Human Renal Cell Carcinoma Cell Lines Research Articles

Suppressed OGT expression inhibits cell proliferation and modulates EGFR expression in renal cell carcinoma.

PurposeO-linked N-acetylglucosamine (O-GlcNAc or O-GlcNAcylation) is a post-translational modification, which plays a vital role in the progression of various cancers. The purpose of the present study was to assess O-GlcNAcylation in human renal cell carcinoma (RCC).MethodsO-GlcNAcylation levels and O-GlcNAc-transferase (OGT) expression in human RCC cell lines and 10 paired clinical tissues were detected by Western blot and Immunohistochemis-try. Then, the effects of O-GlcNAcylation on RCC cell proliferation in vitro were investigated by Cell Counting Kit-8 assay. A xenograft assay was performed to assess the in vivo effects of OGT knockdown in RCC cells. Cell apoptosis and cell cycle assays were performed by flow cytometry. Co-immunoprecipitation assays were used to assess epidermal growth factor receptor (EGFR) O-GlcNAcylation and the interaction between OGT and EGFR.ResultsO-GlcNAcylation levels and OGT expression were increased in RCC, and the high amounts correlated with poor patient prognosis. OGT knockdown significantly suppressed RCC cell proliferation in vitro and in vivo. Notably, EGFR was modulated by O-GlcNAcylation and directly interacted with OGT.ConclusionThese findings provide novel insights into the oncogenic roles of O-GlcNAcylation and OGT in the development of RCC, indicating that OGT might be used as a target for RCC therapy in the future.

Timosaponin AIII inhibits metastasis of renal carcinoma cells through suppressing cathepsin C expression by AKT/miR-129-5p axis.

Timosaponin AIII (TSAIII) is a steroidal saponin that exerts anticancer activity on various cancer cells. In this study, we explore the effects of TSAIII on renal cell carcinoma (RCC) cells. Our findings show that TSAIII treatment (<8 μM) insignificantly influenced cell viability and cell cycle distribution of human RCC cell lines 786-O, A-498, and ACHN. Further observations revealed that TSAIII inhibited migration and invasion of 786-O and A-498 cells, as well as significantly decreased the production and expression of cathepsin C (CTSC) in both the cell types. Kinase cascade analysis exhibited that PI3K/AKT activation was inhibited, but PTEN expression was increased, in response to TSAIII treatments. Combining TSAIII and PI3K inhibitors, LY294002 synergically reduced the migration and invasion of 786-O and A-498 cells, as well as decreased the CTSC expression in both the cell types. We also observed that miR-129-5p bound to CTSC gene and suppressed the expression of CTSC and demonstrated that the miR-129-5p expression was synergically enhanced by TSAIII and LY294002. In addition, pretreatment with antago-miR-129-5p significantly restored the CTSC expression and the migration and invasion of TSAIII-treated 786-O cells. In conclusion, our findings reveal that TSAIII inhibits the metastatic properties of RCC cells, contributing to the inhibition of PI3K/AKT and the increase of miR-129-5p and the subsequent downregulation of CTSC. This suggests that TSAIII has significant antimetastatic activity against RCC cells and may be beneficial to RCC treatments.

Anticancer Action and Mechanism of Ergosterol Peroxide from Paecilomyces cicadae Fermentation Broth.

Isaria cicadae, a medicinal food fungus, is a fruit from Paecilomyces cicadae. In this study, we purified ergosterol peroxide (EP) from the fermentation broth of P. cicadae and investigated its effects on renal cell carcinoma (RCC) cells, in vitro. EP was purified from P. cicadae fermentation broth. The human RCC cell line 786-0 was used to analyze the anticancer mechanism of EP and inhibit its effect on cancer cell proliferation, in vitro. EP with a validated structure showed a yield rate of 20.1 mg/L and a purity of 96%. EP significantly inhibited RCC cell growth and clone formation in vitro. In addition, EP suppressed the migration and invasion, triggered the apoptosis, and modulated the cell cycle of RCC cells, in a dose-dependent manner. It also downregulated β-catenin expression. EP could be routinely produced through P. cicadae. It fights RCC cells in vitro through multiple mechanisms, including suppressing cell growth, colonization, migration, and invasion, arresting the cell cycle, attenuating β-catenin pathways, and triggering apoptosis.

Inhibitor of growth 4 inhibits cell proliferation, migration, and induces apoptosis of renal cell carcinoma cells.

