Growth Of Renal Cancer Cells Research Articles

Methuosis Inducer SGI-1027 Cooperates with Everolimus to Promote Apoptosis and Pyroptosis by Triggering Lysosomal Membrane Permeability in Renal Cancer.

The mTOR inhibitor everolimus has been approved as a sequential or second-line therapy for renal cell carcinoma (RCC). However, the development of drug resistance limits its clinical applications. This study aims to address the challenge of everolimus resistance and provide new insights into the treatment of advanced RCC. Here, the cytotoxicity of the DNA methyltransferase 1 (DNMT1) inhibitor SGI-1027 in inducing cell vacuolation and methuosis is discovered and demonstrated for the first time. Additionally, SGI-1027 exerts synergistic effects with everolimus, as their combination suppresses the growth, migration, and invasion of renal cancer cells. Mechanistically, apoptosis and GSDME-dependent pyroptosis triggered by lysosomal membrane permeability (LMP) are observed. The upregulation of GSDME expression and increased lysosomal activity in renal cancer cells provide a therapeutic window for the combination of these two drugs to treat renal cancer. The combination treatment exhibits effective anti-tumor activity and is well tolerated in a subcutaneous tumor model. Overall, this study validates and reveals the specific cytotoxicity property of SGI-1027 and its potent synergistic effect with everolimus, offering new insights into advanced RCC therapy and everolimus-resistance overcoming.

Ketogenic diet enhances the anti-cancer effects of PD-L1 blockade in renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is characterized by a predominant metabolic reprogramming triggering energy production by anaerobic glycolysis at the expense of oxydative phosphorylation. Ketogenic diet (KD), which consists of high fat and low carbohydrate intake, could bring required energy substrates to healthy cells while depriving tumor cells of glucose. Our objective was to evaluate the effect of KD on renal cancer cell tumor metabolism and growth proliferation. Growth cell proliferation and mitochondrial metabolism of ACHN and Renca renal carcinoma cells were evaluated under ketone bodies (KB) exposure. In vivo studies were performed with mice (nude or Balb/c) receiving a xenograft of ACHN cells or Renca cells, respectively, and were then split into 2 feeding groups, fed either with standard diet or a 2:1 KD ad libitum. To test the effect of KD associated to immunotherapy, Balb/c mice were treated with anti-PDL1 mAb. Tumor growth was monitored. In vitro, KB exposure was associated with a significant reduction of ACHN and Renca cell proliferation and viability, while increasing mitochondrial metabolism. In mice, KD was associated with tumor growth reduction and PDL-1 gene expression up-regulation. In Balb/c mice adjuvant KD was associated to a better response to anti-PDL-1 mAb treatment. KB reduced the renal tumor cell growth proliferation and improved mitochondrial respiration and biogenesis. KD also slowed down tumor growth of ACHN and Renca in vivo. We observed that PDL-1 was significantly overexpressed in tumor in mice under KD. Response to anti-PDL-1 mAb was improved in mice under KD. Further studies are needed to confirm the therapeutic benefit of adjuvant KD combined with immunotherapy in patients with kidney cancer.

Von Hippel Lindau tumor suppressor controls m6A-dependent gene expression in renal tumorigenesis.

N6-Methyladenosine (m6A) is the most abundant posttranscriptional modification, and its contribution to cancer evolution has recently been appreciated. Renal cancer is the most common adult genitourinary cancer, approximately 85% of which is accounted for by the clear cell renal cell carcinoma (ccRCC) subtype characterized by VHL loss. However, it is unclear whether VHL loss in ccRCC affects m6A patterns. In this study, we demonstrate that VHL binds and promotes METTL3/METTL14 complex formation while VHL depletion suppresses m6A modification, which is distinctive from its canonical E3 ligase role. m6A RNA immunoprecipitation sequencing (RIP-Seq) coupled with RNA-Seq allows us to identify a selection of genes whose expression may be regulated by VHL-m6A signaling. Specifically, PIK3R3 is identified to be a critical gene whose mRNA stability is regulated by VHL in a m6A-dependent but HIF-independent manner. Functionally, PIK3R3 depletion promotes renal cancer cell growth and orthotopic tumor growth while its overexpression leads to decreased tumorigenesis. Mechanistically, the VHL-m6A-regulated PIK3R3 suppresses tumor growth by restraining PI3K/AKT activity. Taken together, we propose a mechanism by which VHL regulates m6A through modulation of METTL3/METTL14 complex formation, thereby promoting PIK3R3 mRNA stability and protein levels that are critical for regulating ccRCC tumorigenesis.

