Renal Cell Carcinoma Cell Lines Research Articles

Human circular RNA hsa_circRNA_101705 (circTXNDC11) regulates renal cancer progression by regulating MAPK/ERK pathway

ABSTRACT Circular RNAs (circRNAs) play essential roles in the progression of human tumors, including renal cell carcinoma (RCC). The present study aimed to explore the functions and potential mechanisms of human circular RNA hsa_circRNA_101705 (circTXNDC11) in RCC. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to measure circTXNDC11 expression in RCC tissues and cell lines. RNase R and actinomycin D assays were conducted to analyze the characteristic of circTXNDC11. Cell Counting Kit-8 (CCK-8) assay, colony formation assay, and transwell invasion assay were performed to assess cell proliferation and invasion abilities. Western blotting was applied to assess the levels of MEK and ERK proteins in RCC cells. Murine xenograft model assay was conducted to deduce the role of circTXNDC11 in vivo. The current data showed that circTXNDC11 was overexpressed in RCC tissues and cells. The overexpression of circTXNDC11 is linked to advanced TNM stage and lymph node metastasis of renal cancer. Knocking down circTXNDC11 suppressed cell proliferation and invasion in vitro and reduced tumor growth in vivo. Mechanistically, circTXNDC11 promoted RCC growth and invasion by activating the MAPK/ERK pathway. Thus, the current findings identified circTXNDC11 as a novel regulator of RCC tumorigenesis through the regulation of the MAPK/ERK pathway, offering a potential therapeutic target for RCC treatment.

CircAMOTL1L Suppresses Renal Cell Carcinoma Growth by Modulating the miR-92a-2-5p/KLLN Pathway.

Accumulating evidence indicates that the dysregulation of circular RNAs (circRNAs) contributes to tumor progression; however, the regulatory functions of circRNAs in renal cell carcinoma (RCC) remain largely unknown. In this study, the function and underlying mechanism of circAMOTL1L in RCC progression were explored. qRT-PCR showed the downregulation of circAMOTL1L in RCC tissues and cell lines. The decrease in circAMOTL1L expression correlated with the tumor stage, metastasis, and poor prognosis in patients with RCC. Functional experiments revealed that circAMOTL1L inhibited cell proliferation and increased apoptosis in RCC cells. Subcutaneous implantation with circAMOTL1L-overexpressing cells in nude mice decreased the growth ability of the xenograft tumors. Mechanistically, circAMOTL1L served as a sponge for miR-92a-2-5p in upregulating KLLN (killin, p53-regulated DNA replication inhibitor) expression validated by bioinformatics analysis, oligo pull-down, and luciferase assays. Further, reinforcing the circAMOTL1L–miR-92a-2-5p–KLLN axis greatly reduced the growth of RCC in vivo. Conclusively, our findings demonstrate that circAMOTL1L has an antioncogenic role in RCC growth by modulating the miR-92a-2-5p–KLLN pathway. Thus, targeting the novel circAMOTL1L–miR-92a-2-5p–KLLN regulatory axis might provide a therapeutic strategy for RCC.

Establishment of a Risk Signature Based on m6A RNA Methylation Regulators That Predicts Poor Prognosis in Renal Cell Carcinoma

PurposeN6-methyladenosine (m6A) modifications represent one of the most common methylation modifications, and they are mediated by m6A RNA methylation regulators. However, their functions in renal cell carcinoma (RCC) are not completely understood. The aim of this study was to investigate the effects of the regulators in RCC.Materials and MethodsThe expression levels of the 13 main m6A RNA methylation regulators in RCC were detected and consensus clustering was performed to explore their relationships with RCC. Thereafter, a risk signature based on the regulators was established. This risk model was fully verified by conducting prognostic analyses using two datasets (The Cancer Genome Atlas [TCGA] and Gene Expression Omnibus [GEO] datasets) and a ROC curve analysis.ResultsOf the 13 main m6A regulators, six were significantly upregulated and four were significantly downregulated in 893 RCC cases compared to 128 normal controls in the TCGA database. Consensus clustering based on the regulators identified two clusters of RCC cases, which were significantly associated with a pathological characteristic (T status). Thus, these results indicated that m6A RNA methylation regulators were associated with RCC. Thereafter, a risk model involving two of the regulators (METTL14 and WTAP) was established. The alterations in the mRNA and protein expression levels of these two regulators were further confirmed based on Human Protein Atlas data and real-time PCR in RCC and normal cell lines. The results indicated that the risk model may serve as an independent prognostic marker of overall survival, and it was also associated with clinicopathological characteristics (T status, M status, pathological stage, and gender) in RCC.ConclusionCollectively, the results of this study indicated that the risk model (based on two m6A RNA methylation regulators) may serve as an independent prognostic indicator of RCC, which may aid further investigation into m6A RNA modification in RCC.

