Sunitinib-resistant Cells Research Articles

Atractylenolide I inhibits angiogenesis and reverses sunitinib resistance in clear cell renal cell carcinoma through ATP6V0D2-mediated autophagic degradation of EPAS1/HIF2α

ABSTRACT Clear cell renal cell carcinoma (ccRCC) is tightly associated with VHL (von Hippel-Lindau tumor suppressor) mutation and dysregulated angiogenesis. Accumulating evidence indicates that antiangiogenic treatment abolishing tumor angiogenesis can achieve longer disease-free survival in patients with ccRCC. Atractylenolide I (ATL-I) is one of the main active compounds in Atractylodes macrocephala root extract and exhibits various pharmacological effects, including anti-inflammatory and antitumor effects. In this study, we revealed the potent antitumor activity of ATL-I in ccRCC. ATL-I exhibited robust antiangiogenic capacity by inhibiting EPAS1/HIF2α-mediated VEGFA production in VHL-deficient ccRCC, and it promoted autophagic degradation of EPAS1 by upregulating the ATPase subunit ATP6V0D2 (ATPase H+ transporting V0 subunit d2) to increase lysosomal function and facilitated fusion between autophagosomes and lysosomes. Mechanistically, ATP6V0D2 directly bound to RAB7 and VPS41 and promoted the RAB7-HOPS interaction, facilitating SNARE complex assembly and autophagosome-lysosome fusion. Moreover, ATP6V0D2 promoted autolysosome degradation by increasing the acidification and activity of lysosomes during the later stages of macroautophagy/autophagy. Additionally, we found that ATL-I could decrease the level of EPAS1, which was upregulated in sunitinib-resistant cells, thus reversing sunitinib resistance. Collectively, our findings demonstrate that ATL-I is a robust antiangiogenic and antitumor lead compound with potential clinical application for ccRCC therapy.

Metabolomics Reveals Tyrosine Kinase Inhibitor Resistance-Associated Metabolic Events in Human Metastatic Renal Cancer Cells.

The development of resistance to tyrosine kinase inhibitors (TKIs) is a major cause of treatment failure in metastatic renal cell carcinoma (mRCC). A deeper understanding of the metabolic mechanisms associated with TKI resistance is critical for refining therapeutic strategies. In this study, we established resistance to sunitinib and pazopanib by exposing a parental Caki-1 cell line to increasing concentrations of sunitinib and pazopanib. The intracellular and extracellular metabolome of sunitinib- and pazopanib-resistant mRCC cells were investigated using a nuclear magnetic resonance (NMR)-based metabolomics approach. Data analysis included multivariate and univariate methods, as well as pathway and network analyses. Distinct metabolic signatures in sunitinib- and pazopanib-resistant RCC cells were found for the first time in this study. A common metabolic reprogramming pattern was observed in amino acid, glycerophospholipid, and nicotinate and nicotinamide metabolism. Sunitinib-resistant cells exhibited marked alterations in metabolites involved in antioxidant defence mechanisms, while pazopanib-resistant cells showed alterations in metabolites associated with energy pathways. Sunitinib-resistant RCC cells demonstrated an increased ability to proliferate, whereas pazopanib-resistant cells appeared to restructure their energy metabolism and undergo alterations in pathways associated with cell death. These findings provide potential targets for novel therapeutic strategies to overcome TKI resistance in mRCC through metabolic regulation.

Upregulation of serine metabolism enzyme PSAT1 predicts poor prognosis and promotes proliferation, metastasis and drug resistance of clear cell renal cell carcinoma

Serine metabolic reprogramming is known to be associated with oncogenesis and tumor development. The key metabolic enzyme PSAT1 has been identified as a potential prognostic marker for various cancers, but its role in ccRCC remains unkown. In this study, we investigated expression of PSAT1 in ccRCC using the TCGA database and clinical specimens. Our results showed that PSAT1 exhibited lower expression in tumor tissue compared to adjacent normal tissue, but its expression level increased with advancing stages and grades of ccRCC. Patients with elevated expression level of PSAT1 exhibited an unfavorable prognosis. Functional experiments have substantiated that the depletion of PSAT1 shows an effective activity in inhibiting the proliferation, migration and invasion of ccRCC cells, concurrently promoting apoptosis. RNA sequencing analysis has revealed that the attenuation of PSAT1 can diminish tumor resistance to therapeutic drugs. Furthermore, the xenograft model has indicated that the inhibition of PSAT1 can obviously impact the tumorigenic potential of ccRCC and mitigate lung metastasis. Notably, pharmacological targeting PSAT1 by Aminooxyacetic Acid (AOA) or knockdown of PSAT1 increased the susceptibility of sunitinib-resistant cells. Inhibition of PSAT1 increased the sensitivity of drug-resistant tumors to sunitinib in vivo. Collectively, our investigation identifies PSAT1 as an independent prognostic biomarker for advanced ccRCC patients and as a prospective therapeutic target.

