[Mechanism of nuclear protein 1 in the resistance to axitinib in clear cell renal cell carcinoma].

OBJECTIVE: To explore the potential mechanism of resistance to axitinib in clear cell renal cell carcinoma (ccRCC), with a view to expanding the understanding of axitinib resistance, facilitating the design of more specific treatment options, and improving the treatment effectiveness and survival prognosis of patients. METHODS: By exploring the half maximum inhibitory concentration (IC50) of axitinib on ccRCC cell lines 786-O and Caki-1, cell lines resistant to axitinib were constructed by repeatedly stimulated with axitinib at this concentration for 30 cycles in vitro. Cell lines that were not treated by axitinib were sensitive cell lines. The phenotypic differences of cell proliferation and apoptosis levels between drug resistant and sensitive lines were tested. Genes that might be involved in the drug resistance process were screened from the differentially expressed genes that were co-upregulated in the two drug resistant lines by transcriptome sequencing. The expression level of the target gene in the drug resistant lines was verified by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot (WB). The expression differences of the target gene in ccRCC tumor tissues and adjacent tissues were analyzed in the Gene Expression Profiling Interactive Analysis (GEPIA) public database, and the impact of the target gene on the prognosis of ccRCC patients was analyzed in the Kaplan-Meier Plotter (K-M Plotter) database. After knocking down the target gene in the drug resistant lines using RNA interference by lentivirus vector, the phenotypic differences of the cell lines were tested again. WB was used to detect the levels of apoptosis-related proteins in the different treated cell lines to find molecular pathways that might lead to drug resistance. RESULTS: Cell lines 786-O-R and Caki-1-R resistant to axitinib were successfully constructed in vitro, and their IC50 were significantly higher than those of the sensitive cell lines (10.99 µmol/L, P < 0.01; 11.96 µmol/L, P < 0.01, respectively). Cell counting kit-8 (CCK-8) assay, colony formation, and 5-ethynyl-2 '-deoxyuridine (EdU) assay showed that compared with the sensitive lines, the proliferative ability of the resistant lines decreased, but apoptosis staining showed a significant decrease in the level of cell apoptosis of the resistant lines (P < 0.01). Although resistant to axitinib, the resistant lines had no obvious new replicated cells in the environment of 20 µmol/L axitinib. Nuclear protein 1 (NUPR1) gene was screened by transcriptome sequencing, and its RNA (P < 0.0001) and protein expression levels significantly increased in the resistant lines. Database analysis showed that NUPR1 was significantly overexpressed in ccRCC tumor tissue (P < 0.05); the ccRCC patients with higher expression ofNUPR1had a worse survival prognosis (P < 0.001). Apoptosis staining results showed that knockdown ofNUPR1inhibited the anti-apoptotic ability of the resistant lines to axitinib (786-O, P < 0.01; Caki-1, P < 0.05). WB results showed that knocking downNUPR1decreased the protein level of B-cell lymphoma-2 (BCL2), increased the protein level of BCL2-associated X protein (BAX), decreased the protein level of pro-caspase3, and increased the level of cleaved-caspase3 in the resistant lines after being treated with axitinib. CONCLUSION: ccRCC cell lines reduce apoptosis through theNUPR1 -BAX/ BCL2 -caspase3 pathway, which is involved in the process of resistance to axitinib.

T.H.R.O.B.V.S. Score - A Comprehensive Model to Predict the Surgical Complexity of Renal Cell Carcinoma With Tumor Thrombus.

Background: To propose a quantitative model for predicting the surgical complexity of patients with renal cell carcinoma (RCC) and venous tumor thrombus (VTT). Method: The clinical data of 226 cases of RCC with VTT in Peking University Third Hospital from January 2014 to August 2020 were retrospectively analyzed. Seven indicators were selected to establish the T.H.R.O.B.V.S. system, including alkaline phosphatase, tumor thrombus height, maximum tumor diameter, obesity, bland thrombus, vascular wall invasion, and side. Each indicator was assigned with 0, (1), and 2 points, and the total scores of 0~2, 3~5, and ≥6 were set as the low-, middle-, and high-risk groups, respectively. The surgical complexity was compared and validated among groups. Results: As the risk increased, the proportion of open surgery significantly increased (P<0.001). The operation time (P<0.001), intraoperative blood loss (P<0.001), blood or plasma transfusion (P<0.001), and hospitalization (P<0.001) increased significantly. The postoperative complications (P<0.001), including notable complications (≥Clavein-Dindo II, P<0.001), were significantly different, and similar trends were shown in the validation group. Conclusion: The T.H.R.O.B.V.S. scoring system is a quantifiable and satisfactory model to predict the surgical complexity and perioperative management of RCC with VTT.