Long Non-Coding RNA LOC648987 Promotes Proliferation and Metastasis of Renal Cell Carcinoma by Regulating Epithelial-Mesenchymal Transition.

Renal cell carcinoma (RCC) is a type of urinary tumor with a high incidence and is often associated with tumor metastasis. Long non-coding RNA (lncRNA) regulates tumorigenesis, progression, and metastasis. However, the role and the predictive value of lncRNA in RCC progression and metastasis have not been elucidated. The purpose of this study was to evaluate the effect of a newly discovered lncRNA LOC648987 on RCC proliferation and metastasis. LOC648987 was identified by RT-PCR for high expression in human RCC tissues as well as in metastatic RCC tissues. In the cell experiments, we infected the RCC cell lines ACHN and 786-O cells with LOC648987-shRNA and its negative control (shNC). The results showed that the knockdown of LOC648987 inhibited the proliferation of ACHN and 786-O cells and colony formation. The cell cycle and the apoptosis progression of ACHN and 786-O cells were assessed using flow cytometry. The knockdown of LOC648987 significantly inhibited the progression of ACHN and 786-O cells from G0/G1 to S phase and promoted cell apoptosis. The metastasis promoting effects of LOC648987 on ACHN and 786-O cells were verified by transwell migration assays, which depended on vimentin and MMP-9 to regulate the epithelial–mesenchymal transition. Finally, the promotion of LOC648987 on RCC tumorigenesis was evaluated in BALb/c nude mice. These data confirmed that lncRNA LOC648987 promoted RCC cell proliferation and tumor metastasis and regulated the expression of EMT-related proteins in RCC cells.

TRIP13 is identified as a prognosis biomarker for renal clear cell carcinoma and promotes renal cell carcinoma cell proliferation, migration and invasion

This work aimed to discover new therapeutic targets in renal clear cell carcinoma by bioinformatics and detect the effect of candidate gene TRIP13 in renal cell carcinoma (RCC) cell proliferation, migration, and invasion. Differentially expressed mRNAs were screened based on The Cancer Genome Atlas (TCGA)-Kidney Renal Clear Cell Carcinoma (KIRC) databases, and functional enrichments, survival analysis, receiver operating characteristic curve (ROC), and Protein–Protein Interaction (PPI) protein interaction analysis were performed by R software to screen the candidate gene TRIP13. Then, the expression of candidate gene TRIP13 in 92 pairs of cancer and adjacent normal tissues of renal clear cell carcinoma patients were detected by qRT-PCR, western blotting, and immunochemical analysis. The TRIP13 level and clinicopathological characteristics of patients with renal clear cell carcinoma were analyzed. Using 186-O and ACHN RCC cell lines with TRIP13 overexpressing or downregulating, the effect of TRIP13 on cell viability and proliferation were detected by CCK8 and EdU staining, respectively. The migration and invasion were detected by Transwell assays. A total of 19858 differentially expressed genes, 5823 differentially expressed genes, 3657 up-regulated genes, and 2166 down-regulated genes were identified. TRIP13 was closed associated with cell cycle regulation, and survival and prognosis of renal clear cell carcinoma were selected as a candidate gene. The mRNA and protein levels of TRIP13 in cancer tissues were higher than that in adjacent normal tissues. TRIP13 level was significantly associated with tumor size, tumor stage, Fuhrman grade, and lymph node metastasis. TRIP13 overexpression significantly increased cell viability, proliferation, migration, and invasion, while downregulating of TRIP13 had opposite effects in both 186-O and ACHN cells. Therefore, TRIP13 promotes RCC proliferation and metastasis, which should be a novel biomarker for early diagnosis, treatment, and prognosis of RCC.