N6-methyladenosine-modified TRAF1 promotes sunitinib resistance by regulating apoptosis and angiogenesis in a METTL14-dependent manner in renal cell carcinoma

BackgroundSunitinib resistance can be classified into primary and secondary resistance. While accumulating research has indicated several underlying factors contributing to sunitinib resistance, the precise mechanisms in renal cell carcinoma are still unclear.MethodsRNA sequencing and m6A sequencing were used to screen for functional genes involved in sunitinib resistance. In vitro and in vivo experiments were carried out and patient samples and clinical information were obtained for clinical analysis.ResultsWe identified a tumor necrosis factor receptor-associated factor, TRAF1, that was significantly increased in sunitinib-resistant cells, resistant cell-derived xenograft (CDX-R) models and clinical patients with sunitinib resistance. Silencing TRAF1 increased sunitinib-induced apoptotic and antiangiogenic effects. Mechanistically, the upregulated level of TRAF1 in sunitinib-resistant cells was derived from increased TRAF1 RNA stability, which was caused by an increased level of N6-methyladenosine (m6A) in a METTL14-dependent manner. Moreover, in vivo adeno-associated virus 9 (AAV9) -mediated transduction of TRAF1 suppressed the sunitinib-induced apoptotic and antiangiogenic effects in the CDX models, whereas knockdown of TRAF1 effectively resensitized the sunitinib-resistant CDXs to sunitinib treatment.ConclusionsOverexpression of TRAF1 promotes sunitinib resistance by modulating apoptotic and angiogenic pathways in a METTL14-dependent manner. Targeting TRAF1 and its pathways may be a novel pharmaceutical intervention for sunitinib-treated patients.

Circular RNA circSNX6 promotes sunitinib resistance in renal cell carcinoma through the miR-1184/GPCPD1/ lysophosphatidic acid axis.

Sunitinib resistance is a major challenge in systemic therapy for renal cell carcinoma (RCC). The role of circular RNAs (circRNAs) in regulating sunitinib resistance of RCC is largely unknown. We established sunitinib-resistant RCC cell lines in vivo. Through RNA-sequencing, we identified circSNX6, whose expression is upregulated in sunitinib-resistant cells compared with their parental cells. High circSNX6 expression was correlated with sunitinib resistance and worse oncologic outcomes in a cohort of 81 RCC patients. In vitro and in vivo experiments confirmed that circSNX6 could promote sunitinib resistance in RCC. circSNX6 acts as a molecular "sponge" to relieve the suppressive effect of microRNA (miR)-1184 on its target gene, glycerophosphocholine phosphodiesterase 1 (GPCPD1), which increases intracellular lysophosphatidic acid (LPA) levels and, ultimately, promotes sunitinib resistance in RCC cells. Our findings demonstrated that the circSNX6/miR-1184/GPCPD1 axis had a critical role in regulation of intracellular LPA levels and sunitinib resistance in RCC; they also provide a novel prognostic indicator and promising therapeutic targets.

Overcoming sunitinib resistance with tocilizumab in renal cell carcinoma: Discordance between in vitro and in vivo effects

Sunitinib is one of the first-line multi-tyrosine kinase inhibitors for metastatic renal cell carcinoma, and resistance to sunitinib continues to be a limiting factor for the successful treatment. As interleukin-6 (IL-6) is overexpressed in sunitinib-resistant cells, the purpose of this study was to explore the potential of IL-6 inhibition with tocilizumab, an IL-6 receptor inhibitor, to overcome resistance. In vitro, two sunitinib-resistant renal cell carcinoma cell lines (Caki-1 and SN12K1) were treated with tocilizumab. A mouse subcutaneous xenograft model was also used. Cell viability was studied by MTT assay, and apoptosis by morphology and ApopTag. Expression of IL-6, vascular endothelial growth factor (VEGF), and Bcl-2 was analyzed by qPCR. In vitro, tocilizumab induced significant cell death, and reduced the expression of IL-6, VEGF, and Bcl-2 in sunitinib-resistant cells. However, the in vitro findings could not be successfully translated in vivo, as tocilizumab did not decrease the growth of the tumors.