VHL-HIF-2α axis-induced SMYD3 upregulation drives renal cell carcinoma progression via direct trans-activation of EGFR.

It has been well established that the von Hippel-Lindau/hypoxia-inducible factor α (VHL-HIFα) axis and epidermal growth factor receptor (EGFR) signaling pathway play a critical role in the pathogenesis and progression of renal cell carcinoma (RCC). However, few studies have addressed the relationship between the two oncogenic drivers in RCC. SET and MYND domain-containing protein 3 (SMYD3) is a histone methyltransferase involved in gene transcription and oncogenesis, but its expression and function in RCC remain unclear. In the present study, we found that SMYD3 expression was significantly elevated in RCC tumors and correlated with advanced tumor stage, histological and nuclear grade, and shorter survival. Depletion of SMYD3 inhibited RCC cell proliferation, colony numbers, and xenograft tumor formation, while promoted apoptosis. Mechanistically, SMYD3 cooperates with SP1 to transcriptionally promote EGFR expression, amplifying its downstream signaling activity. TCGA data analyses revealed a significantly increased SMYD3 expression in primary RCC tumors carrying the loss-of-function VHL mutations. We further showed that HIF-2α can directly bind to the SMYD3 promoter and subsequently induced SMYD3 transcription and expression. Taken together, we identify the VHL/HIF-2α/SMYD3 signaling cascade-mediated EGFR hyperactivity through which SMYD3 promotes RCC progression. Our study suggests that SMYD3 is a potential therapeutic target and prognostic factor in RCC.

Integrative genomic study of Chinese clear cell renal cell carcinoma reveals features associated with thrombus.

Clear cell renal cell carcinoma (ccRCC) is a heterogeneous disease with features that vary by ethnicity. A systematic characterization of the genomic landscape of Chinese ccRCC is lacking, and features of ccRCC associated with tumor thrombus (ccRCC-TT) remain poorly understood. Here, we applied whole-exome sequencing on 110 normal-tumor pairs and 42 normal-tumor-thrombus triples, and transcriptome sequencing on 61 tumor-normal pairs and 30 primary-thrombus pairs from 152 Chinese patients with ccRCC. Our analysis reveals that a mutational signature associated with aristolochic acid (AA) exposure is widespread in Chinese ccRCC. Tumors from patients with ccRCC-TT show a higher mutational burden and genomic instability; in addition, mutations in BAP1 and SETD2 are highly enriched in patients with ccRCC-TT. Moreover, patients with/without TT show distinct molecular characteristics. We reported the integrative genomic sequencing of Chinese ccRCC and identified the features associated with tumor thrombus, which may facilitate ccRCC diagnosis, prognosis and treatment.

Selenoprotein M stimulates the proliferative and metastatic capacities of renal cell carcinoma through activating the PI3K/AKT/mTOR pathway.

High-throughput sequencing methods have facilitated the identification of novel selenoproteins, which exert a vital role in the development and progression of tumor diseases. Recently, Selenoprotein M (SELM) is upregulated in several types of cancer. However, the biological roles of SELM in renal cell carcinoma (RCC) remain unclear. In this paper, quantitative reverse transcription PCR (qRT-PCR) and Western blot were used to measure relative levels of SELM in a cohort of RCC tissues with matched normal tissues as well as human RCC cell lines. SELM expression was found to be upregulated in RCC. High level of SELM was related to poor prognosis of RCC. Furthermore, silence of SELM could inhibit the in vitro proliferative, migratory, and invasive capacities of RCC. In addition, downregulated SELM could impede in vivo tumorigenesis of RCC. SELM could activate the PI3K/Akt/mTOR pathway and mediate expressions of matrix metallopeptidase 2 and 9 (MMP2, MMP9). In conclusion, our study reveals the oncogenic function of SELM in RCC, and SELM may be a therapeutic and prognostic target for RCC.