Metabolomic Analysis to Elucidate Mechanisms of Sunitinib Resistance in Renal Cell Carcinoma.

Metabolomics analysis possibly identifies new therapeutic targets in treatment resistance by measuring changes in metabolites accompanying cancer progression. We previously conducted a global metabolomics (G-Met) study of renal cell carcinoma (RCC) and identified metabolites that may be involved in sunitinib resistance in RCC. Here, we aimed to elucidate possible mechanisms of sunitinib resistance in RCC through intracellular metabolites. We established sunitinib-resistant and control RCC cell lines from tumor tissues of RCC cell (786-O)-injected mice. We also quantified characteristic metabolites identified in our G-Met study to compare intracellular metabolism between the two cell lines using liquid chromatography-mass spectrometry. The established sunitinib-resistant RCC cell line demonstrated significantly desuppressed protein kinase B (Akt) and mesenchymal-to-epithelial transition (MET) phosphorylation compared with the control RCC cell line under sunitinib exposure. Among identified metabolites, glutamine, glutamic acid, and α-KG (involved in glutamine uptake into the tricarboxylic acid (TCA) cycle for energy metabolism); fructose 6-phosphate, D-sedoheptulose 7-phosphate, and glucose 1-phosphate (involved in increased glycolysis and its intermediate metabolites); and glutathione and myoinositol (antioxidant effects) were significantly increased in the sunitinib-resistant RCC cell line. Particularly, glutamine transporter (SLC1A5) expression was significantly increased in sunitinib-resistant RCC cells compared with control cells. In this study, we demonstrated energy metabolism with glutamine uptake and glycolysis upregulation, as well as antioxidant activity, was also associated with sunitinib resistance in RCC cells.

LHPP Inhibits the Proliferation and Metastasis of Renal Cell Carcinoma.

Renal cell carcinoma (RCC) is one of the ten most common cancers in the globe. Despite the diagnosis and treatment of renal cell carcinoma that have made great improvements, the morbidity and mortality rates of renal cell carcinoma remain unchanged remarkably. LHPP is a kind of histidine phosphatases, acting as a tumor suppressor in the progression of various cancers. In this study, we found that LHPP was significantly downregulated in RCC tissues and cell lines. Decreased expression of LHPP was closely correlated with tumor size and postoperative metastasis of RCC patients. In addition, overexpression of LHPP inhibited the proliferation and metastasis of RCC. However, suppression of LHPP promoted the proliferation and metastasis of RCC. In conclusion, our results presented the important role of LHPP in the development and progression of RCC.

MiR-30d-5p suppresses proliferation and autophagy by targeting ATG5 in renal cell carcinoma.

Previous reports have shown that miR-30d-5p functions as a tumor suppressor in prostate cancer and gallbladder carcinoma, but its role in renal cell carcinoma (RCC) remains elusive. This study was designed to explore the functional role of miR-30d-5p in proliferation and autophagy of RCC. Our results show that miR-30d-5p is significantly down-regulated in RCC tissues compared with normal tissues. miR-30d-5p overexpression suppressed cell proliferation, cell-cycle G1/S transition and autophagy, but promoted apoptosis in RCC cell lines (786-O and ACHN). Intriguingly, autophagy-related gene 5 (ATG5) was directly targeted by miR-30d-5p, as shown using luciferase reporter assay and biotin-avidin pull-down assay. Moreover, overexpression of ATG5 attenuated the inhibitory effect of miR-30d-5p on proliferation and autophagy in 786-O cells. These results suggest that miR-30d-5p suppresses proliferation and autophagy in RCC cells by targeting ATG5, and this pathway may be a suitable basis for the design of novel cancer therapeutics.

Prognostic role of macrophage migration inhibitory factor in patients with clear cell renal cell carcinoma.

