Sunitinib-resistant Renal Cell Carcinoma Cells Research Articles

Abstract 7584: The solute transported xCT (SLC7A11) modulates lysosome biogenesis and microRNA trafficking in drug resistant renal cell carcinoma

Abstract Multi-tyrosine kinase inhibitors (TKIs) like sunitinib bind to over 50 kinases and inhibit several receptors such as VEGFR-1, VEGFR-2, and VEGFR-3 receptors, PDGFRa and PDGFRb, RET, cKIT, and FLT3. TKIs are effective drugs for the treatment of renal cell carcinoma (RCC) but intrinsic/acquired resistance remains a major hurdle. One of the proposed models for TKI resistance is drug sequestration by lysosomes. The solute transporter xCT is a cystine/glutamate antiporter that plays a role in the production of antioxidants in cancer cells. Our lab has found that SLC7A11, the gene that encodes xCT, is overexpressed in sunitinib resistant RCC cells. LAMP1 expression was also increased in sunitinib resistant RCC cells, suggesting an upregulation of lysosome biogenesis. The biochemical knock down of SLC7A11 by shRNA showed a decrease in LAMP1 expression, suggesting a decrease in lysosome biogenesis. SEC24D is a key catalytic component of COPII complex involved in formation of exosomes and secretory pathways. Our preliminary data showed that reduction in xCT expression was associated with a decrease in SEC24D protein levels which was associated with a reduction in the extracellular export and, consequently, an increase in intracytoplasmic levels of miRNA 605, a known tumor suppressor. We are currently testing the hypothesis that xCT may modulate lysosomes biogenesis and micro-RNA trafficking in several clear cell and translation renal cell carcinoma models. In summary, a better understanding of the role of xCT in the regulation of intracytoplasmic organelles may lead to the identification of a novel therapeutic strategy to overcome TKI resistance in RCC. Citation Format: Abbas Jawadwala, Christopher Rupert, Remi Adelaiye-Ogala, Roberto Pili, Sreenivasulu Chintala, Ashley Orillion, May Elbanna, Nur Damayanti, Ilaria Delle Fontane. The solute transported xCT (SLC7A11) modulates lysosome biogenesis and microRNA trafficking in drug resistant renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7584.

Elucidation and Regulation of Tyrosine Kinase Inhibitor Resistance in Renal Cell Carcinoma Cells from the Perspective of Glutamine Metabolism.

Tyrosine kinase inhibitors (TKIs) play a crucial role in the treatment of advanced renal cell carcinoma (RCC). However, there is a lack of useful biomarkers for assessing treatment efficacy. Through urinary metabolite analysis, we identified the metabolites and pathways involved in TKI resistance and elucidated the mechanism of TKI resistance. To verify the involvement of the identified metabolites obtained from urine metabolite analysis, we established sunitinib-resistant RCC cells and elucidated the antitumor effects of controlling the identified metabolic pathways in sunitinib-resistant RCC cells. Through the analysis of VEGFR signaling, we aimed to explore the mechanisms underlying the antitumor effects of metabolic control. Glutamine metabolism has emerged as a significant pathway in urinary metabolite analyses. In vitro and in vivo studies have revealed the antitumor effects of sunitinib-resistant RCC cells via knockdown of glutamine transporters. Furthermore, this antitumor effect is mediated by the control of VEGFR signaling via PTEN. Our findings highlight the involvement of glutamine metabolism in the prognosis and sunitinib resistance in patients with advanced RCC. Additionally, the regulating glutamine metabolism resulted in antitumor effects through sunitinib re-sensitivity in sunitinib-resistant RCC. Our results are expected to contribute to the more effective utilization of TKIs with further improvements in prognosis through current drug therapies.

Potential therapeutic target secretogranin II might cooperate with hypoxia-inducible factor 1α in sunitinib-resistant renal cell carcinoma.