BIRC6 Modulates the Protein Stability of Axin to Regulate the Growth, Stemness, and Resistance of Renal Cancer Cells via the β-Catenin Pathway.

The mechanism underlying the development of renal cell carcinoma (RCC) remains unclear, and effective prevention and therapeutic measures are lacking. BIRC6, a protein inhibitor of apoptosis, has attracted great interest. Our data indicated that overexpression of BIRC6 elevated cell growth, colony formation, migration, and invasion of cultured RCC cells, while siRNA knockdown of BIRC6 suppressed these processes. Additionally, BIRC6 was highly expressed in RCC clinical samples along with a downregulated level of Axin. Immunoprecipitation assays found that BIRC6 interacted with Axin and the two proteins colocalized within the cytoplasm of RCC cells. Overexpression of BIRC6 promoted the ubiquitination modification of Axin, while genetic knockdown of BIRC6 suppressed it. Furthermore, overexpression of BIRC6 significantly promoted the turnover of Axin, suggesting BIRC6's inhibitory effect on Axin protein stability. BIRC6 was also upregulated in cancer stem-like cells of RCC and increased the drug resistance of RCC cells against sunitinib. Western blotting assays showed that the overexpression of BIRC6 upregulated CXCR4 protein expression and activated the β-catenin pathway. Two cell lines were then constructed with BIRC6 overexpressed by lentiviruses. Pharmacological administration of a Wnt/β-catenin inhibitor, XAV-939, or genetic knockdown of β-catenin inhibited cell growth, tumor sphere formation, colony formation, migration, and invasion of BIRC6-overexpressed cells. In vivo administration of XAV-939 markedly suppressed the tumorigenesis of BIRC6-overexpressed RCC cells in nude mice. In conclusion, we propose that BIRC6 activates the β-catenin signaling pathway via mediating the ubiquitination and degradation of Axin, promoting the growth, stemness, and drug resistance of RCC cells. This project aims to elucidate the role of BIRC6 as a potential therapeutic target and provide new insights into the clinical treatment of RCC.

A novel combination therapy with Cabozantinib and Honokiol effectively inhibits c-Met-Nrf2-induced renal tumor growth through increased oxidative stress

Receptor tyrosine kinase (RTK), c-Met, is overexpressed and hyper active in renal cell carcinoma (RCC). Most of the therapeutic agents mediate cancer cell death through increased oxidative stress. Induction of c-Met in renal cancer cells promotes the activation of redox-sensitive transcription factor Nrf2 and cytoprotective heme oxygenase-1 (HO-1), which can mediate therapeutic resistance against oxidative stress. c-Met/RTK inhibitor, Cabozantinib, has been approved for the treatment of advanced RCC. However, acquired drug resistance is a major hurdle in the clinical use of cabozantinib. Honokiol, a naturally occurring phenolic compound, has a great potential to downregulate c-Met-induced pathways. In this study, we found that a novel combination treatment with cabozantinib + Honokiol inhibits the growth of renal cancer cells in a synergistic manner through increased production of reactive oxygen species (ROS); and it significantly facilitates apoptosis-and autophagy-mediated cancer cell death. Activation of c-Met can induce Rubicon (a negative regulator of autophagy) and p62 (an autophagy adaptor protein), which can stabilize Nrf2. By utilizing OncoDB online database, we found a positive correlation among c-Met, Rubicon, p62 and Nrf2 in renal cancer. Interestingly, the combination treatment significantly downregulated Rubicon, p62 and Nrf2 in RCC cells. In a tumor xenograft model, this combination treatment markedly inhibited renal tumor growth in vivo; and it is associated with decreased expression of Rubicon, p62, HO-1 and vessel density in the tumor tissues. Together, cabozantinib + Honokiol combination can significantly inhibit c-Met-induced and Nrf2-mediated anti-oxidant pathway in renal cancer cells to promote increased oxidative stress and tumor cell death.

Role of ATP Binding Cassette Subfamily A Member 1 (ABCA1) in Chemoradiotherapy-induced Renal Injury