Macrophage migration inhibitory factor (MIF) is a cytokine that mediates the interaction between malignant cells and the innate immune system. Recently, MIF has received attention for its role in tumorigenesis. We evaluated the prognostic role of MIF in clear cell renal cell carcinoma (CCRCC).A total of 152 patients, who underwent nephrectomy for CCRCC were enrolled in this study. Immunohistochemical staining of tissue microarray blocks containing 298 cores—2 cores per CCRCC patient was performed. The relationship between MIF expression and clinicopathological factors was evaluated. Total RNA and protein were extracted from 7 RCC (renal cell carcinoma) cell lines. MIF was knocked down in Caki-2 cells, and a wound healing assay was performed to evaluate migratory activity.Among the 298 cores, 180 (60.4%) were positive for MIF. Multivariate analysis, showed that, CCRCC patients with negative MIF expression exhibited poor disease-free survival (hazard ratio: 2.087, 95% confidence interval: 0.821–5.307, P value: .023) and poor disease-specific survival (hazard ratio: 2.101, 95% confidence interval: 1.009–4.374, P value: .047). The wound healing assay revealed that cell confluence was lower in MIF-deficient Caki-2 cells than in control cells.Negative MIF expression might be an independent prognostic marker for patients with CCRCC.

Parthenolide inhibits the tumor characteristics of renal cell carcinoma.

Parthenolide has been demonstrated to have anticancer effects against various types of cancer. However, the functional role of parthenolid has yet to be clearly reported in renal cell carcinoma (RCC). The aim of the present study was to investigate the effect of parthenolide in RCC 786‑O and ACHN cells. CCK‑8 and colony‑formation assays were used to observe the proliferation of RCC 786‑O and ACHN cells. Migration and invasion abilities were assessed through Transwell assays. The stem cell‑like properties of RCC cell lines were evaluated by mammosphere formation assay. Western blot analysis was used to investigate the metastasis and epithelial‑mesenchymal transition (EMT) induced by parthenolide on the expression levels of MMP2, MMP9, E‑cadherin, N‑cadherin, vimentin and snail. The results revealed that when the cells were treated with various concentrations of parthenolide, the rate of proliferation and growth was decreased in 786‑O and ACHN cells. The number of invasive cells in a field was approximately 170, 90, 40 and 190, 150, 70 in 786‑O and ACHN cells with 0, 4 and 8µM of parthenolide treatment. MMP‑2/‑9 expression (P<0.05) was inhibited by parthenolide. The protein levels of E‑cadherin were increased (P<0.05) and N‑cadherin, vimentin and snail were decreased (P<0.05) by parthenolide treatment. In addition, Parthenolide inhibited the expression of cancer stem cell markers and the PI3K/AKT pathway. The present study confirmed that parthenolide inhibited RCC cell proliferation and metastasis and suppressed the stem cell‑like properties of RCC cell lines, which could be a potential strategy to treat RCC. However, further molecular mechanisms of parthenolide in RCC should be observed and reported in the future.

MiR-106a-5p Functions as a Tumor Suppressor by Targeting VEGFA in Renal Cell Carcinoma

MicroRNAs (miRNAs) regulate progression of different cancers. Nevertheless, there is limited information regarding the role of miR-106a-5p in renal cell carcinoma (RCC). Herein, we demonstrate that miR-106a-5p levels are drastically decreased in clear cell RCC (ccRCC) tissues and cell lines, which subsequently contribute to a poor patient overall survival and a high tumor stage. By screening and analyzing, we found that miR-106a-5p directly targets the 3′-UTR of the VEGFA mRNA and led to a decrease in VEGFA. This process is important for tumor cells' growth and colony formation, and overexpression of miR-106a-5p can especially kill kidney tumor cells. Therefore, our data reveal that miR-106a-5p functions as a tumor suppressor by regulating VEGFA and ccRCC may be susceptible to miR-106a-5p therapy.

Role of Nuclear Claudin-4 in Renal Cell Carcinoma.

