Abstract
The aim of this study was to compare effect of everolimus on growth of different renal cell carcinoma (RCC) populations and develop experimental design to measure the early response of everolimus in clear cell RCC (ccRCC) cell lines including renal cancer stem cells. Effect of everolimus on RCC cell lines which include primary (786-0) and metastatic (ACHN) RCC cell lines as well as heterogenous populations of tumor cells of different histological RCC subtypes (clear cell RCC and papillary RCC) was measured when treated with everolimus in the range of 1–9 µM. Gene expression profiling using microarray was performed to determine the early response to everolimus in ccRCC cell lines after optimizing concentration of drug. Gene Set Enrichment Analysis (GSEA) was done which mainly focused on basic genes related to mTOR, hormonal and metabolic pathways. Everolimus acts on RCC cells in a dose—dependent manner. In all examined cell lines IC50 dose was possible to calculate after the third day of treatment. In ccRCC lines (parental and stem cell) everolimus changes expression of mTOR complexes elements and elements of related pathways when treated with optimized doses of drug. Characteristic expression profile for ccRCC cells at an early exposure time to everolimus is to elucidate. Wevarie include some basic observations derived from data analysis in the context of mechanism of action of drug with a view to better understand biology of renal cancer cells.
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Abbreviations
- 4EBP1:
-
Eukaryotic translation initiation factor 4E-binding protein 1
- 786-O[0]:
-
Primary tumor cell line
- ACHN:
-
Malignant pleural effusion of metastatic renal adenocarcinoma cell line
- ACTH:
-
Adrenocorticotropic hormone
- ASE:
-
Healthy kidney cells
- ccRCC-PCSC:
-
Clear cell renal cell carcinoma - parental cell line
- ccRCC-CSC:
-
Clear cell renal cell carcinoma–stem cell line
- GSEA:
-
Gene Set Enrichment Analysis
- HCG:
-
Human chorionic gonadotropin
- HIF1α:
-
Hypoxia-inducible factor 1- alpha
- HIF2α:
-
Hypoxia-inducible factor 2-alpha
- HKCSC:
-
Human kidney cancer stem cells
- HPKCSC:
-
Human parental kidney cancer stem cells
- mTORC1:
-
Mechanistic target of rapamycin complex 1
- mTORC2:
-
Mechanistic target of rapamycin complex 2
- ppRCC-PCSC:
-
Papillary renal cell carcinoma – parental stem cell line
- ppRCC-CSC:
-
Papillary renal cell carcinoma - stem cell line
- RCC:
-
Renal cell carcinoma
- S6K1:
-
p70 ribosomal protein S6 kinase 1)
References
Chen, F., et al. (2016). Multilevel genomics-based taxonomy of renal cell carcinoma. Cell Reports, 14(10), 2476–2489.
Sircar, K., Rao, P., Jonasch, E., Monzon, F. A., & Tamboli, P. (2013). Contemporary approach to diagnosis and classification of renal cell carcinoma with mixed histologic features. Chinese Journal of Cancer, 32(6), 303–311.
Khan, M. I., Czarnecka, A. M., Helbrecht, I., Bartnik, E., Lian, F., & Szczylik, C. (2015). Current approaches in identification and isolation of human renal cell carcinoma cancer stem cells. Stem Cell Research & Therapy, 6, 178.
Peired, A. J., Sisti, A., & Romagnani, P. (2016). Renal cancer stem cells: characterization and targeted therapies. Stem Cells International, 2016.
Bergmann, L., et al. (2015). Everolimus in metastatic renal cell carcinoma after failure of initial anti-VEGF therapy: final results of a noninterventional study. BMC Cancer, 15, 303.
Escudier, B., et al. (2016). Renal cell carcinoma: ESMO clinical practice guidelines for diagnosis, treatment and follow-up. Annals of Oncology, 27(5), v58–v68.
Formica, R. N. et al. (2004). The evolving experience using everolimus in clinical transplantation. Transplantation Proceedings, 36(2 Suppl), 495S–499S.
Lane, H. A., et al. (2009). mTOR inhibitor RAD001 (Everolimus) has antiangiogenic/vascular properties distinct from a VEGFR tyrosine kinase inhibitor. Clinical Cancer Research, 15(5), 1612–1622.
Racila, R. G., Melchinger, W., Finke, J., & Marks, R. E. (2010). Everolimus enhances immunomodulation of alloreative T cells by multipotent stromal cells due to transforming growth factor - β Dependent Mechanisms. Blood, 116(21), 2545–2545.
Jhanwar-Uniyal, M., Gillick, J. L., Neil, J., Tobias, M., Thwing, Z. E., & Murali, R. (2015). Distinct signaling mechanisms of mTORC1 and mTORC2 in glioblastoma multiforme: a tale of two complexes. Advances in Biological Regulation, 57, 64–74.