The inhibitor of growth 4 (ING4) is known as a tumor suppressor. The expressions of ING4 were markedly reduced in human renal clear cell carcinoma (ccRCC) tissues. However, the role of ING4 in renal cell carcinoma (RCC) remains unknown. The aim of the current study was to detect the ING4 expression level and its potential role in human RCC cell lines. Our results showed that ING4 was lowly expressed in human RCC cell lines compared with that in proximal tubular cell line. Ectopic overexpression of ING4 inhibited the proliferation, migration, and invasion properties, and as well as prevented epithelial-mesenchymal transition (EMT) phenotype of RCC cells. In addition, ING4 overexpression induced cell apoptosis and autophagy in RCC cells. Furthermore, ING4 overexpression suppressed the activation of PI3K/Akt pathway in RCC cells. The activator of PI3K/Akt, insulin-like growth factor 1, abolished the effects of ING4 on RCC cells. These findings indicated that ING4 presented anticancer activity in RCC cells. The effects of ING4 on RCC cells were mediated by regulating the PI3K/Akt pathway. These findings suggested that ING4 could be used for gene therapy of RCC.

Estrogen affects cell growth and IGF-1 receptor expression in renal cell carcinoma.

PurposeBoth obesity and gender are important etiological factors in renal cell carcinoma (RCC) development, suggesting a pivotal role of sex hormone signaling pathway and insulin-like growth factor (IGF) family in RCC carcinogenesis. Here, we aimed to investigate the effect of estrogen on RCC growth and the possible interaction between estrogen/estrogen receptor (ER) signaling pathway and the IGF axis.MethodsER-α and ER-β were detected in four human RCC cell lines. Cells were treated with 17β-estradiol (E2), and cell proliferation was determined using the cell counting kit-8 assay. Using siRNA, ER-β was downregulated in RCC cells and the effect of E2 on cell growth and IGF-1 receptor (IGF-1R) expression was examined.ResultsE2 inhibited 786-O cell but not A498 cell growth significantly. After the downregulation of ER-β, E2 showed no obvious inhibitory role in 786-O cells. E2 stimulation increased the expression of IGF-1R in 786-O cells. Downregulation of ER-β, as well as fulvestrant, attenuated the stimulatory effect of E2 on IGF-1R expression.ConclusionOur results revealed that estrogen induced RCC growth inhibition via an ER-β-dependent pathway. Estrogen also upregulated the expression of IGF-1R, suggesting a link between estrogen/ER and IGF axis.

Long noncoding RNA HOTAIR promotes renal cell carcinoma malignancy through alpha-2, 8-sialyltransferase 4 by sponging microRNA-124.

Accumulating evidence demonstrated that the long noncoding RNA (lncRNA) HOTAIR (Hox transcript antisense intergenic RNA) plays key role in renal cell carcinoma (RCC) malignancy, while microRNA-124 (miR-124) is a tumour suppressor in RCC. The aim of this work was to assess the biological function of HOTAIR and to explore underlying mechanism involved in HOTAIR/miR-124/alpha-2, 8-sialyltransferase 4 (ST8SIA4) axis-regulated progression in RCC. Real-time PCR analyses and western blots were performed to the levels of HOTAIR, miR-124 and ST8SIA4 expression in human RCC tissues and RCC cell lines (ACHN and 786-O). Bioinformatics analysis and dual-luciferase reporter assay were used to illustrate relationship between HOTAIR and miR-124 in RCC. Colony formation assays, EdU assays, Ki67 assays and apoptosis assays were taken to evaluate cell proliferation. Tumour xenograft was created to explore the functions of HOTAIR and ST8SIA4 in tumorigenesis in vivo. Migration assays, invasion assays and cell adhesion assays and were also taken to analyse the carcinoma progression. In this study, HOTAIR level was confirmed to be significantly upregulated in RCC samples and RCC cell lines compared with those in the paired adjacent tissues and normal renal cell line. Overexpression of HOTAIR promoted the capability of proliferation, migration and invasion in RCC cell lines. HOTAIR directly bound to miR-124, while miR-124 mediated the expression of ST8SIA4 in RCC cell lines. ST8SIA4 was upregulated in RCC tissues and RCC cell lines. Ectopic expression of ST8SIA4 modulated the proliferation, migration and invasion of RCC cells. Further results indicated that HOTAIR promoted the proliferation and metastasis as a competing endogenous RNA to regulate ST8SIA4 expression by sponging miR-124 in RCC. Our results demonstrated that HOTAIR mediated RCC progression in part through miR-124/ST8SIA4 axis, which functioned as a new prognostic biomarker in RCC.

Characterisation of the Morphological, Functional and Molecular Changes in Sunitinib-Resistant Renal Cell Carcinoma Cells.