Radiation therapy (RT) alone or combined with chemotherapy reduces cholesterol efflux and causes chronic kidney disease (CKD). There is no treatment for CKD except renal replacement strategies such as dialysis or transplantation. We hypothesize that cancer treatment-induced ABCA1 deficiency leads to dysregulation of cholesterol metabolism, making podocytes susceptible and cancerous cells resistant to treatment-induced injuries. To quantify the cell index of LNCaP, 786-0, and Podcytes were grown in an e-plate reader (Roche) for 24 h and then were treated with either enzalutamide (10µM) or sunitinib (1.0 µM) or cyclophosphamide (10µM) with or without RT (4Gy) or ABCA1 inducer (liver-X-receptors agonist (GW3965; Sigma; 10µM). Podocyte cholesterol efflux assay was measured using NBD-cholesterol efflux assay, and expression of ABCA1 and cleaved caspase-3 were measured by western blotting. 10-14-week-old C57BL/6 male and female mice were given bilateral kidney X-irradiation of 4Gy with or without cisplatin (3mg/kg; IV). Functional kidney parameters, histopathological changes, and ultrastructural changes were measured at baseline and 20 weeks after treatment. GFR was measured using a FITC-sinistrin-based transdermal monitor. In vitro studies suggest that after radiation exposure (4Gy and 8Gy), ABCA1 expression decreases in a dose and time-dependent manner in cultured podocytes (p < 0.05), which coincides with reduced cholesterol efflux (p < 0.05) and increased podocyte apoptosis (p < 0.01). In contrast, LXR agonist treatment in vitro prevents reduced ABCA1 expression and cholesterol efflux and prevents podocytes from radiation-induced apoptosis. Our in vitro studies further suggest that Enzalutamide treatment alone reduces the growth of LNCaP and podocytes, and RT (5Gy) plus enzalutamide combination treatment further decreases the growth of LNCaP and podocytes. However, LXR agonist treatment further increased the efficacy of enzalutamide and RT treatment against LNCaP and increased podocyte growth simultaneously. Similarly, Sunitinib with or without RT reduces the growth of renal cancer cells (786-0) and podocytes. LXR agonist treatment further increases the efficacy of the sunitinib and RT against 786-0 cells and simultaneously increases podocyte growth. Cyclophosphamide reduces the growth of the podocytes, and LXR agonist treatment improves the podocyte growth after CP treatment. In vivo studies observed that focal bilateral kidney irradiation with or without cisplatin of C57BL/6 mice induces lipid accumulation in the kidney cortex and increases GBM thickness (p < 0.001), which correlates with decreased Abca1 expression, podocyte number, and GFR. This study shows that LXR agonist treatment protects podocytopathy in vitro and chemotherapy or radiotherapy-induced kidney injury in vivo. ABCA1 may be an important therapeutic target for chemoradiotherapy-induced kidney injuries in cancer patients.

MiR-4284 Functions as a Tumor Suppressor in Renal Cell Carcinoma Cells by Targeting Glutamate Decarboxylase 1.

MicroRNAs (miRNAs) play a crucial role as oncogenic or tumor suppressors in the pathogenesis and progression of tumors. However, few studies have investigated the exact role of miR-4284 in renal cell carcinoma (RCC). We aimed to investigate the role of miR-4284 as a tumor suppressor in renal cancer cell lines. A498 and Caki-1 were transfected with miR-4284. The Cell Counting Kit-8, colony formation, apoptosis assays, and quantitative reverse transcription-polymerase chain reaction were used to evaluate tumor growth-inhibiting functions. The wound-healing, transwell, and sphere-formation assays were conducted to investigate tumorigenic characteristics. The potential target genes of miR-4284 were predicted and experimentally verified. A xenograft experiment was performed to estimate the tumor-growth-suppressive function of miR-4284. miR-4284 overexpression suppressed proliferation, induced apoptosis, and suppressed tumorigenic features of renal cancer cells. Glutamate decarboxylase 1 (GAD1) was directly targeted by miR-4284. A xenograft mouse model injected with Caki-1 cells transfected with miR-4284 showed significantly decreased tumor growth rate and volume. miR-4284 affected tumor growth, metastasis, and apoptosis of renal cancer cells in vitro and in vivo. These findings highlight the potential of miR-4284 as a target for anticancer miRNA therapeutics in RCC.

Brusatol inhibits the growth of renal cell carcinoma by regulating the PTEN/PI3K/AKT pathway