Multitargeted receptor tyrosine kinase inhibitors, including vascular endothelial growth factor (VEGF) inhibitors, such as sunitinib, have been used as the primary targeted agents for patients with recurrent or distant metastasis of advanced renal cell carcinoma (RCC). However, endogenous or acquired sunitinib resistance has become a significant therapeutic problem. Therefore, we focused on mechanisms of sunitinib resistance in RCC. First, we undertook RNA sequencing analysis using previously established sunitinib-resistant RCC (SUR-Caki1, SUR-ACHN, and SUR-A498) cells. The results showed increased expression of secretogranin II (SCG2, chromogranin C) in SUR-RCC cells compared to parental cells. The Cancer Genome Atlas database showed that SCG2 expression was increased in RCC compared to normal renal cells. In addition, the survival rate of the SCG2 high-expression group was significantly lower than that of the RCC low-expression group. Thus, we investigated the involvement of SCG2 in sunitinib-resistant RCC. In vitro analysis showed that migratory and invasive abilities were suppressed by SCG2 knockdown SUR cells. As SCG2 was previously reported to be associated with angiogenesis, we undertook a tube formation assay. The results showed that suppression of SCG2 inhibited angiogenesis. Furthermore, coimmunoprecipitation assays revealed a direct interaction between SCG2 and hypoxia-inducible factor 1α (HIF1α). Expression levels of VEGF-A and VEGF-C downstream of HIF1α were found to be decreased in SCG2 knockdown SUR cells. In conclusion, SCG2 could be associated with sunitinib resistance through VEGF regulation in RCC cells. These findings could lead to a better understanding of the VHL/HIF/VEGF pathway and the development of new therapeutic strategies for sunitinib-resistant RCC.

Extracellular Vesicle-Mediated Transfer of LncRNA IGFL2-AS1 Confers Sunitinib Resistance in Renal Cell Carcinoma.

Extracellular vesicle-packaged IGFL2-AS1 promotes sunitinib resistance by regulating TP53INP2-triggered autophagy, implicating this lncRNA as a potential therapeutic target in renal cell carcinoma.

Abstract 5384: Cell growth of sunitinib-resistant renal cell carcinoma was inhibited by Shikonin through necroptosis induction and Akt/mTOR signaling inhibition

Abstract Despite the establishment of new therapy options during the last decades, advanced renal cell carcinoma (RCC) remains an uncurable disease, due to evolving drug-resistance. New, more efficient treatment strategies are still in urgent demand. Shikonin (SHI) from Traditional Chinese Medicine has exhibited anti-tumor properties in several tumor entities. Thus, in the current investigation we evaluated the impact of SHI on progressive growth and metastatic behavior, not only in therapy-sensitive (parental) but also in therapy-resistant RCC cells. Parental and sunitinib-resistant RCC cells, Caki-1, 786-O, KTCTL-26, and A498, were treated with SHI. Tumor cell growth, proliferation, clonogenic capacity, cell cycle phase distribution, induction of cell death (apoptosis and necroptosis) and the expression and activity of regulating and signaling proteins were evaluated. Moreover, the adhesion and chemotactic activity of the RCC cells after exposure to SHI were investigated. SHI did not affect cell motility but led to significant inhibition of progressive tumor cell growth in both therapy-sensitive and therapy-resistant RCC cells. Thereby, SHI significantly inhibited the growth, proliferation and clone formation in parental and sunitinib-resistant RCC cells by G2/M phase arrest through down-regulation of cell cycle activating proteins, blocking cell division. Furthermore, SHI induced apoptosis and necroptosis by activating necrosome complex proteins. Concomitantly, SHI impaired the Akt/mTOR pathway, pivotal for cell survival and growth. Thus, SHI may hold promise as an additive option in treating patients with advanced and therapy-resistant RCC. Citation Format: Eva Juengel, Sascha D. Markowitsch, Olesya Vakhrusheva, Yasminn Akele, Jovanna Kitanovic, Patricia Schupp, Kimberly S. Slade, Anita Thomas, Igor Tsaur, Rene Mager, Thomas Efferth, Axel Haferkamp. Cell growth of sunitinib-resistant renal cell carcinoma was inhibited by Shikonin through necroptosis induction and Akt/mTOR signaling inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5384.

Shikonin Inhibits Cell Growth of Sunitinib-Resistant Renal Cell Carcinoma by Activating the Necrosome Complex and Inhibiting the AKT/mTOR Signaling Pathway.