Sunitinib resistance is a major clinical problem hampering the treatment of renal cell carcinoma (RCC). Studies on the comprehensive characterisation of morphological, functional and molecular changes in sunitinib-resistant RCC cells are lacking. The aim of the current study was to develop sunitinib resistance in four human RCC cell lines (786-0, Caki-1, Caki-2 and SN12K1), and to characterise the changed cell biology with sunitinib resistance. RCC cells were made resistant by continuous, chronic exposure to 10 μM of sunitinib over a period of 12 months. Cell proliferation, morphology, transmigration, and gene expression for interleukin-6 (IL-6), interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), Bcl-2 and Bax were studied. There was no significant difference in growth rate or transmigration between the parental and resistant cells. Sunitinib-resistant cells were significantly hypertrophic compared with parental cells as evidenced by increases in the surface areas of the whole cells and the nuclei. IL-6 was significantly increased in all resistant cells. IL-8 was increased in sunitinib-resistant Caki-2 and SN12K1 cells and decreased in 786-0 without any significant changes in Caki-1. VEGF was increased in resistant Caki-2 and SN12K1 cells but not in 786-0 and Caki-1. The Bcl2/Bax ratio was increased in Caki-1, Caki-2 and SN12K1 cells but decreased in 786-0 cells. The increased IL-6 may contribute to sunitinib resistance either via VEGF-mediated angiogenesis or through shifting of the Bcl2/Bax balance in favour of anti-apoptosis.

Abstract 2457: Epithelial-to-mesenchymal transition and stemness: Key targets of HOTAIR-miR-203 interaction in renal cell carcinoma

Abstract Background: Tumor metastasis and recurrence are major obstacles in renal cell carcinoma (RCC) treatment. RCC aggressiveness is highly associated with stemness and epithelial-to-mesenchymal transition (EMT) which involves increased cellular migration, invasion, inhibition of apoptosis and senescence. HOX transcript antisense RNA (HOTAIR), a lncRNA, is reported to be over-expressed, whereas miR-203 has low expression and is a tumor suppressor in various cancers. However, their association and role in RCC is not well understood. The aim of the present study was to examine the interaction of HOTAIR and miR-203 in the regulation of stemness and EMT in RCC. Experimental Procedure: ACHN, Caki-1 (human RCC cell lines), normal renal epithelial cells HK-2 and clinical specimens were used for this study. Profiling of HOTAIR and miR203 expression were done by quantitative real-time PCR and luciferase assay was performed to confirm their interaction. Attenuation of HOTAIR (25nM siRNA) and overexpression of miR-203 (10nM mimic) for 72 hours were used for functional studies. The biological role of miR-203 and its interaction with HOTAIR was also investigated using nude mouse models. Results: HOTAIR was observed to be overexpressed in RCC cell-lines and clinical specimens whereas, miR-203 was significantly under-expressed when compared to normal cells and tissues. Overexpression of miR-203 altered the cell cycle, inhibited epithelial-to-mesenchymal transition (EMT) and decreased cell proliferation along with induction of epithelial marker proteins and decrease in mesenchymal and stemness marker proteins. Conversely, knockdown of miR-203 in non-malignant HK-2 cells induced pro-cancerous characteristics. Direct binding of miR-203 to HOTAIR was shown by RNA immunoprecipitation and luciferase assay. Attenuation of HOTAIR expression reduced cell migration, invasion, and induced apoptosis, mimicking the effects of miR-203 overexpression. Administration of miR-203 mimic to established tumors in nude mice significantly suppressed tumor growth compared to controls. Conclusion: Our results show that HOTAIR-miR-203 interaction inhibits EMT and stemness to regulate RCC progression. Statistical analysis of the clinicopathological data from kidney cancer patients suggests that HOTAIR and miR-203 may be useful in RCC diagnostics and therapeutics. Citation Format: Pritha Dasgupta, Priyanka Kulkarni, Shahana Majid, Varahram Shahryari, Yutaka Hashimoto, Nadeem S. Bhat, Marisa Shiina, Guoren Deng, Sharanjot Saini, Soichiro Yamamura Yamamura, Yuichiro Tanaka, Rajvir Dahiya. Epithelial-to-mesenchymal transition and stemness: Key targets of HOTAIR-miR-203 interaction in renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2457.