Toschi, A., Lee, E., Xu, L., Garcia, A., Gadir, N., & Foster, D. A. (2009). Regulation of mTORC1 and mTORC2 complex assembly by phosphatidic acid: competition with rapamycin. Molecular and Cellular Biology, 29(6), 1411–1420.
Battelli, C., & Cho, D. C. (2011). mTOR inhibitors in renal cell carcinoma. Therapy, 8(4), 359–367.
Shimobayashi, M., & Hall, M. N. (2014). Making new contacts: the mTOR network in metabolism and signalling crosstalk. Nature Reviews Molecular Cell Biology, 15(3), 155–162.
Galardi, S., et al. (2016). Resetting cancer stem cell regulatory nodes upon MYC inhibition. EMBO Reports, 17(12), 1872–1889.
Simon, M. Metabolic outcomes of c-MYC, p53 and mTOR regulation by HIF. Grantome.
Fagnocchi, L., et al. (2016). A Myc-driven self-reinforcing regulatory network maintains mouse embryonic stem cell identity. Nature Communications, 7.
Cancer Genome Atlas Research Network (2013). Comprehensive molecular characterization of clear cell renal cell carcinoma. Nature, 499(7456), 43–49.
Altwein, J. (1983). Is renal cancer a hormone-dependent tumour and how does it respond to hormonal treatment? Round table report. In Cancer of the prostate and kidney (pp. 705–709). Boston, MA: Springer.
Czarnecka, A. M., Niedzwiedzka, M., Porta, C., & Szczylik, C. (2016). Hormone signaling pathways as treatment targets in renal cell cancer (Review). International Journal of Oncology, 48(6), 2221–2235.
Bojar, H. (1984). “Hormone responsiveness of renal cancer. World Journal of Urology, 2(2), 92–98.
Khan, M. I., et al. (2016) Comparative gene expression profiling of primary and metastatic renal cell carcinoma stem cell-like cancer cells. PLoS ONE, 11(11).
Al-Nasiry, S., Geusens, N., Hanssens, M., Luyten, C., & Pijnenborg, R. (2007). The use of Alamar Blue assay for quantitative analysis of viability, migration and invasion of choriocarcinoma cells. Human Reproduction (Oxford, England), 22(5), 1304–1309.
Majewska, A., Gajewska, M., Dembele, K., Maciejewski, H., Prostek, A., & Jank, M. (2016). Lymphocytic, cytokine and transcriptomic profiles in peripheral blood of dogs with atopic dermatitis. BMC Veterinary Research, 12(1).
Bohler, A., et al. (2016). Reactome from WIKIpathways perspective. PLOS.
Subramanian, A., et al. (2005). Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proceedings of the National Academy of Sciences of the United States of America, 102(43), 15545–15550.
Zhang, H., et al. (2013). A comparison of Ku0063794, a dual mTORC1 and mTORC2 inhibitor, and temsirolimus in preclinical renal cell carcinoma models. PloS One, 8(1), e54918.
Liu, Y., Zhang, X., Liu, J., Hou, G., Zhang, S., & Zhang, J. (2014). Everolimus in combination with letrozole inhibit human breast cancer MCF-7/Aro stem cells via PI3K/mTOR pathway: an experimental study. Tumour Biology, 35(2), 1275–1286.
Zhao, Y., & Sun, Y. (2012). Targeting the mTOR-DEPTOR Pathway by CRL E3 ubiquitin ligases: therapeutic application. Neoplasia, 14(5), 360–367.
Malaguarnera, R., & Belfiore, A. (2014). The emerging role of insulin and insulin-like growth factor signaling in cancer stem cells. Frontiers in Endocrinology, 5, 10.
Masola, V., Zaza, G., Granata, S., Gambaro, G., Onisto, M., & Lupo, A. (2013). Everolimus-induced epithelial to mesenchymal transition in immortalized human renal proximal tubular epithelial cells: key role of heparanase. Journal of Translational Medicine, 11, 292.
Shen, Y.-A., Wang, C.-Y., Hsieh, Y.-T., Chen, Y.-J., & Wei, Y.-H. (2015). Metabolic reprogramming orchestrates cancer stem cell properties in nasopharyngeal carcinoma. Cell Cycle (Georgetown, Texas), 14(1), 86–98.
Russell, R. C., Fang, C., & Guan, K.-L. (2011). An emerging role for TOR signaling in mammalian tissue and stem cell physiology. Development (Cambridge, England), 138(16), 3343–3356.