Simple SummaryAdvanced renal cell carcinoma (RCC) remains an incurable disease, despite the establishment of new therapeutic options during the last decades. Unfortunately, drug resistance inevitably evolves, making better treatment strategies essential. Shikonin (SHI) from traditional Chinese medicine has shown promising antitumor properties with other tumor entities. Thus, in the current investigation, we evaluated the impact of SHI on therapy-sensitive and therapy-resistant RCC cells. SHI led to significant inhibition of progressive tumor cell growth in both therapy-sensitive and therapy-resistant RCC cells. This was accompanied by cell cycle arrest and reduction in cell cycle activating proteins, which results in blockage of cell division. SHI also activated necrosome complex proteins, leading to necroptosis, a programmed cell death. Furthermore, SHI inhibited the AKT/mTOR pathway, pivotal for cell survival and growth. Thus, SHI may hold promise in improving the treatment of RCC, even in its advanced form.Therapy resistance remains a major challenge in treating advanced renal cell carcinoma (RCC), making more effective treatment strategies crucial. Shikonin (SHI) from traditional Chinese medicine has exhibited antitumor properties in several tumor entities. We, therefore, currently investigated SHI’s impact on progressive growth and metastatic behavior in therapy-sensitive (parental) and therapy-resistant Caki-1, 786-O, KTCTL-26, and A498 RCC cells. Tumor cell growth, proliferation, clonogenic capacity, cell cycle phase distribution, induction of cell death (apoptosis and necroptosis), and the expression and activity of regulating and signaling proteins were evaluated. Moreover, the adhesion and chemotactic activity of the RCC cells after exposure to SHI were investigated. SHI significantly inhibited the growth, proliferation, and clone formation in parental and sunitinib-resistant RCC cells by G2/M phase arrest through down-regulation of cell cycle activating proteins. Furthermore, SHI induced apoptosis and necroptosis by activating necrosome complex proteins. Concomitantly, SHI impaired the AKT/mTOR pathway. Adhesion and motility were cell line specifically affected by SHI. Thus, SHI may hold promise as an additive option in treating patients with advanced and therapy-resistant RCC.

CircRNA_001895 promotes sunitinib resistance of renal cell carcinoma through regulation of apoptosis and DNA damage repair

Sunitinib, an inhibitor of receptor tyrosine kinase, possesses anti-tumor activity in renal cell carcinoma (RCC) through its anti-angiogenic effects. However, patients with advanced RCC are resistant to sunitinib. Dysregulated circRNAs has been shown to be associated with drug resistance in various tumors. However, little is known about the effect of circRNA_001895 on sunitinib resistance of RCC. First, the expression of circRNA_001895 was found to be higher in sunitinib-resistant RCC tissues than chemosensitive tumor tissues. Half maximal inhibitory concentration of sunitinib-resistant RCC cells (786-O/R and ACHN/R) was higher than sunitinib-sensitive 786-O and ACHN cells. CircRNA_001895 was also upregulated in 786-O/R and ACHN/R cells. Second, data from colony formation and flow cytometry analysis showed that knockdown of circRNA_001895 suppressed cell proliferation and promoted cell apoptosis in 786-O/R and ACHN/R cells. Moreover, the protein expression of phosphorylated histone H2AX (γH2AX) was enhanced, while phosphorylated DNA-dependent protein kinase (p-DNA-PK) and Rad51 were reduced in 786-/R and ACHN/R by knockdown of circRNA_001895. Lastly, knockdown of circRNA_001895 conferred sensitivity of in vivo tumor growth to sunitinib. In conclusion, circRNA_001895 was implicated in the sunitinib resistance in RCC through regulation of apoptosis and DNA damage repair, suggesting that circRNA_001895 might be a potential therapeutic target for advanced RCC.

Lefty A is involved in sunitinib resistance of renal cell carcinoma cells via regulation of IL-8.

Renal cell carcinoma (RCC) is the third most frequent malignancy within urological oncology. Sunitinib has been used as the standard of treatment for first-line RCC therapy. Understanding mechanisms of sunitinib resistance in RCC cell is important for clinical therapy and drug development. We established sunitinib resistant RCC cells by treating cells with increasing concentrations of sunitinib and named resistant cells as RCC/SR. Lefty A, animportant embryonic morphogen, was increased in RCC/SR cells. Targeted inhibition of Lefty via its siRNAs restored the sensitivity of renal resistant cells to sunitinib treatment. It was due to that si-Lefty can decrease the expression of interleukin-8 (IL-8) in RCC/SR cells. Knockdown of IL-8 abolished Lefty-regulated sunitinib sensitivity of RCC cells. Mechanistically, Lefty can regulate IL-8 transcription via activation of p65, one major transcription factor of IL-8. Collectively, our present revealed that LeftyAcan regulate sunitinib sensitivity of RCC cells of via NF-κB/IL-8 signals. It indicated that targeted inhibition of Lefty might be a potent approach to overcome sunitinib resistance of RCC.