Claudin-4 (CLDN4) is a tight junction protein to maintain the cancer microenvironment. We recently reported the role of the CLDN4 not forming tight junction in the induction of epithelial-mesenchymal transition (EMT). Herein, we investigated the role of CLDN4 in renal cell carcinoma (RCC), focusing on CLDN4. CLDN4 expression in 202 RCCs was examined by immunostaining. CLDN4 phosphorylation and subcellular localization were examined using high metastatic human RCC SN12L1 and low metastatic SN12C cell lines. In 202 RCC cases, the CLDN4 expression decreased in the cell membrane and had no correlation with clinicopathological factors. However, CLDN4 was localized in the nucleus in 5 cases (2%), all of which were pT3. Contrastingly, only 6 of 198 nuclear CLDN4-negative cases were pT3. CLDN4 was found in the nuclear fraction of a highly metastatic human RCC cell line, SN12L1, but not in the low metastatic SN12C cells. In SN12L1 cells, phosphorylation of tyrosine and serine residues was observed in cytoplasmic CLDN4, but not in membranous CLDN4. In contrast, phosphorylation of serine residues was observed in nuclear CLDN4. In SN12L1 cells, CLDN4 tyrosine phosphorylation by EphA2/Ephrin A1 resulted in the release of CLDN4 from tight junction and cytoplasmic translocation. Furthermore, protein kinase C (PKC)-ε phosphorylated the CLDN4 serine residue, resulting in nuclear import. Contrarily, in SN12C cells that showed decreased expression of EphA2/Ephrin A1 and PKCε, the activation of EphA2/EphrinA1 and PKCε induced cytoplasmic and nuclear translocation of CLDN4, respectively. Furthermore, the nuclear translocation of CLDN4 promoted the nuclear translocation of Yes-associated protein (YAP) bound to CLDN4, which induced the EMT phenotype. These findings suggest that the release of CLDN4 by impaired tight junction might be a mechanism underlying the malignant properties of RCC. These findings suggest that the release of CLDN4 by impaired tight junction might be one of the mechanisms of malignant properties of RCC.

Investigation of ALLO-316: A Fratricide-Resistant Allogeneic CAR T Targeting CD70 As a Potential Therapy for the Treatment of AML

CD70, a member of the TNF superfamily, is a type II transmembrane glycoprotein that interacts with its receptor (CD27) to promote survival of primed T cells and leads to formation of effector and memory T cells. Expression of CD70 in normal tissues is restricted to activated T and B lymphocytes and mature dendritic cells. CD70 is also widely expressed in various malignancies, including renal cell carcinoma (RCC) and acute myeloid leukemia (AML). The restricted expression pattern of CD70 in normal tissues makes it an attractive target for cancer therapeutics. Adoptive transfer of T cells expressing chimeric antigen receptors (CARs) is an exciting new therapeutic modality showing great promise in hematologic malignancies. Approval of two CD19-targeting autologous CAR Ts, Kymriah® and Yescarta®, has been followed with promising results from BCMA autologous CAR T clinical trials, showing that activity can extend to other targets. We have previously described the functional screening of a library of anti-CD70 scFv-based CARs and the identification of lead CD70 allogeneic CAR T cells (AlloCAR TTM) with robust activity against RCC cell lines both in vitro and in vivo. Here, we evaluate the anti-tumor activity and safety of a lead CD70 AlloCAR T (ALLO-316) for the treatment of AML. CD70 expression was evaluated and detected on three AML cell lines and in six primary AML patient samples, with 5/6 patient samples showing expression on 24%-99% of cells. CD70 expression will be profiled in a broader subset of AML patients and preliminary data will be presented. Despite the expression of CD70 on activated T cells it was possible to generate CD70 AlloCAR T cells. No CD70 expression was observed on CAR T cells after generation, suggesting either cells are succumbing to fratricide or are being "masked" by the CAR. CD70 was also not detected on Jurkat cells expressing CARs and this data, in combination with results showing CAR expression is protective when overexpressed in RCC cells support the phenomenon of "masking". Cellectis' TALEN® gene-editing was used to inactivate the TRAC and CD52 loci with the intent to minimize the risk of graft-versus-host disease and to confer resistance to ALLO-647, an anti-CD52 antibody that can be used as part of the conditioning regimen to deplete host alloreactive immune cells potentially leading to increased persistence and efficacy of the infused allogeneic cells. ALLO-316 cells were highly effective at lysing CD70-expressing target cells and eliminated greater than 99% of cells at the high effector to target (E:T) ratio and were unable to lyse AML cells in which CD70 was knocked out. Moreover, ALLO-316 cells were able to kill primary AML blasts with CD70 expression ex vivo. An orthotopic in vivo model utilizing the AML cell line MV4-11 was developed and anti-tumor activity was observed. In addition to evaluating efficacy against AML cell lines and tumors we also explored potential toxicity liabilities related to ALLO-316 treatment. Previous studies have reported that certain AML tumor antigens can also be expressed on normal hematopoietic progenitors and such expression could potentially lead to toxicity with targeted therapeutics. No detectable CD70 was observed by flow cytometry on purified CD34+ cells from 14 healthy donors. Taken together, our results support clinical development of CD70 AlloCAR T therapy for the treatment of AML. Disclosures Srinivasan: Allogene Therapeutics: Current Employment. Tan:Allogene Therapeutics: Current Employment. Cheng:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Zhang:Allogene Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tacheva-Grigorova:Allogene Therapeutics: Current Employment, Current equity holder in publicly-traded company. Van Blarcom:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Sommer:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Nguyen:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Sasu:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Panowski:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company.