PLIN3 is up-regulated and correlates with poor prognosis in clear cell renal cell carcinoma

BackgroundPLIN3, one of the members of the perilipin family, has been reported to be involved in the formation and accumulation of lipid droplets. However, the expression levels and diagnostic and prognostic value of PLIN3 in renal cell carcinoma (RCC) remain unclear. MethodsBioinformatic analysis was used to assess the levels of PLIN3 and the correlation between PLIN3 levels and clinicopathological parameters in renal cancer. The expression levels of PLIN3 were determined in human RCC tissues and cell lines by western blot, immunofluorescence and immunohistochemistry assays. Receiver operating characteristic curves and Kaplan-Meier curves were used to analyze the diagnostic and prognostic significance of PLIN3 in RCC. ResultsThe expression level of PLIN3 was elevated in RCC tissues and cell lines, which was consistent with the analysis of the TCGA and Oncomine cancer database. The receiver operating characteristic curve indicated that high PLIN3 expression can distinguish cancer tissues from normal kidney tissues (area under the curve = 0.7270, P<0.0001). Kaplan-Meier curves revealed that elevated PLIN3 predicted poor disease-free survival and overall survival. ConclusionsPLIN3 is highly expressed in kidney cancer, and high expression of PLIN3 can serve as a useful diagnostic and prognostic biomarker. PLIN3 functional inhibition can be used as a new clinical treatment option.

VHL loss predicts response to Aurora kinase A inhibitor in renal cell carcinoma cells.

The majority of molecular targets of anticancer agents are limited to a subset of patients, and therefore identification of more specific biomarkers that can be used to improve clinical outcomes is of increasing interest. The present study showed that von Hippel‑Lindau tumor suppressor (VHL) tumor‑suppressor activity may influence the therapeutic response to Aurora kinase A (AURKA) inhibitors in human renal cell carcinoma (RCC). VHL protein (pVHL) expression was evaluated by immunoblotting in the human RCC cell lines CAKI, ACHN, 786‑O, 769‑P and A498. The anti‑tumor activities of alisertib, an AURKA‑specific chemical inhibitor, were detected by Cell Counting Kit‑8 assay invitro and mouse xenograft model invivo. Additionally, the VHL‑mediated anti‑tumor activity was assessed in 769‑P and CAKI cells via the loss or gain of VHL. The results revealed that VHL‑deficient 786‑O, 769‑P and A498 cells were sensitive to alisertib. By contrast, alisertib‑resistant CAKI and ACHN cells expressed the wild type VHL gene. In addition, rescue or knockdown of VHL was observed to increase or decrease alisertib anti‑proliferation activity, respectively, in RCC cells. The inverse correlation between the VHL gene expression profile and alisertib sensitivity was further confirmed in human cancer xenografts models. Taken together, these results suggested that VHL loss could potentially serve as a biomarker for predicting the efficacy of AURKA inhibitors.

MiR-488 inhibits cell growth and metastasis in renal cell carcinoma by targeting HMGN5.

PurposemicroRNAs are thought to play crucial roles in tumorigenesis. Dysregulation of miR-488 has been implicated to be involved in several cancer progressions. However, the biological functions of miR-488 in renal cell carcinoma (RCC) remain unclear. This study aimed to explore the molecular mechanism underlying the role of miR-488 in RCC development.Materials and methodsThe expression levels of miR-488 were detected in 38 paired RCC tumor samples and cell lines by quantitative real-time polymerase chain reaction method. miR-488 was upregulated by mimics transfection in RCC cell lines. MTT, colony formation, transwell assay, flow cytometry assay, and a xenograft model were performed to determine cell proliferation, invasion, migration, epithelial-to-mesenchymal transition, and apoptosis in vitro and in vivo. Moreover, the potential target of miR-488 was verified by dual-luciferase reporter assay, quantitative real-time polymerase chain reaction, and Western blot. The correlation between miR-488 expression and its target gene expression was confirmed by Spearman’s correlation analysis in 38 selected RCC tissue samples.ResultsWe found that miR-488 was remarkably downregulated in human RCC samples and cell lines compared with paired normal tissues and cell lines. Functional investigations revealed that overexpression of miR-488 significantly suppressed cell proliferation, invasion, and migration, and promoted cell apoptosis in RCC cells. Nucleosome binding protein 1 (high-mobility group nucleosome binding domain 5 [HMGN5]) was identified as a direct target of miR-488, and an inverse relationship was found between miR-488 expression and HMGN5 mRNA levels in RCC specimens. Rescue experiments suggested that restoration of HMGN5 partially abolished miR-488-mediated cell proliferation and invasion inhibition in RCC cells through regulating phosphatidylinositol 3-kinase/protein kinase B/the mammalian target of rapamycin and epithelial-to-mesenchymal transition signaling pathways.ConclusionThese data indicated that miR-488 acted as a tumor suppressor in RCC proliferation and invasion by targeting HMGN5, which might provide potential therapeutic biomarker for RCC patients.