Ethnopharmacological relevanceBrucea javanica (L.) Merr. is a medicinal herb used in China for the prevention and treatment of diseases such as cancer and malaria. Brusatol was isolated from the seeds of Brucea javanica (L.) Merr, brusatol has a wide range of pharmacological effects, including anti-inflammation and anti-cancer effects. Aim of the studyRenal cell carcinoma is one of the most common urinary system tumours and seriously threatens the lives of patients. We aimed to study the mechanism by which brusatol regulates the growth of renal cancer cells through the PTEN/PI3K/AKT signalling pathway. Materials and methodsWe chose the A498, ACHN, and OSRC-2 cell lines as experimental models. After intervention with brusatol, CCK-8 experiments and plate cloning experiments were used to detect the cell proliferation ability; flow cytometry was used to detect the cell apoptosis rate; scratch and transwell invasion assays were used to detect the cell migration and invasion ability; qRT-PCR and Western blotting was used to detect PTEN, p-PI3K/PI3K, p-AKT/AKT, Bax, Bcl2, E-cadherin, N-cadherin, and vimentin relative expression. Then, we knocked down the PTEN gene in the three cell lines and again tested the proliferation, apoptosis, migration, and invasion capabilities of each group of cells. ResultsBrusatol significantly inhibited the proliferation, migration and invasion and increased the rate of apoptosis of the A498, ACHN, and OSRC-2 cell lines, and brusatol significantly increased the expression of PTEN mRNA and protein, and inhibited the expression of p-PI3K and p-AKT. Moreover, knockdown of PTEN significantly reduced the inhibitory effect of brusatol on the growth of renal cancer cells. ConclusionOur research results show that brusatol has an effective inhibitory effect on the growth of A498, ACHN, and OSRC-2 renal cancer cell lines, and this effect is likely to be produced by regulating the PTEN/PI3K/AKT signalling pathway.

Isothiocyanate From Moringa oleifera Seeds Inhibits the Growth and Migration of Renal Cancer Cells by Regulating the PTP1B-dependent Src/Ras/Raf/ERK Signaling Pathway.

Moringa oleifera Lam. is a tropical and subtropical plant that has been used for centuries as both food and traditional medicine. 4-[(α-L-Rhamnosyloxy) benzyl] isothiocyanate (MIC-1) is an active substance in M. oleifera, with anti-cancer activity. However, whether MIC-1 exerts anti-renal cancer effects is unknown. Therefore, the aim of the present study was to evaluate the effects of MIC-1 on the growth and migration of renal cell carcinoma (RCC) cells and to identify the putative underlying mechanism. We found that, among 30 types of cancer cells, MIC-1 exerted the strongest growth inhibitory effects against 786-O RCC cells. In addition, MIC-1 (10 μM) significantly inhibited the growth of five RCC cell lines, including 786-O, OSRC-2, 769-P, SK-NEP-1, and ACHN cells, but was not toxic to normal renal (HK2) cells. Also, MIC-1 suppressed 786-O and 769-P cell migration and invasion abilities, and reduced the expression of matrix metalloproteinase (MMP)-2 and MMP-9. Furthermore, MIC-1 induced apoptosis and cell cycle arrest, increased Bax/Bcl-2 ratio, and decreased cell cycle-related protein expression in 786-O cells and 769-P cells. Molecular docking and small-molecule interaction analyses with PTP1B both showed that MIC-1 inhibited PTP1B activity by binding to its active site through hydrogen bonding and hydrophobic interactions. Additionally, MIC-1 could suppress the growth and migration of 786-O cells by inhibiting PTP1B-mediated activation of the Src/Ras/Raf/ERK signaling pathway. In vivo experiments further showed that MIC-1 markedly inhibited the growth of xenograft tumors in mice, and greatly increased Bax/Bcl-2 ratio in tumor tissues. In addition, MIC-1 had no effect on the PTP1B-dependent Src/Ras/Raf/ERK signaling pathway in HCT-116 cells, Hep-G2 cells, and A431 cells. Overall, our data showed that MIC-1 could be a promising, non-toxic, natural dietary supplement for the prevention and treatment of renal cancer.

TRIM27 interacts with I\u03bab\u03b1 to promote the growth of human renal cancer cells through regulating the NF-\u03baB pathway

BackgroundThe tripartite motif (TRIM) family proteins exhibit oncogenic roles in various cancers. The roles of TRIM27, a member of the TRIM super family, in renal cell carcinoma (RCC) remained unexplored. In the current study, we aimed to investigate the clinical impact and roles of TRIM27 in the development of RCC.MethodsThe mRNA levels of TRIM27 and Kaplan–Meier survival of RCC were analyzed from The Cancer Genome Atlas database. Real-time PCR and Western blotting were used to measure the mRNA and protein levels of TRIM27 both in vivo and in vitro. siRNA and TRIM27 were exogenously overexpressed in RCC cell lines to manipulate TRIM27 expression.ResultsWe discovered that TRIM27 was elevated in RCC patients, and the expression of TRIM27 was closely correlated with poor prognosis. The loss of function and gain of function results illustrated that TRIM27 promotes cell proliferation and inhibits apoptosis in RCC cell lines. Furthermore, TRIM27 expression was positively associated with NF-κB expression in patients with RCC. Blocking the activity of NF-κB attenuated the TRIM27-mediated enhancement of proliferation and inhibition of apoptosis. TRIM27 directly interacted with Iκbα, an inhibitor of NF-κB, to promote its ubiquitination, and the inhibitory effects of TRIM27 on Iκbα led to NF-κB activation.ConclusionsOur results suggest that TRIM27 exhibits an oncogenic role in RCC by regulating NF-κB signaling. TRIM27 serves as a specific prognostic indicator for RCC, and strategies targeting the suppression of TRIM27 function may shed light on future therapeutic approaches.