Repurposing ketoconazole as an exosome directed adjunct to sunitinib in treating renal cell carcinoma

Renal Cell Carcinoma (RCC) is the most common form of kidney cancer, with clear cell RCC (ccRCC) representing about 85% of all RCC tumors. There are limited curable treatments available for metastatic ccRCC because this disease is unresponsive to conventional targeted systemic pharmacotherapy. Exosomes (Exo) are small extracellular vesicles (EVs) secreted from cancer cells with marked roles in tumoral signaling and pharmacological resistance. Ketoconazole (KTZ) is an FDA approved anti-fungal medication which has been shown to suppress exosome biogenesis and secretion, yet its role in ccRCC has not been identified. A time-course, dose-dependent analysis revealed that KTZ selectively decreased secreted Exo in tumoral cell lines. Augmented Exo secretion was further evident by decreased expression of Exo biogenesis (Alix and nSMase) and secretion (Rab27a) markers. Interestingly, KTZ-mediated inhibition of Exo biogenesis was coupled with inhibition of ERK1/2 activation. Next, selective inhibitors were employed and showed ERK signaling had a direct role in mediating KTZ’s inhibition of exosomes. In sunitinib resistant 786-O cells lines, the addition of KTZ potentiates the efficacy of sunitinib by causing Exo inhibition, decreased tumor proliferation, and diminished clonogenic ability of RCC cells. Our findings suggest that KTZ should be explored as an adjunct to current RCC therapies.

G-Protein-coupled Estrogen Receptor 1 Agonist G-1 Perturbs Sunitinib Resistance-related Phosphoproteomic Signatures in Renal Cell Carcinoma.

Metastatic renal cell carcinoma (RCC) often develops resistance to first-line targeted therapy such as sunitinib. G-Protein-coupled estrogen receptor 1 (GPER1) agonist G-1 was recently reported to regulate RCC physiology but the role of G-1 in RCC tumorigenesis and sunitinib resistance remains largely unknown. Parental and sunitinib-resistant 786-O cells were treated with GPER1 agonist G-1, and quantitative phosphoproteomics was performed. Bioinformatic analyses and validations, including immunoblotting, cell migration, and cell cycle distribution, were performed. G-1 repressed cell proliferation and migration in both parental and sunitinib-resistant 786-O cells. Phosphoproteomic signatures, including phosphoinositide 3-kinase and protein kinase B (PI3K-AKT) as well as other pathways, were up-regulated in sunitinib-resistant cells but application of G-1 reversed this effect. Among phosphoprotein candidates, activating transcription factor 2 (ATF2) Thr69/71 phosphorylation was antagonistically regulated by sunitinib resistance and G-1. Our results open up the possibility for managing RCC and sunitinib resistance by GPER1 agonist G-1 and its regulated pathways.

Long Non-coding RNA CCAT1 Acts as an Oncogene and Promotes Sunitinib Resistance in Renal Cell Carcinoma.

Although sunitinib contributes to prolonging the progression-free survival of metastatic renal cell carcinoma significantly, the universal presence of resistance limits the initial response rate and restricts durable responses. The mechanisms involved in sunitinib resistance vary and need further investigation. We found long non-coding RNA (lncRNA) colon cancer-associated transcript-1 (CCAT1) overexpressed in sunitinib-resistant cells while declined in the parental cells. Moreover, lncRNA CCAT1 increased significantly in samples with resistance to sunitinib compared with those with responses to sunitinib. The reduction of CCAT1 suppressed cell growth and colony formation while triggering apoptosis. Inversely, the ectopic expression of c-Myc reversed the inhibition of cell growth and enhancement of apoptosis by the knockdown of CCAT1. We also verified that anti-apoptosis protein B-cell lymphoma 2 (Bcl-2) and myeloid cell leukemia 1 (Mcl-1) decreased along with the deregulation of CCAT1, whereas the expression of Bcl-2 and Mcl-1 restored in cells that were transfected sh-CCAT1 and c-Myc simultaneously. Apart from the in vitro experiments, we demonstrated that knockdown of CCAT1 boosted response to sunitinib by performing sunitinib-resistant ACHN mouse models. Briefly, lncRNA CCAT1 conferred renal cell carcinoma resistance to sunitinib in a c-Myc-dependent manner, providing a novel target for improvement of sunitinib therapy.

