRCC cells by upregulating sterol regulatory element-binding protein 1 (SREBP1). E2F1 knockdown or SREBP1 suppression attenuated fatty acid (FA) de novo synthesis, cell proliferation and epithelial-mesenchymal transition (EMT) in ccRCC cells. Furthermore, overexpression of E2F1 promoted lipid storage, tumor growth and metastasis in a mouse xenograft model, whereas E2F1 downregulation or SREBP1 inhibition reversed these effects. In ccRCC patients, high levels of E2F1 and SREBP1 were associated with increased lipid accumulation and correlated with poor prognosis. Our results demonstrate that E2F1 can increase proliferation and metastasis through SREBP1-induced aberrant lipid metabolism, which is a novel critical signaling mechanism driving human ccRCC progression.

A Novel Preoperative Nomogram for Predicting Lymph Node Invasion in Renal Cell Carcinoma Patients Without Metastasis.

OBJECTIVE: To provide a preoperative predictive model to support clinical decision-making regarding the selection of in renal cell carcinoma (RCC) patients who will benefit the most from lymph node dissection. METHODS: This retrospective analysis enrolled 374 RCC patients without distant metastasis who underwent surgical treatment from January 2006 to December 2017. The relationships between lymph node invasion (LNI) and age at surgery; gender; body mass index(BMI); the presence of clinical symptoms such as flank pain, hematuria or a palpable mass; clinical T stage (cT stage); clinical N stage (cN stage); and the results of routine hematological and serum biochemical analyses were investigated. All the variables were included in univariate and multivariate logistic regression analyses, and the significant variables were then included in a novel nomogram to predict the probability of LNI. Then, we calibrated the nomogram with an internal validation set. RESULTS: Six of eighteen variables were significant in the univariate logistic regression analysis. After multivariate logistic regression analysis, age at surgery (OR=0.643, 95% CI: 0.421-0.975), cT stage (OR=3.034, 95% CI: 1.541-5.926), cN stage (OR=6.353, 95% CI: 3.273-12.456), lymphocyte percentage (OR=0.481, 95% CI: 0.256-0.894), and the presence of clinical symptoms (OR=2.045, 95% CI: 1.065-3.924) were independent predictors of LNI and were included in the nomogram. The C-index of this nomogram was 0.824. CONCLUSION: Preoperative basic laboratory findings combined with the results of a physical examination and radiological examination can indicate the probability of LNI in RCC patients.

Up-regulation of miR-181a in clear cell renal cell carcinoma is associated with lower KLF6 expression, enhanced cell proliferation, accelerated cell cycle transition, and diminished apoptosis.

OBJECTIVES: Dysregulated expression of miR-181a accompanies tumorigenesis in many human cancers. However, in clear cell renal cell carcinoma (ccRCC), the role of miR-181a remains unclear. The aim of this study was to investigate biological functions of miR-181a and its expression levels in ccRCC tissues and cancer cell lines. MATERIAL AND METHODS: Expression levels of miR-181a in samples of ccRCC tumors and adjacent nontumor tissues from 42 patients as well as in 786-O, 769-P, A498, and CAKI-1 ccRCC cell lines were determined by quantitative real-time polymerase chain reaction. Potential targets of miR-181a were predicted using bioinformatic approaches and then verified by using the luciferase reporter assay. The effects of miR-181a on cell proliferation, colony formation, cell cycle progression, and apoptosis were investigated in ccRCC cell lines transfected with specific miR-181a mimic and inhibitor. RESULTS: We found that miR-181a expression was up-regulated in ccRCC tissues and cell lines. The expression level of miR-181a significantly correlated with the tumor size, tumor/node/metastasis staging, and Fuhrman grade. Luciferase assays showed that KLF6 was a target of miR-181a. KLF6 expression was inversely correlated with the level of miR-181a. Overexpression of miR-181a led to reduced KLF6 mRNA and protein levels, whereas mutations of the potential miR-181a binding sites in the KLF6 gene abrogated this inhibitory effect. Furthermore, overexpression of miR-181a promoted proliferation and G1/S cell cycle transition, as well as inhibited apoptosis by down-regulating KLF6 in ccRCC cells. CONCLUSIONS: miR-181a is up-regulated in ccRCC and may act as a tumor promoting factor by targeting KLF6 expression. Manipulating miR-181a may provide a beneficial effect in the treatment of ccRCC.

Upregulation of Far Upstream Element-Binding Protein 1 (FUBP1) Promotes Tumor Proliferation and Tumorigenesis of Clear Cell Renal Cell Carcinoma.