Upregulated circPDK1 Promotes RCC Cell Migration and Invasion by Regulating the miR-377-3P-NOTCH1 Axis in Renal Cell Carcinoma.

BackgroundCircular RNAs (circRNAs) are novel clusters of endogenous noncoding RNAs (ncRNAs) that are involved in the regulation of multiple biological processes in diverse types of cancers. However, the roles and precise mechanisms of circRNAs in renal cell carcinoma (RCC) occurrence and progression have not been clearly elucidated.MethodsWe identified the aberrantly expressed circRNAs in RCC by high-throughput RNA-seq assay and used qRT-PCR to test the expression level of circRNAs in RCC tissues. Loss-of-function experiments were executed to detect the biological roles of circPDK1 in the RCC cells both in vivo and in vitro. RNA Fish, luciferase reporter assays and Western blotting were used to explore the molecular mechanism of circPDK1 function. All data were expressed as the means ± standard error of the mean (SEM). Student’s t-test, one-way ANOVA, Cox regression, an LSD-t-test, Pearson’s chi-squared test, a Log-rank test, and linear regression analyses were used to evaluate the group differences. P < 0.05 was considered significant.ResultsCircPDK1 was overexpressed in RCC tissues and positively associated with patient tumor metastasis and renal cell invasion. The in vivo functional assays also revealed that circPDK1 drove RCC xenograft metastasis. CircPDK1 was mainly located in the cytoplasm, serving as a sponge of miR-377-3P to regulate RCC invasion and metastasis through NOTCH1 (Notch Homolog 1). Ectopic express of NOTCH1 in RCC cell lines will block the metastasis inhibition effect after circPDK1 knockdown.ConclusionCircPDK1 is aberrantly expressed in RCC and promotes the metastasis of RCC cells mainly through sponging miR-377-3P and reducing its negative regulation of NOTCH1. Thus, circPDK1 may act as a therapeutic target and biomarker for RCC.

Downregulation of long non-coding RNA DUXAP10 inhibits proliferation, migration, and invasion of renal cell carcinoma.

Renal cell carcinoma (RCC) is the most common kidney malignancy that frequently leads to metastasis. Increasing evidence has shown that long non-coding RNAs (lncRNAs) play crucial roles affecting the progression of RCC. The role of lncRNA DUXAP10 in the evolution of RCC has not been defined yet. This project was designed to clarify the effects of DUXAP10 on the proliferation and tumorigenesis of RCC. We examined the expression of DUXAP10 in the Cancer Genome Atlas (TCGA) and ONCOMINE oncology databases. Then, we performed quantitative reverse-transcription polymerase chain reaction (qRT-PCR) to evaluate DUXAP10 expression in human RCC tissues and cell lines. The correlation between the expression of DUXAP10 and clinical characteristics of RCC patients was analyzed by univariate and Kaplan-Meier analyses. To unveil the biological function of DUXAP10 in cell cycle progression, cell growth, and invasion of RCC, we conducted knockdown experiments in vitro. qRT- PCR and western blotting assays were performed to further investigate the function of DUXAP10 in cancer biology. The data from TCGA showed that the expression of DUXAP10 was upregulated in tissues of RCC compared with normal tissues. Moreover, ONCOMINE database analysis indicated that high DUXAP10 levels were correlated with high clinical stages, inferior TNM classification, and poor overall survival. Furthermore, the results indicated that knockdown of DUXAP10 remarkably inhibited the RCC cell growth, mobility, and invasion, in association with the upregulation of E-cadherin and downregulation of cyclin D, cyclin E, CDK4, N-cadherin, and vimentin. Our findings highlight the oncogenic role of DUXAP10 in RCC and that DUXAP10 may serve as a novel predictive biomarker and therapeutic target for RCC.