Lee, K.-W., et al. (2010). Rapamycin promotes the osteoblastic differentiation of human embryonic stem cells by blocking the mTOR pathway and stimulating the BMP/Smad pathway. Stem Cells and Development, 19(4), 557–568.
Chlenski, A., et al. (2006). SPARC expression is associated with impaired tumor growth, inhibited angiogenesis and changes in the extracellular matrix. International Journal of Cancer, 118(2), 310–316.
Sakai, N., et al. (2001). SPARC expression in primary human renal cell carcinoma: upregulation of SPARC in sarcomatoid renal carcinoma. Human Pathology, 32(10), 1064–1070.
Efeyan, A., & Sabatini, D. M. (2010). mTOR and cancer: many loops in one pathway. Current Opinion in Cell Biology, 22(2), 169–176.
Song, W., et al. (2015). Infiltrating neutrophils promote renal cell carcinoma (RCC) proliferation via modulating androgen receptor (AR) → c-Myc signals. Cancer Letters, 368(1), 71–78.
Acknowledgements
Research supported by Ministry of Science and Higher Education “Diamond grant” no. DI2012007842.
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Contributions
The study was designed and developed by AK. Experiments were performed by AK, PK, AVK. Figures were prepared by AK and MIK. Design of experiments was based on AC and CS concepts and previous projects. Creating research design was performed by AK and supported by AMC and MIK. Literature search was performed by AK and supported by AMC. Design of data analysis was supported by MIK and AVK. The manuscript was written and drafted by AK. Draft of manuscript was edited by AK, AMC, AVK, MIK. Scientific work was supported and guided by CS.
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Electronic Supplementary material
Below is the link to the electronic supplementary material.
12015_2018_9804_MOESM3_ESM.xlsx
Supplementary table 3 (S3): List of common differentially expressed genes in everolimus treated HPKCSC and HKCSC cells. (XLSX 1623 KB)
12015_2018_9804_MOESM6_ESM.xlsx
Supplementary table 6 (S6): List of GO terms in common differentially altered genes between everolimus treated HPKCSC and HKCSC cells. (XLSX 10 KB)
12015_2018_9804_MOESM10_ESM.xlsx
Supplementary table 10 (S10): List of differentially expressed genes in everolimus treated HPKCSC cells with statistical data. (XLSX 288 KB)
12015_2018_9804_MOESM11_ESM.xlsx
Supplementary table 11(S11): List of differentially expressed genes in everolimus treated HKCSC cells with statistical data. (XLSX 652 KB)
12015_2018_9804_MOESM12_ESM.pdf
Supplementary table 12 (S12) Effects of everolimus on expression of genes in mTOR pathway in parental cell line. (PDF 39 KB)
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Supplementary table 14 (S14) Effects of everolimus on expression of genes in hormonal pathways in parental cell line. (PDF 35 KB)
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Supplementary table 15 (S15) Effects of everolimus on expression of genes in hormonal pathways in stem cells. (PDF 39 KB)
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Supplementary table 16 (S16) Effects of everolimus on expression of genes in metabolic pathways in parental cell line. (PDF 35 KB)
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Supplementary table 17 (S17) Effects of everolimus on expression of genes in metabolic pathways in stem cells. (PDF 32 KB)
12015_2018_9804_MOESM18_ESM.pdf
Supplementary table 18 (S18) Effects of everolimus on expression of genes in angiogenic pathways in parental cell line. (PDF 35 KB)
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Supplementary table 19 (S19) Effects of everolimus on expression of genes in angiogenic pathways in stem cells. (PDF 33 KB)
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Supplementary table 20 (S20) Effects of everolimus on expression of genes in calcium/bone metabolism pathways in parental cell line. (PDF 32 KB)
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Supplementary table 21 (S21) Table 21. Effects of everolimus on expression of genes in calcium/bone metabolism pathways in stem cell line. (PDF 30 KB)
12015_2018_9804_MOESM22_ESM.pdf
Figure S1. Effect of everolimus in 5 first subsequent days on a) primary tumor (786-O) b) lung metastases (ACHN). (PDF 218 KB)
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Figure S2. Effect of everolimus in 5 first subsequent days on a) clear cell carcinoma parental cell line (ccRCC-PCSC) b) papillary parental cell line (ppRCC-PCSC) c) cancer stem cells (CSC) d) healthy kidney (ASE). (PDF 413 KB)
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Kornakiewicz, A., Czarnecka, A.M., Khan, M.I. et al. Effect of Everolimus on Heterogenous Renal Cancer Cells Populations Including Renal Cancer Stem Cells. Stem Cell Rev and Rep 14, 385–397 (2018). https://doi.org/10.1007/s12015-018-9804-2
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DOI: https://doi.org/10.1007/s12015-018-9804-2