RRM2 Regulates Sensitivity to Sunitinib and PD-1 Blockade in Renal Cancer by Stabilizing ANXA1 and Activating the AKT Pathway.

Renal cell carcinoma (RCC) is a malignant tumor of the kidneys. Approximately 70% of RCC cases are clear cell renal cell carcinoma with von Hippel–Lindau (VHL) gene mutation and activation of the vascular endothelial growth factor (VEGF) pathway. Tyrosine kinase inhibitors (TKIs) targeting VEGF have emerged as promising agents for RCC treatment. Apart from primary resistance, acquired resistance to TKIs after initial tumor regression is common in RCC. Recently, immune checkpoint inhibition, including PD‐1/PD‐L1 blockade, alone or in combination with TKIs has improved the overall survival of patients with RCC. Ribonucleotide reductase subunit M2 (RRM2) has been reported in many types of cancer and has been implicated in tumor progression. However, the role of RRM2 in TKIs resistance in RCC remains unclear. In this study, the authors have demonstrated that RRM2 is upregulated in sunitinib‐resistant RCC cells and patient tissues. They also find that RRM2 stabilizes ANXA1 and activates the AKT pathway independent of its ribonucleotide reductase activity, promoting sunitinib resistance in RCC. Moreover, RRM2 regulated antitumor immune responses, and knockdown of RRM2 enhance the anti‐tumor efficiency of PD‐1 blockade in renal cancer. Collectively, these results suggest that aberrantly expressed RRM2 may be a promising therapeutic target for RCC.

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

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

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

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

Linkage of Drug Resistance and Metabolome Shift in Renal Cell Carcinoma Cells

Metabolome analysis is an approach to investigate cell characteristics from the metabolites that are constantly produced and changed by those cells. We conducted a metabolome analysis of the response of 786-O renal cell carcinoma (RCC) cells to histone deacetylase (HDAC) inhibitors, which are expected to increase anticancer drug sensitivity, and compared the response with that of drug-resistant cells. Trichostatin A (TSA), an HDAC inhibitor, increased the sensitivity of 786-O cells to sunitinib. Moreover, TCA cycle and nucleotide metabolism of the cells were promoted. The findings that acetylated p53 (active form) and early apoptotic cells were increased suggests that the mechanism involved enhancement of mitochondrial metabolism and function. In addition, established sunitinib-resistant RCC cells were exposed to a combination of sunitinib and TSA, resulting in significant growth inhibition. Principal component analysis revealed that the parent and resistant cells were obviously different, but approximately half their fluctuations were illustrated by the same pathways. In summary, it was suggested that TSA reduced sunitinib resistance by triggering intracellular metabolome shifts in energy metabolism. This was the first recognized mechanism of action of TSA as an HDAC inhibitor.

Long noncoding RNA SNHG12 promotes tumour progression and sunitinib resistance by upregulating CDCA3 in renal cell carcinoma

Renal cell carcinoma (RCC) is one of the most frequently observed malignant tumours in the urinary system and targeted drug resistance is quite common in RCC. Long noncoding RNA SNHG12 (lncRNA SNHG12) has emerged as a key molecule in numerous human cancers, but its functions in renal cell carcinoma (RCC) sunitinib resistance remain unclear. In this study, we found SNHG12 was highly expressed in RCC tissues and in sunitinib-resistant RCC cells and was associated with a poor clinical prognosis. SNHG12 promoted RCC proliferation, migration, invasion and sunitinib resistance via CDCA3 in vitro. Mechanically, SNHG12 bound to SP1 and prevented the ubiquitylation-dependent proteolysis of SP1. Stabilised SP1 bound to a specific region in the promoter of CDCA3 and increased CDCA3 expression. Furthermore, in vivo experiments showed that SNHG12 increased tumour growth and that knocking down SNHG12 could reverse RCC sunitinib resistance. Our study revealed that the lncRNA SNHG12/SP1/CDCA3 axis promoted RCC progression and sunitinib resistance, which could provide a new therapeutic target for sunitinib-resistant RCC.

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.

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 < 0.05 was considered statistically significant. As for label-free phosphoproteomics, false discovery rate <0.01 and phosphosite possibility >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.