Expression Profile of S100A2 and its Clinicopathological Significance in Renal Cell Carcinoma.

Dysregulated expression of S100 protein family members, including S100A2, has been reported in various types of human malignant tumors; however, the functional role of S100A2 in renal cell carcinoma (RCC) remains unknown. The objective of this study was to assess the S100A2 expression pattern and its clinicopathological significance in RCC. This study investigated the expression profiles of S100A2 mRNA, S100A2 protein and p53 mRNA in addition to S100A2 DNA methylation levels in 3 human RCC cell lines and 81 surgically resected RCC specimens. These findings were analyzed according to several clinicopathological parameters. In all RCC cell lines, both S100A2 mRNA and protein were barely detectable, and the S100A2 promoter was considered to be methylated. S100A2 mRNA expression levels in RCC tissues were markedly lower than those in normal kidney tissues. The rate in clear cell RCC (ccRCC) specimens with higher expression of S100A2 mRNA was significantly lower than that in non-ccRCC specimens (29.9 versus 71.4%, respectively). Furthermore, S100A2 expression in ccRCC specimens was significantly correlated with p53 mRNA expression, but not conventional clinicopathological parameters. These findings suggest limited roles of S100A2 in the progression of RCC, particularly ccRCC.

Artesunate Inhibits Growth of Sunitinib-Resistant Renal Cell Carcinoma Cells through Cell Cycle Arrest and Induction of Ferroptosis.

Simple SummaryRenal cell carcinoma (RCC) is the most common kidney malignancy. Due to development of therapy resistance, efficacy of conventional drugs such as sunitinib is limited. Artesunate (ART), a drug originating from Traditional Chinese Medicine, has exhibited anti-tumor effects in several non-urologic tumors. ART inhibited growth, reduced metastatic properties, and curtailed metabolism in sunitinib-sensitive and sunitinib–resistant RCC cells. In three of four tested cell lines, ART’s growth inhibitory effects were accompanied by cell cycle arrest and modulation of cell cycle regulating proteins. In a fourth cell line, KTCTL-26, ART evoked ferroptosis, an iron-dependent cell death, and exhibited stronger anti-tumor effects than in the other cell lines. The regulatory protein, p53, was only detectable in the KTCTL-26 cells, possibly making p53 a predictive marker of cancer that may respond better to ART. ART, therefore, may hold promise as an additive therapy option for selected patients with advanced or therapy-resistant RCC.Although innovative therapeutic concepts have led to better treatment of advanced renal cell carcinoma (RCC), efficacy is still limited due to the tumor developing resistance to applied drugs. Artesunate (ART) has demonstrated anti-tumor effects in different tumor entities. This study was designed to investigate the impact of ART (1–100 µM) on the sunitinib-resistant RCC cell lines, Caki-1, 786-O, KTCTL26, and A-498. Therapy-sensitive (parental) and untreated cells served as controls. ART’s impact on tumor cell growth, proliferation, clonogenic growth, apoptosis, necrosis, ferroptosis, and metabolic activity was evaluated. Cell cycle distribution, the expression of cell cycle regulating proteins, p53, and the occurrence of reactive oxygen species (ROS) were investigated. ART significantly increased cytotoxicity and inhibited proliferation and clonogenic growth in both parental and sunitinib-resistant RCC cells. In Caki-1, 786-O, and A-498 cell lines growth inhibition was associated with G0/G1 phase arrest and distinct modulation of cell cycle regulating proteins. KTCTL-26 cells were mainly affected by ART through ROS generation, ferroptosis, and decreased metabolism. p53 exclusively appeared in the KTCTL-26 cells, indicating that p53 might be predictive for ART-dependent ferroptosis. Thus, ART may hold promise for treating selected patients with advanced and even therapy-resistant RCC.

Dual inhibition of DNA-PKcs and mTOR by CC-115 potently inhibits human renal cell carcinoma cell growth.