Bluetongue Viruses Act as Novel Oncolytic Viruses to Effectively Inhibit Human Renal Cancer Cell Growth In Vitro and In Vivo.

BackgroundThe bluetongue virus (BTV) is the prototype virus in the genus Orbivirus within the family Reoviridae. Recent studies indicate that BTVs are capable of infecting and selectively lysing human hepatic carcinoma cells (Hep-3B) and prostate carcinoma cells (pc-3). This study was designed to evaluate the oncolytic potential of BTV in experimental models of human renal cancer in vitro and in vivo.Material/MethodsFive human renal cancer cell lines, ACHN, CAKI-1, OS-RC-2, 786-O, and A498, were used in this study to analyze BTV replication. These cells were lysed by oncolysis compared to normal control. Xenograft models were used to assess the efficacy and toxicity of BTVs in vivo. Data were analyzed by one-way ANOVA or two-sided unpaired t tests.ResultsThe results showed HPTEC cells to be relatively resistant to cytotoxic effects of BTVs and exhibited normal growth rate even at high dose of BTVs. Nonetheless, the renal cancer cells showed a remarkably higher sensitivity to BTVs. Moreover, the ultramicroscopic subcellular changes were also detected in the renal cells. The viral particles were observed in all the RCC cell lines, but not in HPTEC cells. Intratumoral injections of BTVs significantly decreased the tumor volume as compared to animals that received no virus treatment. Infection with BTVs significantly increased the percentage of apoptotic renal cancer cells but not the HPTEC cells. Moreover, BTV triggered apoptosis in renal cancer cells via a mitochondria-mediated pathway.ConclusionsThis study for the first time demonstrated the oncolytic potential of BTV in experimental models of human renal cancer. BTV exhibits the potential to inhibit human renal cancer cell growth in vitro and in vivo.

Emodin Induced Necroptosis and Inhibited Glycolysis in the Renal Cancer Cells by Enhancing ROS

Renal cell carcinoma (RCC) is a tumor with unpredictable presentation and poor clinical outcome. RCC is always resistant to chemotherapy and radiation, and weakly sensitive to immunotherapeutic agents. Therefore, novel agents and approaches are urgently needed for the treatment of RCC. Emodin, an anthraquinone compound extracted from rhubarb and other traditional Chinese herbs, has been implicated in a wide variety of pharmacological effects, such as anti-inflammatory, antiviral, and antitumor activities. However, its role in RCC remains unknown. In this study, we found that emodin effectively killed renal cancer cells without significant toxicity to noncancerous cell HK-2. Flow cytometry assay with Annexin V-FITC and PI demonstrated that emodin induces necroptosis, but not apoptosis, in renal cancer cells. Meanwhile, the phosphorylation levels of RIP1 and MLKL, the key necroptosis-related proteins, were significantly increased. To explore how emodin inhibits kidney tumor growth, we tested reactive oxygen species (ROS) levels and found that the levels of ROS increased upon emodin treatment in a dose-dependent manner. Further studies demonstrated that emodin induces necroptosis through ROS-mediated activation of JNK signaling pathway and also inhibits glycolysis by downregulation of GLUT1 through ROS-mediated inactivation of the PI3K/AKT signaling pathway. Our findings revealed the potential mechanisms by which emodin suppresses renal cancer cell growth and will help develop novel therapeutic approaches for patients with JNK- or PI3K/AKT-dysregulated renal cancer.

Novel Honokiol-eluting PLGA-based scaffold effectively restricts the growth of renal cancer cells.