Sunitinib induces early histomolecular changes in a subset of renal cancer cells that contribute to resistance.

Sunitinib is the standard-of-care, first-line treatment for advanced renal cell carcinoma (RCC). Characteristics of treatment-resistant RCC have been described; however, complex tumor adaptation mechanisms obstruct the identification of significant operators in resistance. We hypothesized that resistance is a late manifestation of early, treatment-induced histomolecular alterations; therefore, studying early drug response may identify drivers of resistance. We describe an epithelioid RCC growth pattern in RCC xenografts, which emerges in sunitinib-sensitive tumors and is augmented during resistance. This growth modality is molecularly and morphologically related to the RCC spheroids that advance during in vitro treatment. Based on time-lapse microscopy, mRNA and microRNA screening, and tumor behavior-related characteristics, we propose that the spheroid and adherent RCC growth patterns differentially respond to sunitinib. Gene expression analysis indicated that sunitinib promoted spheroid formation, which provided a selective survival advantage under treatment. Functional studies confirm that E-cadherin is a key contributor to the survival of RCC cells under sunitinib treatment. In summary, we suggest that sunitinib-resistant RCC cells exist in treatment-sensitive tumors and are histologically identifiable.-Lichner, Z., Saleeb, R., Butz, H., Ding, Q., Nofech-Mozes, R., Riad, S., Farag, M., Varkouhi, A. K., dos Santos, C. C., Kapus, A., Yousef, G. M. Sunitinib induces early histomolecular changes in a subset of renal cancer cells that contribute to resistance.

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.

MP72-11 TARGETING SUNITINIB RESISTANT CELLS BY TUMOR SUPPRESSIVE MIRNA-99A-3P THROUGH RIBONUCLEOTIDE REDUCTASE REGULATION IN CLEAR CELL RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology II1 Apr 2018MP72-11 TARGETING SUNITINIB RESISTANT CELLS BY TUMOR SUPPRESSIVE MIRNA-99A-3P THROUGH RIBONUCLEOTIDE REDUCTASE REGULATION IN CLEAR CELL RENAL CELL CARCINOMA Youichi Osako, Hirofumi Yoshino, Takashi Sakaguchi, Satoshi Sugita, Shuichi Tatarano, Takashi Yamane, Hideki Enokida, and Masayuki Nakagawa Youichi OsakoYouichi Osako More articles by this author , Hirofumi YoshinoHirofumi Yoshino More articles by this author , Takashi SakaguchiTakashi Sakaguchi More articles by this author , Satoshi SugitaSatoshi Sugita More articles by this author , Shuichi TataranoShuichi Tatarano More articles by this author , Takashi YamaneTakashi Yamane More articles by this author , Hideki EnokidaHideki Enokida More articles by this author , and Masayuki NakagawaMasayuki Nakagawa More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2295AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Sunitinib is the most common targeting drug for metastatic renal cell carcinoma (RCC), however resistance to sunitinib is a large problem in clinical aspect. In this study, we focused on miR-99a-3p, which showed decreased expression in sunitinib-resistant RCC based on previous screening analyses, and we investigated the mechanism of sunitinib tolerance associated with miR-99a-3p and the molecular network controlled by miR-99a-3p. METHODS Expression levels of miR-99a-3p and its candidate target genes in the RCC cell lines (786-o, ACHN, and our sunitinib-resistant cell (SU-R-786-o)) and RCC clinical specimens were evaluated by qRT-PCR. For the gain of function study, cell proliferation, apoptosis, and colony formation assay were performed in miR-99a-3p transfected RCC cell lines. Putative target genes were determined by the analyses with publically available database such as GEO and TargetScan, as well as RNA sequencing expression analysis using SU-R-786-o. Loss of functional studies of the target gene was performed using si-RNA. Overall survival between high and low expression of the target gene was analyzed by the Kaplan-Meier method using the database of The Cancer Genome Atlas (TCGA). RESULTS The expression levels of miR-99a-3p were down regulated in RCC clinical specimens and the RCC cell lines compared to normal kidneys. In addition, its expression in SU-R-786-o was much lower compared to the parent cell. Restoration of miR-99a-3p in the RCC cells including SU-R-786-o significantly inhibited cell proliferation (p<0.0001) through induction of apoptosis (p<0.0001). RRM2, involved in the synthesis of deoxynucleotides that is necessary for polymerization and repair of DNA, was identified as a direct target by miR-99a-3p based on target analyses. Loss of functional studies using si-RRM2 showed that cell proliferation was significantly inhibited through induction of apoptosis especially in SU-R-786-o (p<0.0001). Kaplan-Meier analysis revealed that the patients (n= 534) with high expression of RRM2 showed lower overall survival time than those with low expression according to the TCGA database (p<0.0001). CONCLUSIONS miR-99a-3p might have tumor suppressive function through induction of cell apoptosis in sunitinib-resistant RCC cells. To the best of our knowledge, this is the first report demonstrating that miR-99a-3p directly regulate RRM2. The identification of novel target gene regulated by tumor-suppressive miR-99a-3p in sunitinib resistant RCC cells may lead to a better understanding of resistant mechanism and the development of new therapeutic strategies. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e956 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Youichi Osako More articles by this author Hirofumi Yoshino More articles by this author Takashi Sakaguchi More articles by this author Satoshi Sugita More articles by this author Shuichi Tatarano More articles by this author Takashi Yamane More articles by this author Hideki Enokida More articles by this author Masayuki Nakagawa More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Molecular mechanism mediating cytotoxic activity of axitinib in sunitinib-resistant human renal cell carcinoma cells.