OBJECTIVE: The far upstream element (FUSE)-binding protein 1 (FUBP1) is a transactivator of human c-myc proto-oncogene transcription, with important roles in carcinogenesis. However, the expression pattern and potential biological function of FUBP1 in clear cell renal cell carcinoma (ccRCC) is yet to be established. METHODS: FUBP1 expression was detected in ccRCC tissues and cell lines by real-time RT-PCR, Western blot analysis, and immunohistochemistry. The correlations of FUBP1 mRNA expression levels with clinicopathological factors were evaluated. The biological function of FUBP1 during tumor cell proliferation was studied by MTS, colony formation, and soft-agar colony formation. The effects of FUBP1 on cell cycle distribution and apoptosis were analyzed by flow cytometry. Western blot analysis was used to identify the potential mechanism of FUBP1 regulating cell cycle and apoptosis. RESULTS: The levels of FUBP1 mRNA and protein expression were upregulated in human ccRCC tissues compared with adjacent noncancerous tissues. High levels of FUBP1 mRNA expression were associated with higher tumor stage and tumor size. FUBP1 knockdown inhibited cell proliferation and induced cell cycle arrest and apoptosis. Meanwhile, the expression levels of c-myc and p21 mRNA were correlated with that of FUBP1 mRNA. CONCLUSIONS: FUBP1 acts as a potential oncogene in ccRCC and may be considered as a novel biomarker or an attractive treatment target of ccRCC.

Upregulation of long noncoding RNA PVT1 predicts unfavorable prognosis in patients with clear cell renal cell carcinoma.

BACKGROUND: Renal cell carcinoma (RCC) is one of the most malignant genitourinary diseases worldwide. Long noncoding RNAs (lncRNAs) are a class of noncoding RNAs in the human genome that are involved in RCC initiation and progression. OBJECTIVE: To investigate the expression of PVT1 in ccRCC and evaluate its correlation with clinicopathologic characteristics and patients' survival. METHODS: Quantitative real-time PCR was performed to examine PVT1 expression in 129 ccRCC tissue samples and matched adjacent normal tissue samples. The relationship of PVT1 expression with clinicopathologic characteristics and clinical outcome was evaluated. RESULTS: We identified the lncRNA PVT1, which was upregulated in clear cell renal cell carcinoma (ccRCC) tissues when compared with corresponding controls. Furthermore, PVT1 expression was positively associated with gender, tumor size, pT stage, TNM stage, and Fuhrman grade. Kaplan-Meier survival analysis showed that patients with high PVT1 expression had shorter disease-free survival (DFS) and overall-survival (OS) than those with low PVT1 expression, and multivariate analysis identified PVT1 as an independent prognostic factor in ccRCC. CONCLUSIONS: PVT1 may be an oncogene as well as may promote metastasis in ccRCC and could serve as a potential biomarker to predict the prognosis of ccRCC patients.

[Overexpression of SEPP1 inhibits the proliferation and induces cell cycle G2/M arrest of 786-O and 769-P human renal carcinoma cells].

Objective To establish selenoprotein P, plasma 1 (SEPP1) gene recombinant lentiviral vector and investigate the effect of SEPP1 on the proliferation of human clear cell renal cell carcinoma (ccRCC) cells. Methods cDNA sequence of SEPP1 was cloned from the total cDNA of HEK293T cells by PCR. Then, the cDNA fragment was combined with the pLV-EGFP(2A)Puro vector and the constructed plasmid pLV-EGFP(2A)Puro-SEPP1 was transfected into HEK293T cells for packaging the virus. Forty-eight hours after transfected with the virus supernatant, the level of SEPP1 protein in 769-P and 786-O cells were tested by Western blotting. Cells were divided into recombinant lentivirus-infected cells, empty vector lentivirus-infected cells and the blank control cells. Cell proliferation rate was detected by MTS assay, colony forming ability was evaluated by plate clony formation assay and cell cycle change was assayed by flow cytometry after transfected with pLV-EGFP(2A)Puro-SEPP1 or empty pLV-EGFP(2A)Puro vector. Results Enzyme digestion analysis and DNA sequencing showed that the recombinant plasmid pLV-EGFP(2A)Puro-SEPP1 was constructed successfully. After being infected by the virus supernatant, the 786-O and 769-P cells expressed EGFP. Compared with the empty vector group and the blank control group, expression level of SEPP1 in the experimental group was much higher. The cell proliferative ability was inhibited in the cells overexpressing SEPP1, and the colony forming ability of SEPP1-overexpressed cells evidently decreased. Cell cycle was arrested in G2/M phase in 786-O cells overexpressing SEPP1. Conclusion The recombinant plasmid pLV-EGFP(2A)Puro-SEPP1 has been constructed successfully. Overexpression of SEPP1 could significantly reduce the proliferation rate of 786-O and 769P cells, and cause G2/M phase arrest of 786-O cells.