Renal Cell Carcinoma (RCC) often becomes resistant to targeted therapies, and in addition, dose-dependent toxicities limit the effectiveness of therapeutic agents. Therefore, identifying novel drug delivery approaches to achieve optimal dosing of therapeutic agents can be beneficial in managing toxicities and to attain optimal therapeutic effects. Previously, we have demonstrated that Honokiol, a natural compound with potent anti-tumorigenic and anti-inflammatory effects, can induce cancer cell apoptosis and inhibit the growth of renal tumors in vivo. In cancer treatment, implant-based drug delivery systems can be used for gradual and sustained delivery of therapeutic agents like Honokiol to minimize systemic toxicity. Electrospun polymeric fibrous scaffolds are ideal candidates to be used as drug implants due to their favorable morphological properties such as high surface to volume ratio, flexibility and ease of fabrication. In this study, we fabricated Honokiol-loaded Poly(lactide-co-glycolide) (PLGA) electrospun scaffolds; and evaluated their structural characterization and biological activity. Proton nuclear magnetic resonance data proved the existence of Honokiol in the drug loaded polymeric scaffolds. The release kinetics showed that only 24% of the loaded Honokiol were released in 24hr, suggesting that sustained delivery of Honokiol is feasible. We calculated the cumulative concentration of the Honokiol released from the scaffold in 24hr; and the extent of renal cancer cell apoptosis induced with the released Honokiol is similar to an equivalent concentration of direct application of Honokiol. Also, Honokiol-loaded scaffolds placed directly in renal cell culture inhibited renal cancer cell proliferation and migration. Together, we demonstrate that Honokiol delivered through electrospun PLGA-based scaffolds is effective in inhibiting the growth of renal cancer cells; and our data necessitates further in vivo studies to explore the potential of sustained release of therapeutic agents-loaded electrospun scaffolds in the treatment of RCC and other cancer types.

MiR-155-5p promotes metastasis and epithelial-mesenchymal transition of renal cell carcinoma by targeting apoptosis-inducing factor.

Although renal cell carcinoma remains one of the most malignant cancers, our understanding of progression and recurrence of this disease is limited. The present study explored the precise role of miR-155-5p in renal cancer metastasis. The expression of miR-155-5p in renal carcinoma clinical tissues and cells was determined using quantitative real time-polymerase chain reaction. The role of miR-155-5p on tumor cell growth were examined using CCK-8 and colony formation assays. Transwell assay was utilized to identify the role of miR-155-5p on the invasion and migration of renal cancer cells. Markers of epithelial-mesenchymal transition were determined using western blot. The in vivo effects of miR-155-5p on renal cancer cell growth, apoptosis, and metastasis were explored using xenograft mice. Luciferase reporter assay was performed to identify the potential target of miR-155-5p. Levels of miR-155-5p were significantly elevated in renal cancer tissues and cell lines. Suppression of miR-155-5p decreased the growth, colony formation, migration, and invasiveness of renal cancer cells. In contrast, overexpression of miR-155-5p led to opposite effects on renal cancer cells. Mechanically, the apoptosis-inducing factor was identified as the target of miR-155-5p. Interference of miR-155-5p significantly increased mRNA and protein expression of the apoptosis-inducing factor, whereas overexpression of miR-155-5p remarkably suppressed the apoptosis-inducing factor levels in renal cancer cells. The xenograft model identified that suppression of miR-155-5p restrained tumor growth and promoted apoptosis, whereas overexpression of miR-155-5p decreased apoptosis and accelerated tumor growth. Moreover, the number of lung metastasis nodules were decreased following injection with anti-miR-155-5p transfected cells, whereas the nodules were remarkably increased after overexpression of miR-155-5p. In addition, in vitro and in vivo assays both confirmed that suppression of miR-155-5p increased the expression of E-cadherin and decreased levels of N-cadherin and Snail, whereas overexpression of miR-155-5p accelerated epithelial-mesenchymal transition progression in renal cancer cells. These findings demonstrate that miR-155-5p enhances metastasis and epithelial-mesenchymal transition by targeting the apoptosis-inducing factor, suggesting that miR-155-5p represents a novel therapeutic target for renal cancer.

Induction of apoptosis in renal cell carcinoma by cinobufotalin through inhibition of Notch1 signal activation*

Abstract Objective The aim of this study was to investigate the effect of cinobufotalin on apoptosis in renal cell carcinoma and its possible mechanism of action. Methods The expression levels of Notch1 in renal cancer cells, as well as in adjacent and normal tissues were assessed in 64 patients with renal cellcarcinoma. The 769-P cells were treated with 0, 10, 20 and 40 mg/L cinobufotalin and the proliferation activity and apoptotic rate of the cells were measured. The expression levels of Notch1, Bcl-2, and Pro-caspase 3 were detected by RT-PCR and Western-blot. Results (1) The rates of Notch1 expression in renal cancer cells, adjacent tissues, and normal tissues were 75.0%, 45.3%, and 9.4%, respectively. Notch1 expression had significant effects on tumor, node and metastasis (TNM) staging, Fuhrman grade, and tumor size in patients with renal cell carcinoma (P &lt; 0.05); (2) The inhibition rates of cinobufotalin on 769-P cells were 0%, 6.85%, 11.37%, and 16.33% at 24 h; 0%, 13.57%, 20.14%, and 31.69% at 48 h; 0%, 19.97%, 28.53% and 51.42% at 72 h. At 24 h, the apoptotic rates were 8.2 ± 3.1%, 19.8 ± 5.6%, 33.7 ± 5.0%, and 51.5 ± 6.8%.The effect of cinobufotalin on apoptosis of 769-P cells was dose-dependent; (3) RT-PCR assay showed that protein expression levels for Notch1, Bcl-2, and Pro-caspase 3 were significantly decreased with the increase of drug concentration. Western-blot analysis also showed that Notch1, Bcl-2 and Pro-caspase 3 protein levels showed a significant downward trend with the increase of drug concentration. Conclusion Cinobufotalin inhibits the growth of renal cancer cells and induces apoptosis in renal cell carcinoma, which may be related to the inhibition of Notch1 signal activation.