Knockdown of TACC3 Inhibits the Proliferation and Invasion of Human Renal Cell Carcinoma Cells.

Transforming acidic coiled-coil protein 3 (TACC3) is a member of the TACC family and plays an important role in regulating cell mitosis, transcription, and tumorigenesis. However, the expression pattern and roles of TACC3 in renal cell carcinoma (RCC) remain unclear. The aim of this study was to investigate the role of TACC3 in RCC. We demonstrated overexpression of TACC3 in human RCC cell lines at both RNA and protein levels. Moreover, knockdown of TACC3 repressed RCC cell proliferation, migration, and invasion in vitro. In addition, knockdown of TACC3 inactivated PI3K/Akt signaling in RCC cells. Furthermore, knockdown of TACC3 significantly reduced tumor growth in xenograft tumor-bearing mice. Taken together, our findings showed that TACC3 was increased in human RCC cell lines, and knockdown of TACC3 inhibited the ability of cell proliferation, migration, invasion, and tumorigenesis in vivo. Therefore, TACC3 may act as a therapeutic target for the treatment of human RCC.

Knockdown of ubiquitin‑specific peptidase 39 inhibits the malignant progression of human renal cell carcinoma.

Ubiquitin specific peptidase 39 (USP39) serves important roles in mRNA processing and is involved in tumorigenesis of multiple solid malignancies. However, the influence and underlying mechanism of USP39 on human renal cell carcinomas (RCC) remain to be elucidated. The current study investigated the functional roles of USP39 in human RCC cell lines. siRNA‑mediated RNA interference was used to downregulate USP39 in RCC cells. CCK‑8, wound healing and invasion assays were performed to assess the proliferative ability and metastatic potential. The cell cycle distribution and apoptosis were evaluated by flow cytometry. The activity of signaling pathways and the expression of cell cycle‑related proteins were detected by western blot analysis. The siRNA‑directed RNA interference targeting USP39 could effectively downregulate the expression level of USP39 in two RCC cell lines. Depletion of USP39 by siRNA significantly suppressed cell growth and decreased invasive capacity of RCC cells. Silencing of USP39 induced cell apoptosis and cell cycle arrest at G2/M phase. Additionally, the expression levels of apoptotic and G2/M phase‑related proteins were notably decreased following depletion of USP39. Mechanistically, downregulation of USP39 blocked the activation of Akt and extracellular signal regulated kinase signaling pathways in RCC cells. These findings indicate that USP39 may serve as an oncogenic factor in RCC and could be a potential therapeutic candidate for human RCCs.

Down-regulation of transient receptor potential melastatin member 7 prevents migration and invasion of renal cell carcinoma cells via inactivation of the Src and Akt pathway.

PurposeTransient receptor potential melastatin member 7 (TRPM7), an ion channel and serine/threonine protein kinase, has been linked with distinct human malignancies. However, the role of TRPM7 in renal cell carcinoma (RCC) has not been investigated. The aim of this study is to determine whether TRPM7 regulates the migration and invasion of RCC cells. Its relationship with signal transduction pathways was also studied.Materials and MethodsThe human RCC cell lines ACHN and SN12C were chosen for this study. The molecular mechanisms of TRPM7 action were studied using Western blot analysis and small interfering RNA (siRNA)-based knockdown. The effect of TRPM7 knockdown on RCC cells was measured by using Transwell invasion and wound healing migration assays.ResultssiRNA-induced silencing of TRPM7 notably decreased the migration and invasion of ACHN and SN12C RCC cells. The phosphorylation levels of Src in both cells were obviously reduced after TRPM7 silencing compared with that of the control ACHN and SN12C cells. Furthermore, the phosphorylation levels of Akt were greatly decreased in ACHN cells after siRNA-induced knockdown of TRPM7. Additionally, the treatment of cells with Src and Akt inhibitors clearly limited the migration and invasion of RCC cells.ConclusionsOur data show that TRPM7 regulated ACHN and SN12C RCC cell invasion via the Src/Akt signaling pathway. Therefore, targeting the Src/Akt signaling pathway and/or the expression or function of TRPM7 could be a potential beneficial treatment for patients with RCC.