LncRNA HOTAIR induces sunitinib resistance in renal cancer by acting as a competing endogenous RNA to regulate autophagy of renal cells.

BackgroundCell autophagy has been proposed to be involved in drug resistance therapy. However, how the long non-coding RNA (lncRNA) reduces risks of drug resistance in renal cancer (RC) cells needs a thorough inquiry. This study was assigned to probe the effect and mechanism of HOTAIR on sunitinib resistance of RC.MethodsClinical RC tissues and para-carcinoma tissues were obtained to detect the expressions of miR-17-5p, HOTAIR and Beclin1. Sunitinib-resistant cells (786-O-R and ACHN-R) were constructed using parental RC cells (786-O and ACHN). The resistance of 786-O-R and ACHN-R cells to sunitinib was examined. Western blot and qRT-PCR were assayed to obtain the expressions of miR-17-5p, HOTAIR and Beclin1. The effects of HOTAIR knockdown or miR-17-5p overexpression/knockdown on cell autophagy and sunitinib resistance were measured by MDC staining, immunofluorescence and Western blot. The sensitivity of RC cells to sunitinib and change in cell clone formation after sunitinib treatment were assessed by CCK-8 assay and colony formation assay, respectively. The relationships among HOTAIR, miR-17-5p and Beclin1 were verified by dual-luciferase reporter gene and RIP assay. The role of HOTAIR knockdown in sunitinib resistance was verified in nude mice.ResultsHOTAIR expression in sunitinib-resistant cells is higher than that in parental cells. Knockdown of HOTAIR in sunitinib-resistant cells lead to refrained sunitinib resistance and cell autophagy both in vivo and in vitro. Activation of autophagy could raise resistance to sunitinib in RC cells, while inhibition of autophagy could improve the sensitivity of sunitinib-resistant cells to sunitinib. HOTAIR could compete with miR-17-5p to regulate Beclin1 expression. Knockdown of miR-17-5p in parental cells increases cell resistant to sunitinib, and overexpression of miR-17-5p in sunitinib-resistant cells increases cell sensitive to sunitinib.ConclusionHOTAIR negatively targets miR-17-5p to activate Beclin1-mediated cell autophagy, thereby enhancing sunitinib resistance in RC cells.

Wogonin Induces Apoptosis and Reverses Sunitinib Resistance of Renal Cell Carcinoma Cells via Inhibiting CDK4-RB Pathway.

Wogonin, an active component derived from Scutellaria baicalensis, has shown anti-tumor activities in several malignancies. However, the roles of wogonin in RCC cells remain elusive. Here, we explored the effects of wogonin on RCC cells and the underlying mechanisms. We found that wogonin showed significant cytotoxic effects against RCC cell lines 786-O and OS-RC-2, with much lower cytotoxic effects on human normal embryonic kidney cell line HEK-293 cells. Wogonin treatment dramatically inhibited the proliferation, migration, and invasion of RCC cells. We further showed that by inhibiting CDK4-RB pathway, wogonin transcriptionally down-regulated CDC6, disturbed DNA replication, induced DNA damage and apoptosis in RCC cells. Moreover, we found that the levels of p-RB, CDK4, and Cyclin D1 were up-regulated in sunitinib resistant 786-O, OS-RC-2, and TK-10 cells, and inhibition of CDK4 by palbociclib or wogonin effectively reversed the sunitinib resistance, indicating that the hyperactivation of CDK4-RB pathway may at least partially contribute to the resistance of RCC to sunitinib. Together, our findings demonstrate that wogonin could induce apoptosis and reverse sunitinib resistance of RCC cells via inhibiting CDK4-RB pathway, thus suggesting a potential therapeutic implication in the future management of RCC patients.