A Novel Combination Treatment with Honokiol and Rapamycin Effectively Restricts c-Met-Induced Growth of Renal Cancer Cells, and also Inhibits the Expression of Tumor Cell PD-L1 Involved in Immune Escape.

The mTOR inhibitor Rapamycin has tumor inhibitory properties; and it is also used as an immunosuppressive agent after organ transplantation. However, prolonged Rapamycin treatment re-activates Akt and can promote cancer growth. Honokiol is a natural compound with both anti-tumorigenic and anti-inflammatory properties. Here, we assessed the anti-tumor effects of Rapamycin and Honokiol combination in renal cell carcinoma (RCC). Receptor tyrosine kinase c-Met-mediated signaling plays a major role in RCC growth. We observed that compared with Rapamycin alone, Rapamycin + Honokiol combination can effectively down-regulate c-Met-induced Akt phosphorylation in renal cancer cells; and it markedly inhibited Ras activation and cell proliferation and promoted G1 phase cell cycle arrest. The combination treatment significantly induced ROS generation and cancer cell apoptosis even when c-Met is activated. Importantly, Honokiol, but not Rapamycin, decreased c-Met-induced expression of the co-inhibitory molecule PD-L1, implied in the immune escape of renal cancer cells. In mouse renal cancer cells and Balb/c splenocytes co-culture assay, Rapamycin + Honokiol markedly potentiated immune-cell-mediated killing of cancer cells, possibly through the down-regulation of PD-L1. Together, Honokiol can effectively overcome the limitation of Rapamycin treatment alone; and the combination treatment can markedly restrict the growth of RCC, with particular importance to post-transplantation renal cancer.

Effects of miR-222 on cisplatin resistance of renal cancer cell strains and related mechanisms

Cisplatin is widely employed in combating renal cancers. However, a major challenge is the resistance of cisplatin by renal cancer cells. Increased miR-222 expression is known to promote this resistance and so ways of downregulating the expression of miR-222 is widely sought after as a means of combating the resistance. The study was performed to explore the effects of miR-222 on cisplatin resistance of renal cancer cell strains and related mechanisms. The expression of miR-222 and Dickkopf-3 (DKK3) was regulated to explore the effects of miR-222 and DKK3 on cisplatin chemotherapy in renal cancer cisplatin-resistant cell strains, and to figure out the regulatory relationship between miR-222 and DKK3. In renal cancer tissues and cell lines, miR-222 was highly expressed and DKK3 was lowly expressed. Renal cancer cisplatin-resistant cells had markedly higher miR-222 expression and lower DKK3 expression than normal renal cancer cells, suggesting a potential involvement of miR-222 and DKK3 in the cisplatin resistance in renal cancer cells. Down-regulated miR-222 expression led to stronger inhibition of the renal cancer cell growth by cisplatin and markedly higher DKK3 expression. Dual-luciferase reporter assay results showed that miR-222 had a targeted inhibition on DKK3. Co-transfection of miR-222 mimic and shDKK3, together with the up-regulation of miR-222 and DKK3 expressions, resulted in a higher sensitivity of renal cancer cells to cisplatin chemotherapy than the up-regulation of miR-222 expression alone. Down-regulated miR-222 expression can remove the inhibition of DKK3 expression and increase the sensitivity of renal cancer cells to cisplatin chemotherapy.

Lopinavir-Ritonavir Combination Induces Endoplasmic Reticulum Stress and Kills Urological Cancer Cells.

Induction of endoplasmic reticulum (ER) stress is a novel approach to cancer treatment. This study investigated the ability of the clinically feasible combination of the human immunodeficiency virus protease inhibitors lopinavir and ritonavir to induce ER stress killing urological cancer cells. Renal cancer cells (769-P, 786-O) and bladder cancer cells (UMUC-3, T-24) were used to investigate the ability of the combination to induce ER stress and its mechanism of action. The combination inhibited the growth of both renal and bladder cancer cells synergistically by inducing ER stress. The combination-induced ER stress increased the expression of AMP-activated protein kinase and suppressed the mammalian target of rapamycin pathway. It also increased the expression of a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor and thereby sensitized the cancer cells to TRAIL. The combination of lopinavir and ritonavir acts against urological cancer cells by inducing ER stress synergistically.