Abstract B141: Repurposing of ketorolac for the treatment of renal cell carcinoma (RCC)

Abstract Renal cell carcinoma (RCC) is the most common type of kidney cancer, accounting for more than 90% of malignant kidney tumors. The incidence of RCC in the eight major markets will increase by 63% over the forecast period of 2013 to 2023 (1). The currently available treatment options include antiangiogenic agents and tyrosine kinase inhibitors (TKIs), while immunotherapies are under development. The 5-year survival rate is approximately 90% (2); however, the 5-year overall survival for patients with distant metastatic RCC is only 12% (2). Thus, despite advances in the treatment of RCC, the prognosis for patients remains poor when the disease has metastasized beyond the kidneys (3), signifying a clear unmet medical need for new treatment options, particularly for high-risk stage II and III RCC patients undergoing surgery. Repurposing existing drugs is an attractive alternative as conventional drug discovery is expensive and time-consuming. In addition, preclinical and clinical data are widely available for such drugs, in turn reducing the time and resources required to bring a candidate drug to clinical trial. Using this strategy we have established the ability of ketorolac as a potential anticancer agent against RCC. Ketorolac is a nonsteroidal anti-inflammatory drug currently indicated for the short-term management of moderately severe acute pain, usually in a postoperative setting. We have tested the in vitro antiproliferative activity of Ketorolac in a panel of ten human RCC cell lines in 2D and 3D models. Ketorolac showed average IC50 value of 3.75 mM and 2.08 mM in the 2D and 3D models respectively. Interestingly, however, the combination of ketorolac with sunitinib, a standard-of-care, showed additive to synergistic effect as seen using the Bliss independence index. The data show that in RXF 1183L cells the combination shows additive effect while synergistic effect was seen in A-498 cells at 125 µM to 500 µM of ketorolac and 0.625 µM to 2.5 µM of sunitinib. Further, we validated the in vitro combination data by performing in vivo efficacy study in athymic nude mice. Ketorolac at 5 mg/kg p.o. and 10 mg/kg p.o. and sunitinib at 20 mg/kg p.o. inhibited tumor formation by up to 51%, 73%, and 50%, respectively. A combination of ketorolac at 5 mg/kg p.o. and 10 mg/kg p.o. with sSunitinib at 20 mg/kg p.o. inhibited tumor formation by 81% and 85%, respectively, clearly indicating superior antitumor efficacy as compared to drug alone. While understanding the mechanism of action (MOA) of ketorolac we astoundingly invented a novel role for ketorolac as an inducer of prostate apoptosis response-4 (PAR-4), a proapoptotic tumor suppressor protein. This indicates the unique use of Ketorolac as a secretagogue causing release of PAR-4 from normal cells; further, this released PAR-4 can cause apoptosis specifically in cancer cells. We thus conclude that this remarkable efficacy can be attributed to the dual MOA of ketorolac as a COX inhibitor and PAR-4 inducer, making it an interesting candidate for repurposing against renal cell carcinoma. 1. PharmaPoint. Renal Cell Carcinoma - Global Drug Forecast and Market Analysis to 2023. 2. National Cancer Institute. SEER Stat Fact Sheets: Kidney and Renal Pelvis. Available from: http://seer.cancer.gov/statfacts/html/kidrp.html. Accessed February 2016. 3. Ljungberg B, Campbell S, Cho H. The epidemiology of renal cell carcinoma. Eur Urol. 2011;60:615-621. Citation Format: Jeevan Deepak Ghosalkar, Siddhika Rajiv Raut, Mariamma Charles Thekkumpurath, Geena Malhotra, Kalpana Sanjay Joshi. Repurposing of ketorolac for the treatment of renal cell carcinoma (RCC) [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B141.

Overexpression of CAPN2 promotes cell metastasis and proliferation via AKT/mTOR signaling in renal cell carcinoma.

The calpain 2 (CAPN2) is upregulated in various malignant carcinomas. Previous studies have reported that CAPN2 functioned as an oncogenic factor in human cancers. However, its clinical role and potential effects on cell metastasis and proliferation in renal cell carcinoma (RCC) remain unknown. In this study, we evaluated the mRNA and protein levels of CAPN2 in human RCC specimens, matched normal specimens, and RCC cell lines using quantitative Real-time PCR (RT-PCR) and western blot. Immunohistochemistry of 74 RCC tissues in a tissue microarrays (TMAs) and normal kidney tissues were performed. Kaplan-Meier survival curve analyses were conducted to measure the correlation between CAPN2 and tumor prognosis. Cell migration, invasion and proliferation were detected by transwell assays and Cell Counting Kit-8 (CCK-8) assays. CAPN2 exhibited a significant overexpression in human RCC tissues and cell lines compared with adjacent non-tumor tissues and normal human proximal tubule epithelial cell line HK-2. Strong staining of CAPN2 was associated with higher clinical stage and histological grade. In addition, sh-CAPN2 could significantly inhibit migration, invasion and proliferation of 769-P and CAKI-1 cells. Conversely, increased cell biological behaviors were observed in CAPN2-OV CAKI-2 cells. Moreover, the subsequent mechanism investigation suggested that CAPN2 promoted tumor progression by activating AKT/mTOR signaling, enhancing epithelial mesenchymal transition (EMT) and MMP9 levels. The present study indicates that CAPN2 may act as a prominent indicator for RCC progression and a novel therapeutic target for RCC patients.