MP18-18 P53 IS INVOLVED IN RESISTANCE TO SUNITINIB IN RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology II (MP18)1 Apr 2020MP18-18 P53 IS INVOLVED IN RESISTANCE TO SUNITINIB IN RENAL CELL CARCINOMA Yohei Sekino*, Xiangrui Han, Keisuke Goto, Shogo Inoue, Tetsutaro Hayashi, Jun Teishima, Taniyama Daiki, Naoya Sakamoto, Kazuhiro Sentani, Naohide Oue, Wataru Yasui, and Akio Matsubara Yohei Sekino*Yohei Sekino* More articles by this author , Xiangrui HanXiangrui Han More articles by this author , Keisuke GotoKeisuke Goto More articles by this author , Shogo InoueShogo Inoue More articles by this author , Tetsutaro HayashiTetsutaro Hayashi More articles by this author , Jun TeishimaJun Teishima More articles by this author , Taniyama DaikiTaniyama Daiki More articles by this author , Naoya SakamotoNaoya Sakamoto More articles by this author , Kazuhiro SentaniKazuhiro Sentani More articles by this author , Naohide OueNaohide Oue More articles by this author , Wataru YasuiWataru Yasui More articles by this author , and Akio MatsubaraAkio Matsubara More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000843.018AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Although approximately 70% of the patients show initial response with sunitinib in renal cell carcinoma (RCC), eventually sunitinib resistance occurred. Therefore there is an urgent need to clarify the mechanisms of sunitinib resistance. The tumor protein p53 is involved in chemo-resistance in several cancer. The aim of this study is to examine the expression and functional role of P53 on sunitinib resistance and a possible role of combination with MDM2 inhibitor: nutlin3 and sunitinib in RCC. METHODS: We established sunitinib resistance RCC cell lines. We analyzed the expression of P53 in sunitinib resistance cell lines and examined the effect of P53 knockout on sunitinib resistance. We also investigated the effect of combination with sunitinib and nutlin3. Expression of P53 in 49 RCC tissues treated with sunitinib was determined by immunohistochemisty. RESULTS: We established sunitinib resistance cells in Caki-1 and ACHN cells by culturing cells with increasing concentrations of sunitinib. Western blotting showed that the expression of P53 was decreased in sunitinib resistant cells compared with that in parental cells. Then, we generated the P53 knockout cells in Caki-1 and ACHN cells by CRISPR-cas9 system. Knockout of P53 promoted sunitinib resistance. Mdm2 is an important negative regulator of the p53. Therefore we examined the effect of combination therapy with sunitinib and nutlin3. Sunitinib alone had little effect on cell viability in the sunitinib resistant cell lines. However, the combination of sunitinib and nutlin3 significantly reduced cell viability in the sunitinib resistant cells. Western blotting revealed that the expression of P53 was induced by combination therapy with suniitnib and nutlin3 compared to nutlin3 treatment alone in sunitinib resistant cells. Additionally, we investigated the expression of P53 in 52 ccRCC cases treated with sunitinib treatment. A Kaplan-Meier analysis showed that the positive cases of P53 were associated with poor prognosis after sunitinib treatment (P = 0.04) (Fig. 1). CONCLUSIONS: The expression of P53 was downregulated in sunitinib resistant RCC cell lines. Knockout of P53 promoted sunitinib resistance. Nutlin3 re-sensitized sunitinib resistant cells to sunitinib treatment. These results suggest that the combination therapy with sunitinib and nutlin3 may be promising in RCC. Source of Funding: I have nothing to disclose © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e242-e242 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yohei Sekino* More articles by this author Xiangrui Han More articles by this author Keisuke Goto More articles by this author Shogo Inoue More articles by this author Tetsutaro Hayashi More articles by this author Jun Teishima More articles by this author Taniyama Daiki More articles by this author Naoya Sakamoto More articles by this author Kazuhiro Sentani More articles by this author Naohide Oue More articles by this author Wataru Yasui More articles by this author Akio Matsubara More articles by this author Expand All Advertisement PDF downloadLoading ...

LINC00461 affects the survival of patients with renal cell carcinoma by acting as a competing endogenous RNA for microRNA‑942.

