Skip to main content

Effect of Everolimus on Heterogenous Renal Cancer Cells Populations Including Renal Cancer Stem Cells

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.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

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

  1. Chen, F., et al. (2016). Multilevel genomics-based taxonomy of renal cell carcinoma. Cell Reports, 14(10), 2476–2489.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  2. 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.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  3. 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.

    Article  CAS  Google Scholar 

  4. Peired, A. J., Sisti, A., & Romagnani, P. (2016). Renal cancer stem cells: characterization and targeted therapies. Stem Cells International, 2016.

  5. 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.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  6. 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.

    Article  PubMed  CAS  Google Scholar 

  7. Formica, R. N. et al. (2004). The evolving experience using everolimus in clinical transplantation. Transplantation Proceedings, 36(2 Suppl), 495S–499S.

    Article  PubMed  CAS  Google Scholar 

  8. 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.

    Article  PubMed  CAS  Google Scholar 

  9. 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.

    Google Scholar 

  10. 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.

    Article  PubMed  CAS  Google Scholar 

  11. 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.

    Article  PubMed  CAS  Google Scholar 

  12. Battelli, C., & Cho, D. C. (2011). mTOR inhibitors in renal cell carcinoma. Therapy, 8(4), 359–367.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. 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.

    Article  PubMed  CAS  Google Scholar 

  14. Galardi, S., et al. (2016). Resetting cancer stem cell regulatory nodes upon MYC inhibition. EMBO Reports, 17(12), 1872–1889.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  15. Simon, M. Metabolic outcomes of c-MYC, p53 and mTOR regulation by HIF. Grantome.

  16. Fagnocchi, L., et al. (2016). A Myc-driven self-reinforcing regulatory network maintains mouse embryonic stem cell identity. Nature Communications, 7.

  17. Cancer Genome Atlas Research Network (2013). Comprehensive molecular characterization of clear cell renal cell carcinoma. Nature, 499(7456), 43–49.

    Article  CAS  Google Scholar 

  18. 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.

  19. 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.

    Article  PubMed  CAS  Google Scholar 

  20. Bojar, H. (1984). “Hormone responsiveness of renal cancer. World Journal of Urology, 2(2), 92–98.

    Article  Google Scholar 

  21. 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).

  22. 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.

    Article  CAS  Google Scholar 

  23. 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).

  24. Bohler, A., et al. (2016). Reactome from WIKIpathways perspective. PLOS.

  25. 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.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. 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.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. 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.

    Article  PubMed  CAS  Google Scholar 

  28. Zhao, Y., & Sun, Y. (2012). Targeting the mTOR-DEPTOR Pathway by CRL E3 ubiquitin ligases: therapeutic application. Neoplasia, 14(5), 360–367.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. 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.

    Article  PubMed  PubMed Central  Google Scholar 

  30. 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.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. 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.

    Article  Google Scholar 

  32. 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.

    Article  CAS  Google Scholar 

  33. 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.

    Article  PubMed  CAS  Google Scholar 

  34. 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.

    Article  PubMed  CAS  Google Scholar 

  35. 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.

    Article  PubMed  CAS  Google Scholar 

  36. Efeyan, A., & Sabatini, D. M. (2010). mTOR and cancer: many loops in one pathway. Current Opinion in Cell Biology, 22(2), 169–176.

    Article  PubMed  CAS  Google Scholar 

  37. 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.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

Research supported by Ministry of Science and Higher Education “Diamond grant” no. DI2012007842.

Author information

Authors and Affiliations

Authors

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.

Corresponding author

Correspondence to Anna Kornakiewicz.

Ethics declarations

Conflict of Interest

The authors indicate no potential conflicts of interest.

Electronic Supplementary material

Below is the link to the electronic supplementary material.

Supplementary table 1 (S1): List of differentially expressed genes in everolimus treated HPKCSC cells. (XLSX 186 KB)

Supplementary table2 (S2): List of differentially expressed genes in everolimus treated HKCSC cells. (XLSX 225 KB)

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)

Supplementary table 4 (S4): List of GO terms in everolimus treated HPKCSC cells. (XLSX 32 KB)

Supplementary table 5 (S5): List of GO terms in everolimus treated HKCSC cells. (XLSX 37 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)

Supplementary table 7 (S7): Pathways enrichment in everolimus treated HPKCSC cells. (XLSX 11 KB)

Supplementary table 8 (S8): Pathways enrichment in everolimus treated HKCSC cells. (XLSX 13 KB)

Supplementary table 9 (S9): Common pathways enriched between everolimus treated HPKCSC and HKCSC cells. (XLSX 12 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)

Supplementary table 13 (S13) Effects of everolimus on expression of genes in mTOR pathway in stem cells. (PDF 33 KB)

12015_2018_9804_MOESM14_ESM.pdf

Supplementary table 14 (S14) Effects of everolimus on expression of genes in hormonal pathways in parental cell line. (PDF 35 KB)

12015_2018_9804_MOESM15_ESM.pdf

Supplementary table 15 (S15) Effects of everolimus on expression of genes in hormonal pathways in stem cells. (PDF 39 KB)

12015_2018_9804_MOESM16_ESM.pdf

Supplementary table 16 (S16) Effects of everolimus on expression of genes in metabolic pathways in parental cell line. (PDF 35 KB)

12015_2018_9804_MOESM17_ESM.pdf

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)

12015_2018_9804_MOESM19_ESM.pdf

Supplementary table 19 (S19) Effects of everolimus on expression of genes in angiogenic pathways in stem cells. (PDF 33 KB)

12015_2018_9804_MOESM20_ESM.pdf

Supplementary table 20 (S20) Effects of everolimus on expression of genes in calcium/bone metabolism pathways in parental cell line. (PDF 32 KB)

12015_2018_9804_MOESM21_ESM.pdf

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)

12015_2018_9804_MOESM23_ESM.pdf

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)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12015-018-9804-2

Keywords

  • Everolimus
  • Renal cell carcinoma
  • Clear cell renal cell carcinoma
  • Renal cancer stem cells
  • Experimental design