Abstract 2351: AXL and c-MET cross-talk in the regulation of PD-L1 expression on renal cancer cells

Abstract Purpose: The purpose of this research is to study the cross-talk between receptor tyrosine kinases (RTKs) c-MET and AXL in renal cancer cells; and to explore if the blockade of AXL-c-MET pathways can restrict PD-L1-mediated growth and immune escape of renal cancer cells. Background: c-MET and AXL are overexpressed in renal cell carcinoma (RCC). Upon binding with their respective ligands hepatocyte growth factor (HGF) and GAS6, c-MET and AXL activate a wide range of pathways, including tumor promoting events. Renal tumors show increased T cell infiltration, yet the T cells are rendered incapable of tumor rejection. The co-inhibitory molecules PD-1 and PD-L1 belong to cell surface glycoprotein B7 family that governs T cell function and proliferation. In RCC, tumor-infiltrating T cells express PD-1 and are associated with poor outcomes for patients. Similarly, PD-L1 expression on renal cancer cells hinders prognosis and indicates tumor aggressiveness. Therefore, PD-L1 in renal tumors may play a significant role in down-regulating host anti-tumor immune responses. Our recent report suggests that c-MET activation induces overexpression of PD-L1 in renal cancer cells; and the blockade of PD-L1 promotes immune-mediated killing of renal cancer cells. However, the cross talk between AXL and c-MET in the regulation of PD-L1 has not been studied in RCC. Results: We have confirmed that both AXL and c-MET are overexpressed in human renal cancer cells (786-O and ACHN) when compared with normal renal tubular epithelial cells. We performed co-immunoprecipitation assays with c-MET antibody and found that c-MET forms a complex with AXL in renal cancer cells. We utilized siRNA-mediated gene silencing approach and found that the knock-down of AXL increased the sensitivity of 786-O and ACHN cells to apoptosis following treatment with XL-184 (a small molecule inhibitor of c-MET, AXL and VGFRs, obtained from Exelixis) as measured by flow cytometry in Annexin V/PI stained cells. We also found that GAS6 treatment enhanced HGF-induced expression of cytoprotective molecule Heme oxygenase-1 (HO-1) and anti-apoptotic molecules Bcl-2 and Bcl-xL in renal cancer cells. In both 786-O and ACHN cells, c-MET activation increased the cell surface expression of PD-L1 as measured by flow cytometry. Interestingly, GAS6 treatment further increased the expression of PD-L1, suggesting a synergistic action of RTKs AXL and c-MET in renal cancer cells. We also utilized a PD-L1 promoter-luciferase plasmid to measure PD-L1 promoter activity and found that GAS6 treatment enhanced HGF-induced effects on PD-L1 transcription. In mouse renal cancer cells (RENCA), which express very high levels of PD-L1, we observed a significant decrease in the expression of PD-L1 when AXL was knocked down in cells. Conclusion: Together, our results demonstrate that AXL-mediated signaling enhances c MET-induced cytoprotection and PD-L1 expression on renal cancer cells. Citation Format: Murugabaskar Balan, Toni Choueiri, Soumitro Pal. AXL and c-MET cross-talk in the regulation of PD-L1 expression on renal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2351.

The Transcription Factor Creb is Involved in Sorafenib-Inhibited Renal Cancer Cell Proliferation, Migration and Invasion

Our previous reports showed that the cyclic-AMP-response element-binding protein (CREB) served as a proto-oncogene in the process of tumorigenesis and mediated the growth and metastatic activity of renal cancer cells. Our study, therefore, explored the role of CREB in sorafenib- -inhibited cell proliferation, migration and invasion. Renal cancer cells were cultured in medium containing sorafenib for 12, 24, 48 and 72 h. The MTT assay was used to study the cytotoxic effects of sorafenib. Cell invasion and migration were assayed in wound healing and transwell experiments, respectively. Protein expression levels were evaluated by western blotting. The results show that sorafenib treatment decreased cell viability in a dose- and time-dependent manner. Sorafenib inhibited cell migration and invasion and decreased the expression of MMP-2 and MMP-9. Moreover, addition of the recombinant plasmid pCI-neo/ CREB (PN) reversed the sorafenib-induced inhibition of cell proliferation, migration and invasion. These results show that CREB is associated with the sorafenib-induced inhibition of proliferation, migration and invasion.