This study aimed to clarify the molecular mechanism mediating the cytotoxicity of axitinib, a selective inhibitor of the vascular endothelial growth factor receptor (VEGFR), in sunitinib-resistant renal cell carcinoma (RCC). In our previous study (Sakai et al. in BJU Int 112:E211-E220, 2013), a human RCC cell line, ACHN, resistant to sunitinib (ACHN/R), was developed from a parental cell line (ACHN/P). Differences in molecular phenotypes following treatment with sunitinib or axitinib between these two cell lines were compared. ACHN/R showed an approximately fivefold higher IC50 of sunitinib than ACHN/P; however, there was no significant difference in the sensitivity to axitinib between these two cell lines. In ACHN/R, despite the lack of a difference in the phosphorylated (p)-Akt or STAT-3 expression between treatment with sunitinib and axitinib, the expression of p-p44/42 mitogen-activated protein kinase (MAPK) and p-VEGFR-2 after treatment with axitinib was markedly down-regulated compared with those after treatment with sunitinib. Furthermore, additional treatment of ACHN/R with an inhibitor of MAPK kinase significantly enhanced the cytotoxic activity of sunitinib, but not that of axitinib. In vivo growth of ACHN/R in nude mice after treatment with axitinib was significantly inhibited compared with that following treatment with sunitinib, accompanying the marked inhibition of angiogenesis. Antitumor activity of axitinib in RCC cells even after the acquisition of resistance to sunitinib could be explained, at least in part, by the inactivation of p44/42 MAPK and VEGFR-2, which were persistently phosphorylated in sunitinib-resistant RCC cells under treatment with sunitinib.

Molecular analysis of sunitinib resistant renal cell carcinomacells after sequential treatment with RAD001(everolimus) or sorafenib.

Sequential application of target drugs is standard procedure after renal cell carcinoma (RCC) patients develop resistance. To optimize the sequence, antitumour effects of the mTOR inhibitor RAD001 or the tyrosine kinase inhibitor (TKI) sorafenib on RCC cells with acquired resistance to the TKI sunitinib was evaluated. RCC cells were exposed to 1 μM sunitinib for 24 hrs (as control) and for 8 weeks (to induce resistance) and then switched to RAD001 (5 nM) or sorafenib (5 μM) for a further 8 weeks. Tumour cell growth, cell cycle progression, cell cycle regulating proteins and intracellular signalling were then investigated. Short-term application of sunitinib (24 hrs) induced cell growth blockade with accumulation in the G2/M phase. RCC cells became resistant to sunitinib after 8 weeks, demonstrated by accelerated cell growth along with enhanced cdk1, cdk2, loss of p27, activation of Akt, Rictor and Raptor. Switching to sorafenib only slightly reduced growth of the sunitinib resistant RCC cells and molecular analysis indicated distinct cross-resistance. In contrast, full response was achieved when the cancer cells were treated with RAD001. p19 and p27 strongly increased, phosphorylated Akt, Rictor and Raptor decreased and the tumour cells accumulated in G0/G1. It is concluded that an mTOR-inhibitor for second-line therapy could be the strategy of choice after first-line sunitinib failure.