The present study aimed to investigate the potential mechanisms of human miR-942 in the sunitinib-resistance of renal cell carcinoma (RCC). A sunitinib-resistant OS-RC-2 cell line was established by continuous exposure to increasing concentrations of sunitinib for ~12 weeks. The expression levels of four miRNAs were determined by reverse transcription-quantitative (RT-q)PCR. miR-942 mimics were transfected into OS-RC-2 cells and RNA sequencing was performed on the miR-942- and negative control-transfected cells. Downregulated genes, including those of long non-coding RNAs (lncRNAs) and mRNAs, were identified. The target genes of miR-942 were predicted, followed by protein-protein interaction network construction and functional enrichment analyses of miR-942 target genes. In addition, RCC RNA-seq and miRNA-seq data were downloaded from The Cancer Genome Atlas (TCGA) database. The contributions of lncRNA and/or mRNAs to survival prediction were assessed and a competing endogenous RNA (ceRNA) network consisting of miR-942, lncRNA and mRNAs was constructed. The expression levels of LINC00461, miR-942, spalt-like transcription factor 1 (SALL1), methionyl aminopeptidase 1 (METAP1) and DDB1 and CUL4 associated factor 1 (DCAF11) were verified using RT-qPCR. The role of LINC00461 in cell viability was detected by MTT assay. The expression level of miR-942 was significantly increased in sunitinib-resistant cells. A total of seven lncRNAs and 155 mRNAs were predicted as target genes of miR-942 in the miR-942 mimic-treated samples, compared with the mimic control-treated group. These potential target genes were significantly associated with ‘protein binding’, ‘TNF-β signaling pathway’, ‘negative transcriptional regulation’ and ‘RNA binding’. Through the integrated analysis of RNA-sequencing and TCGA data, an miR-942-related ceRNA network, which was predicted to significantly affect the survival of patients with RCC, was constructed. The expression levels of lncRNA LINC00461 and the genes SALL1, METAP1, and DCAF11 were further verified. The viability of OS-RC-2 cells was decreased following co-transfection with miR-942 mimics and LINC00641 siRNA, and was comparable to that of wild type OS-RC-2 cells (P>0.05). Therefore, lncRNA LINC00461 may act as an miR-942 ceRNA, and affect the survival of patients with RCC by regulating the expression of SALL1, METAP1 and DCAF11.

Abstract 3805: Artesunate reduces tumor growth in sunitinb-resistant renal cell carcinoma cells

Abstract Introduction: Renal cell carcinoma (RCC) is the most common cancer of the kidney. Due to therapy resistance effect of the approved compounds, like the tyrosine kinase inhibitor (TKI) sunitinib, is limited. Thus, new treatment approaches are urgently needed. Artesunate (ART) is derived from Traditional Chinese Medicine (TCM), showing anti-tumor activity, also in urologic tumors. However, little is known about the efficiency of ART on therapy-resistant RCC. Therefore, we investigated the effect of ART on sunitinib-resistant RCC cell lines. Material and Methods: Parental (=sensitive) and sunitinib-resistant (1 µM) RCC cell lines, KTC-26, A498, and Caki-1, were exposed to ART (1-100 µM) for 24, 48 and 72 hours. ART untreated cells served as controls. Tumor cell growth, proliferation, cell cycling and the expression of cell cycle regulating proteins were then evaluated. Aside, the appearance of apoptotic and necrotic effects was determined. Results: Exposure to different concentrations of ART significantly reduced the tumor cell growth and proliferation of parental and sunitinib-resistant cells in a time- and dose-dependent manner, compared to the controls. At the same time, exposure to ART resulted in a cell cycle arrest in the G0/G1 phase. Notably, this effect was more prominent in the sunitinib-resistant cells. G0/G1 phase elevation was associated with a decrease in the percentage of S and G2/M phase cells. The observed changes in cell growth and cell cycle phases were accompanied by distinct modulations of the cell cycle regulating proteins, indicating for an inhibitory regulation of ART through cell cycle arrest. Indeed, neither necrotic nor apoptotic effects events were detectable after ART treatment, corroborating this assumption. Conclusion: Due to our data we conclude that ART has anti-tumor effects not only in parental but also in sunitinib-resistant RCC cells. Thus, adding ART to the standard therapies might be a promising treatment strategy for patients with advanced RCC. Further investigations are necessary to verify our findings. Citation Format: Eva Juengel, Sascha Markowitsch, Holger H. Erb, Thomas Efferth, Axel Haferkamp. Artesunate reduces tumor growth in sunitinb-resistant renal cell carcinoma 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 3805.

Significance of cyclooxygenase-2, prostaglandin E2 and CD133 levels in sunitinib-resistant renal cell carcinoma.