CC-115 is a dual inhibitor of DNA-PKcs and mTOR, both are valuable therapeutic targets for renal cell carcinoma (RCC). Our results showed that CC-115 inhibited survival and proliferation of established RCC cell lines (786-O and A489) and primary human RCC cells. The dual inhibitor induced selective apoptosis activation in RCC cells, as compared to no cytotoxicity nor apoptotic effects toward normal renal epithelial cells. CC-115 inhibited DNA-PKcs and mTORC1/2 activation in RCC cells. It was however ineffective in DNA-PKcs-mTOR double knockout (DKO) 786-O cells. CC-115 induced feedback autophagy activation in RCC cells. Autophagy inhibitors or Beclin-1/Light chain 3 (LC3) silencing potentiated CC-115-induced anti-RCC cell activity. Conversely, ectopic overexpression of Beclin-1 inhibited CC-115-induced cytotoxicity. At last CC-115 oral administration inhibited 786-O subcutaneous xenograft growth in nude mice. Taken together, dual inhibition of DNA-PKcs and mTOR by CC-115 potently inhibited RCC cell growth.

Role of Tissue Transglutaminase Catalytic and Guanosine Triphosphate-Binding Domains in Renal Cell Carcinoma Progression.

Tissue transglutaminase (TG2) is a multifunctional protein that can act as a cross-linking enzyme, GTPase/ATPase, protein kinase, and protein disulfide isomerase. TG2 is involved in cell adhesion, migration, invasion, and growth, as well as epithelial–mesenchymal transition (EMT). Our previous findings indicate that the increased expression of TG2 in renal cell carcinoma (RCC) results in tumor metastasis with a significant decrease in disease- and cancer-specific survival outcome. Given the importance of the prometastatic activity of TG2 in RCC, in the present study, we aim to investigate the relative contribution of TG2’s transamidase and guanosine triphosphate (GTP)-binding/GTPase activity in the cell migration, invasion, EMT, and cancer stemness of RCC. For this purpose, the mouse RCC cell line RenCa was transduced with wild-type-TG2 (wt-TG2), GTP-binding deficient-form TG2-R580A, transamidase-deficient form with low GTP-binding affinity TG2-C277S, and transamidase-inactive form TG2-W241A. Our results suggested that predominantly, GTP-binding activity of TG2 is responsible for cell migration and invasion. In addition, CD marker analysis and spheroid assay confirmed that GTP binding/GTPase activity of TG2 is important in the maintenance of mesenchymal character and the cancer stem cell profile. These findings support a prometastatic role for TG2 in RCC that is dependent on the GTP binding/GTPase activity of the enzyme.

Long noncoding RNA SNHG4 promotes renal cell carcinoma tumorigenesis and invasion by acting as ceRNA to sponge miR-204-5p and upregulate RUNX2

BackgroundLong noncoding RNAs (lncRNAs) are involved in the tumorigenesis and progression of human cancers, including renal cell carcinoma (RCC). Small nucleolar RNA host gene 4 (SNHG4) is reported to play an essential role in tumor growth and progression. However, the molecular mechanisms and function of SNHG4 in RCC remain undocumented.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) was performed to examine expression levels of SNHG4 in RCC tissue samples and cell lines. Cell counting kit-8, western blotting, activities of caspase-3, -8, and -9, wound-healing, and transwell invasion assays were performed to explore cell proliferation, apoptosis, migration, and invasion. The interaction among SNHG4, miR-204-5p, and RUNX2 was verified by bioinformatic analysis, a luciferase gene report, qRT-PCR, western blot analysis, and RNA immunoprecipitation assays. Xenograft mouse models were carried out to examine the role of SNHG4 in RCC in vivo.ResultsSNHG4 was highly expressed in RCC tissue samples and cell lines, and its upregulation was significantly involved in node involvement, distant metastasis, and reduced overall and relapse-free survival of patients with RCC. SNHG4 acted as an oncogenic lncRNA with promoted RCC cell proliferation, migration, invasion, and inhibited apoptosis. SNHG4 boosted tumor growth in xenograft mouse models. Mechanistically, SNHG4 functioned as a competing endogenous RNA (ceRNA) for sponging miR-204-5p, leading to the upregulation of its target RUNX2 to promote RCC cell proliferation and invasion.ConclusionSNHG4 and miR-204-5p might be indicated in RCC progression via RUNX2, suggesting the potential use of SNHG4/miR-204-5p/RUNX2 axis in RCC treatment.