Dendritic-cell-based immunotherapy evokes potent anti-tumor immune responses in CD105+ human renal cancer stem cells.

Cancer stem cells (CSCs) are responsible for tumor initiation, progression, and resistance to therapeutic agents; they are usually less sensitive to conventional cancer therapies, and could cause tumor relapse. An ideal therapeutic strategy would therefore be to selectively target and destroy CSCs, thereby preventing tumor relapse. The aim of the present study was to evaluate the effectiveness of dendritic cells (DCs) pulsed with antigen derived from CD105+ human renal cell carcinoma (RCC) CSCs against renal cancer cells in vitro and in vivo. We identified "stem-like" characteristics of CD105+ cells in two human RCC cell lines: A498 and SK-RC-39. Loading with cell lysates did not change the characteristics of the DCs. However, DCs loaded with lysates derived from CD105+ CSCs induced more functionally specific active T cells and specific antibodies against CSCs, and clearly depressed the tumor growth in mice. Our results could form the basis for a novel strategy to improve the efficacy of DC-based immunotherapy for human RCC.

MiR-338-3p inhibits the invasion of renal cell carcinoma by downregulation of ALK5.

BackgroundThe current study aims to elucidate the role of miRNA-338-3p (miR-338-3p) in the invasion of renal cell carcinoma (RCC).MethodsQuantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed to detect the expression of miR-338-3p in human RCC cell lines with high metastatic potential (Caki-1) and low metastatic potential (786-O), respectively. The Caki-1 and 786-O cells were transfected with miR-338-3p mimic or inhibitor. Wound healing assay, Transwell assay and western blotting were performed to analyze the invasive ability and expression of activin receptor-like kinase 5 (ALK5) in the RCC cell lines. During the 36-month follow-up, we detected the expressions of miR-338-3p and ALK5 in 22 RCC cases with metastasis and 60 cases achieving a remission.ResultsmiR-339-3p was significantly downregulated in the Caki-1 cells as compared with the 786-O cells. The transfection with miR-338-3p inhibitor caused an increased invasive ability of both two cell lines. However, the transfection with miR-338-3p mimic caused a reduction of the invasiveness. In RCC cells, the expression of ALK5 was negatively correlated to miR-338-3p. Upregulation of ALK5 partially counteracted the miR-338-3p-induced invasiveness of RCC cells. We subsequently found the negative correlations between miR-338-3p and metastasis/ALK5 expression could be also observed in human RCC tissues.ConclusionTaken together, these results indicate that miR-338-3p acts as a novel tumor suppressor to inhibit the invasion of RCC by regulating ALK5 expression.

Abstract 4170: Targeting androgen receptor overcomes resistance to tyrosine kinase inhibitors in advanced clear cell renal cell carcinoma

Abstract Background: Advanced renal cell carcinoma (RCC) responds initially to anti-angiogenic therapies but eventually develop resistance which may be driven by the expression of key intratumoral pro-survival protein and overall kinome reprogramming. Androgen receptor (AR) expression has been reported in clear cell renal cell carcinoma (ccRCC) but its biological role remains elusive and its association with resistance to tyrosine kinase inhibitors (TKIs) has not been studied. Here we report the role of AR and its association with resistance to TKIs in advanced RCC. Methods: Human RCC cell lines; 786-0, 786-0R (with induced sunitinib resistance), ACHN, URMC2 (with intrinsic sunitinib resistance) were used to assess the combination effect of sunitinib and the AR antagonist enzalutamide in vitro. In vivo studies utilizing 786-0 tumors assessed the combination effect of enzalutamide and sunitinib following acquired resistance to sunitinib. AR expression was detected by qRT-PCR and Western blot analysis, and AR localization by immunofluorescence in treated and non-treated cells. Gene array was used to assess 93 AR associated genes in sensitive and resistant cells. Results: Quantitative RT-PCR data revealed a significant increase in AR expression with sunitinib resistance (&amp;gt;100 folds; p&amp;gt;0.001). In vitro and in vivo studies indicated significant increase in nuclear and cytoplasmic expression of AR with sunitinib treatment in resistant cells which was abrogated with single agent enzalutamide and combination treatment. More interestingly, combination treatment of enzalutamide and sunitinib significantly decreased cell growth and induced tumor regression in vitro and in vivo, respectively. Conclusion: Our data suggest the role of AR both in intrinsic and acquired sunitinib resistance in ccRCC and provide a rationale for combination strategies to restore TKI sensitivity that can be tested in the clinical setting. Citation Format: Remi M. Adelaiye-Ogala, Sreenivasulu Chintala, Ashley Orillion, May Elbanna, Ben Elzey, Nur Damayanti, Kiersten M. Miles, Chinghai Kao, Piergiorgio Pettazzoni, Giulio F. Draetta, Roberto Pili. Targeting androgen receptor overcomes resistance to tyrosine kinase inhibitors in advanced clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4170. doi:10.1158/1538-7445.AM2017-4170

