Comparative Gene Expression Profiling of Primary and Metastatic Renal Cell Carcinoma Stem Cell-Like Cancer Cells.
BACKGROUND
Recent advancement in cancer research has shown that tumors are highly heterogeneous, and multiple phenotypically different cell populations are found in a single tumor. Cancer development and tumor growth are driven by specific types of cells-stem cell-like cancer cells (SCLCCs)-which are also responsible for metastatic spread and drug resistance. This research was designed to verify the presence of SCLCCs in renal cell cancer cell lines. Subsequently, we aimed to characterize phenotype and cell biology of CD105+ cells, defined previously as renal cell carcinoma tumor-initiating cells. The main goal of the project was to describe the gene-expression profile of stem cell-like cancer cells of primary tumor and metastatic origin.


METHODS
Real-time PCR analysis of stemness genes (Oct-4, Nanog and Ncam) and soft agar colony formation assay were conducted to check the stemness properties of renal cell carcinoma (RCC) cell lines. FACS analysis of CD105+ and CD133+ cells was performed on RCC cells. Isolated CD105+ cells were verified for expression of mesenchymal markers-CD24, CD146, CD90, CD73, CD44, CD11b, CD19, CD34, CD45, HLA-DR and alkaline phosphatase. Hanging drop assay was used to investigate CD105+ cell-cell cohesion. Analysis of free-floating 3D spheres formed by isolated CD105+ was verified, as spheres have been hypothesized to contain undifferentiated multipotent progenitor cells. Finally, CD105+ cells were sorted from primary (Caki-2) and metastatic (ACHN) renal cell cancer cell lines. Gene-expression profiling of sorted CD105+ cells was performed with Agilent's human GE 4x44K v2 microarrays. Differentially expressed genes were further categorized into canonical pathways. Network analysis and downstream analysis were performed with Ingenuity Pathway Analysis.


RESULTS
Metastatic RCC cell lines (ACHN and Caki-1) demonstrated higher colony-forming ability in comparison to primary RCC cell lines. Metastatic RCC cell lines harbor numerous CD105+ cell subpopulations and have higher expression of stemness genes (Oct-4 and Nanog). CD105+ cells adopt 3D grape-like floating structures under handing drop conditions. Sorted CD105+ cells are positive for human mesenchymal stem cell (MSC) markers CD90, CD73, CD44, CD146, and alkaline phosphatase activity, but not for CD24 and hematopoietic lineage markers CD34, CD11b, CD19, CD45, and HLA-DR. 1411 genes are commonly differentially expressed in CD105+ cells (both from primary [Caki-2] and metastatic RCC [ACHN] cells) in comparison to a healthy kidney epithelial cell line (ASE-5063). TGF-β, Wnt/β-catenine, epithelial-mesenchymal transition (EMT), Rap1 signaling, PI3K-Akt signaling, and Hippo signaling pathway are deregulated in CD105+ cells. TGFB1, ERBB2, and TNF are the most significant transcriptional regulators activated in these cells.


CONCLUSIONS
All together, RCC-CD105+ cells present stemlike properties. These stem cell-like cancer cells may represent a novel target for therapy. A unique gene-expression profile of CD105+ cells could be used as initial data for subsequent functional studies and drug design.
Molecular Oncology Laboratory, Department of Oncology, Military Institute of Medicine, Warsaw, Poland.
Mohammed I
http://orcid.org/0000-0003-3020-1324
MI
Khan
Molecular Oncology Laboratory, Department of Oncology, Military Institute of Medicine, Warsaw, Poland.
Anna M
AM
Czarnecka
Department of Regenerative Medicine, Military Institute of Hygiene and Epidemiology, Warsaw, Poland.
Sławomir
S
Lewicki
Molecular Oncology Laboratory, Department of Oncology, Military Institute of Medicine, Warsaw, Poland.|Institute of Genetics and Biotechnology, Faculty of Biology, Warsaw University, Warsaw, Poland.
Igor
I
Helbrecht
Molecular Oncology Laboratory, Department of Oncology, Military Institute of Medicine, Warsaw, Poland.
Klaudia
K
Brodaczewska
Department of Pathomorphology, Institute of Mother and Child, Warsaw, Poland.
Irena
I
Koch
Department of Regenerative Medicine, Military Institute of Hygiene and Epidemiology, Warsaw, Poland.
Robert
R
Zdanowski
Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences-WULS, Warsaw, Poland.
Magdalena
M
Król
Molecular Oncology Laboratory, Department of Oncology, Military Institute of Medicine, Warsaw, Poland.
Cezary
C
Szczylik
D000071916:AC133 Antigen / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000378:metabolism; D002292:Carcinoma, Renal Cell / Q000235:genetics* / Q000473:pathology*; D000071063:Endoglin / Q000378:metabolism*; D058750:Epithelial-Mesenchymal Transition; D020869:Gene Expression Profiling; D006801:Humans; D007680:Kidney Neoplasms / Q000235:genetics* / Q000473:pathology*; D000071317:Nanog Homeobox Protein / Q000378:metabolism; D014411:Neoplastic Stem Cells / Q000166:cytology*; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D018874:Spheroids, Cellular; D014407:Tumor Cells, Cultured
D000071916:AC133 Antigen; D014408:Biomarkers, Tumor; C064206:ENG protein, human; D000071063:Endoglin; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; C000606023:PROM1 protein, human