The current study investigated the mechanism underlying sunitinib resistance. The parental human renal cell carcinoma (RCC) cell line 786-O was continuously exposed to various doses of sunitinib to obtain sunitinib-resistant cells (786-O/S). Cell proliferation and colony formation assays were performed to assess the survival of 786-O/S cells. The half-inhibitory concentration for the drug-resistant cells was calculated. 786-O/S cells demonstrated notably morphological changes compared with parental cells. Compared with 786-O cells, 786-O/S cells exhibited stronger proliferative and colony-forming abilities. Western blot analysis was performed to measure the levels of cyclooxygenase 2 (COX-2) and prostaglandin E2 (PGE2). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of COX-2 and cluster of differentiation (CD) 133 in both 786-O and 786-O/S cells. Following incubation of the two cell lines with celecoxib, a COX-2 inhibitor, RT-qPCR was performed to detect the expression of COX-2 and CD133, and western blot analysis was used to assess the expression of CD133. The results revealed that the levels of COX-2 and PGE2 were significantly higher in 786-O/S cells compared with 786-O cells (P<0.01). Similarly, the expression of CD133 was 24-fold higher in 786-O/S compared with the parental cells (P<0.01). When celecoxib was incubated with the two cell lines, the expression of COX-2 and CD133 decreased significantly (P<0.0001). In summary, the results indicate that activation of the COX-2-PGE2 pathway in RCC leads to the development of sunitinib resistance and may serve an important role in the maintenance of the characteristics of stem cells that are closely associated with drug resistance.

Label-free Quantitative Phosphoproteomics Reveals the Role of Beta-Estradiol in Sunitinib-Resistant Renal Cell Carcinoma Growth Via Perturbing Transforming Growing Factor-Beta Pathway

Context: Sunitinib is the first-line targeted therapy for metastatic renal cell carcinoma (RCC). However, resistance to sunitinib often occurred in patients receiving sunitinib treatment. On the other hand, 17-beta-estradiol (estrogen or E2) has been demonstrated to repress RCC growth in vitro, whether E2 can also affect the growth of sunitinib-resistant RCC remains unknown. Aims: In this study, the role of E2 in inhibiting sunitinib-resistant RCC growth and the underlining acting mechanisms was explored. Settings and Design: Sunitinib resistance was first induced in vitro in ACHN cells. The effect of E2 on cellular growth was then assayed. Label-free phosphoproteomics was also conducted. Subjects and Methods: ACHN cells were first challenged with 10-μM sunitinib up to 4 months to induce drug resistance. Then, E2 at different concentrations were tested in both parental and sunitinib-resistant ACHN cells. To conduct phosphoproteomics study, the total cell lysates from E2-treated ACHN cells were harvested, trypsin digested, and the phosphopeptides were enriched by Fe-IMAC. Statistical Analysis Used: For comparing the E2-induced cell growth inhibition, Student's t-test was used, and P &lt; 0.05 was considered statistically significant. As for label-free phosphoproteomics, false discovery rate &lt;0.01 and phosphosite possibility &gt;0.75 were considered as positive identifications. Results: E2 at the physiological concentration, that is, 10 nM, can repress the cell growth in both parental and sunitinib-resistant ACHN cells. Further, label-free phosphoproteomics revealed that transforming growth factor beta (TGF-β) pathway, cell cycle, and cytoskeleton bindings were enhanced in sunitinib-resistant cells but can be reduced by E2 treatment. On the other hand, programmed cell death and apoptosis were repressed in sunitinib-resistant cells, and E2 at 10 nM did not reverse the effect. We further validated the expression of SMAD3, an important molecule in TGF-β pathway, and found that SMAD3 decreased in sunitinib-resistant cells but can be upregulated by E2 treatment. Conclusions: Our study demonstrated that E2 can inhibit the cell growth in sunitinib-resistant RCC cells at physiological concentration by upregulating SMAD3 in the TGF-β pathway, which may lead to growth inhibition in RCC.

Resistance to lysosomotropic drugs used to treat kidney and breast cancers involves autophagy and inflammation and converges in inducing CXCL5.

Lysosomotropic agents such as sunitinib, lapatinib, and chloroquine belong to a drug family that is being used more frequently to treat advanced cancers. Sunitinib is standard care for metastatic renal cell carcinomas (mRCC) and lapatinib is used for trastuzumab/pertuzumab-refractory cancers. However, patients ineluctably relapse with a delay varying from a few months to a few years. To improve reactivity prior to relapse it is essential to identify the mechanisms leading to such variability. We showed previously that sunitinib became sequestered in lysosomes because of its basic pKa.Methods: Modifications to gene expression in response to sunitinib and in sunitinib resistant cells were analyzed by transcriptomic and proteomic analysis. ROS production was evaluated by FACS. Nuclear Factor kappa B (NFkB)-dependent transcriptional regulation of inflammatory gene expression was evaluated with a reporter gene. Correlation of CXCL5 with survival was analyzed with an online available data base (TCGA) and using a cohort of patients enrolled in the SUVEGIL clinical trial (NCT00943839).Results: We now show that sunitinib sequestration in lysosomes induced an incomplete autophagic process leading to activation of the NFkB inflammatory pathway. We defined a subset of inflammatory cytokines that were up-regulated by the drug either after an acute or chronic stimulus. One of the most up-regulated genes in sunitinib-resistant cells was the CXCL5 cytokine. CXCL5 was also induced in RCC by chloroquine and in a model of HER2 positive breast cancer cell lines after acute or chronic treatment with lapatinib. CXCL5 correlated to shorter survival in RCC and to the most aggressive forms of breast cancers. The levels of CXCL5 present in the plasma of patients treated with sunitinib were predictive of the efficacy of sunitinib but not of the VEGF-directed antibody bevacizumab.Conclusion: This translational study identified CXCL5 as a biomarker of efficacy of lysosomotropic drugs, a potential asset for personalized medicine.