Abstract 4120: Acquisition of chemoresistance to mTORC1 inhibition due to activation of the GSK-3 β/4EBP1 pathway might predict poor prognosis of mRCC patients

Abstract Background: PI3K/Akt/mTORC1 signaling pathway is aberrantly activated in renal cell carcinoma (RCC). We previously demonstrated that glycogen synthase kinase-3β (GSK-3β) positively regulated RCC proliferation. Herein, we aimed to investigate how the mTORC1 downstream substrate 4EBP1 is directly regulated by GSK-3β leading to acquisition of clinical chemoresistance to mTORC1 inhibitor (mTORi). Methods: The expression and phosphorylation of molecules in the subcellular pathways were examined by immunoblotting in surgically resected RCC tissues and human RCC cell lines including ACHN. Rapamycin-resistant ACHN cells (ACHN/RR) were generated by chronic exposure to an mTORi, rapamycin. Cell viability was investigated by MTS assay. The direct role of GSK-3β in regulating 4EBP1 was evaluated by an in vitro kinase assay. Pharmacological inhibition of GSK-3β, PI3K/Akt/mTORC1 pathway, and mTORC1 was achieved by treatment with AR-A014418, LY294002, and rapamycin, respectively. The effects of drug combination were determined using CompuSyn software. GSK-3β expression was evaluated in pathological RCC tissues obtained by nephrectomy from patients with metastatic RCC (mRCC), nine of whom were treated with mTORi as systemic therapies. GSK-3β expression was immunohistochemically graded as high and low expression using a semi-quantitative method based on positive staining intensity. Survival intervals were evaluated using the Kaplan-Meier method and log-rank test. Significance level was set as 0.05 in statistical analysis using a freeware R. Results: Inhibition of PI3K/Akt/mTORC1 pathway and mTORi treatment sufficiently decreased pS6RP, but only moderately decreased p4EBP1. In contrast to the effect of rapamycin, AR- A014418 remarkably inhibited cell proliferation, and rapidly suppressed p4EBP1 in ACHN and ACHN/RR. In vitro kinase assays showed that recombinant GSK-3β phosphorylated recombinant 4EBP1 at Thr37, Thr46, Thr70 and Ser65, and these phosphorylations were blocked by GSK-3 inhibitor. A combination of AR- A014418 and rapamycin produced an additive effect at lower concentrations, and were antagonistic at higher concentrations. Immunoblotting demonstrated that 4EBP1 and p4EBP1 expression in RCC tissues was positively correlated with GSK-3β expression. Overall survival was significantly short in patients with high GSK-3β expression, compared with those having low expression (n = 25 and 14, respectively, p = 0.04). In addition, high GSK-3β expression in tumors tended to shorten progression-free intervals in mRCC patients treated with mTORi. Conclusions: High GSK-3ββ expression and direct GSK-3β/4EBP1 pathway can be a key mechanism for RCC acquiring clinical chemoresistance to mTORCi. Activation of the direct pathway might be predictive of poor prognosis of mRCC patients. Citation Format: Hiromi Ito, Sei Naito, Osamu Ichiyanagi, Hidenori Kanno, Yuta Kurota, Atushi Yamagishi, Vladimir Bilim, Yoshihiko Tomita, Tomoyuki Kato, Akira Nagaoka, Norihiko Tsuchiya. Acquisition of chemoresistance to mTORC1 inhibition due to activation of the GSK-3 β/4EBP1 pathway might predict poor prognosis of mRCC patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4120. doi:10.1158/1538-7445.AM2017-4120