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

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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)

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Acknowledgements

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

Author information

Authors and Affiliations

  1. Molecular Oncology Laboratory, Department of Oncology, Military Institute of Medicine, ul. Szaserów 128, 04-141, Warsaw, Poland

    Anna Kornakiewicz, Anna M. Czarnecka, Mohammed I. Khan, Paweł Krasowski & Cezary Szczylik

  2. Postgraduate School of Molecular Medicine, Warsaw Medical University, Żwirki i Wigury 61, 02-091, Warsaw, Poland

    Anna Kornakiewicz

  3. Institute of Biochemistry and Biophysics Polish Academy of Sciences, Laboratory of RNA Biology and Functional Genomics, Pawińskiego 5A, 02-106, Warsaw, Poland

    Anna V. Kotrys

  4. Warsaw Medical University, Żwirki i Wigury 61, 02-091, Warsaw, Poland

    Cezary Szczylik

Authors
  1. Anna Kornakiewicz
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  3. Mohammed I. Khan
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  4. Paweł Krasowski
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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.

Corresponding author

Correspondence to Anna Kornakiewicz.

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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)

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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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