Overexpression of FZD1 is Associated with a Good Prognosis and Resistance of Sunitinib in Clear Cell Renal Cell Carcinoma.

Frizzled class receptor 1 (FZD1), a receptor for Wnt signaling pathway. Overexpression of FZD1 has been detected in many cancer tissues and cells resulting in tumor development and drug resistance. However, its expression status and prognostic merit in renal cancer still remains unclear. We screened the FZD1 mRNA in clear cell renal cell carcinoma (ccRCC) and papillary renal cell carcinoma (pRCC) from TCGA database and Oncomine database. We then detected FZD1 mRNA expression in sunitinib-resistant cells and the corresponding parental cells by qRT-PCR. FZD1 level was significantly upregulated in renal cancer tissues, renal cancer cell lines and their corresponding sunitinib-resistant cells. FZD1 level was also associated with the clinicopathological characteristics of ccRCC patients that could discriminate metastasis, pathological stage, recurrence and prognosis in ccRCC patients. The Kaplan-Meier survival curve and the log-rank test revealed FZD1 was higher in lower clinical stage and grade that correlated with better overall survival (OS) and disease-free survival (DFS) in total and subgroups of ccRCC patients. Both univariate and multivariate cox regression analysis indicated that high FZD1 level was an independent predictor of good prognosis for OS (HR 0.569, P=0.001) and DFS (HR 0.559, P=0.036) in ccRCC patients. Using cBioportal program, less than 1% mutation in the patients with renal cancer was observed, the alterations in FZD1 were correlated with better OS (P=0.0404) in ccRCC patients. Finally, the result of KEGG pathway analysis predicted seven potential pathways that FZD1 and its related genes got involved in ccRCC, including Hippo signaling pathway. This indicated potential therapeutic targets of ccRCC. In conclusion, our results suggested that expression status of FZD1 had a diagnostic value and prognostic value in ccRCC patients, it also may serve as a potential drug target to relieve sunitinib resistance in renal cancer patients.

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.

Therapeutic Targeting of Sunitinib-Induced AR Phosphorylation in Renal Cell Carcinoma.

Androgen receptor (AR) plays a crucial role in the development and progression of prostate cancer. AR expression has also been reported in other solid tumors, including renal cell carcinoma (RCC), but its biological role here remains unclear. Through integrative analysis of a reverse phase protein array, we discovered increased expression of AR in an RCC patient-derived xenograft model of acquired resistance to the receptor tyrosine kinase inhibitor (RTKi) sunitinib. AR expression was increased in RCC cell lines with either acquired or intrinsic sunitinib resistance in vitro An AR signaling gene array profiler indicated elevated levels of AR target genes in sunitinib-resistant cells. Sunitinib-induced AR transcriptional activity was associated with increased phosphorylation of serine 81 (pS81) on AR. Additionally, AR overexpression resulted in acquired sunitinib resistance and the AR antagonist enzalutamide-induced AR degradation and attenuated AR downstream activity in sunitinib-resistant cells, also indicated by decreased secretion of human kallikrein 2. Enzalutamide-induced AR degradation was rescued by either proteasome inhibition or by knockdown of the AR ubiquitin ligase speckle-type POZ protein (SPOP). In vivo treatment with enzalutamide and sunitinib demonstrated that this combination efficiently induced tumor regression in a RCC model following acquired sunitinib resistance. Overall, our results suggest the potential role of AR as a target for therapeutic interventions, in combination with RTKi, to overcome drug resistance in RCC.Significance: These findings highlight the therapeutic potential of targeting the androgen receptor to overcome RCC resistance to receptor tyrosine kinase inhibitors. Cancer Res; 78(11); 2886-96. ©2018 AACR.