Int J Stem Cells.  2019 Nov;12(3):430-439. 10.15283/ijsc19067.

Reprogramming of Cancer Cells into Induced Pluripotent Stem Cells Questioned

• Bang JS
• Choi NY
• Lee M
• Ko K
• Park YS
• Ko K

• KMID: 2465883
• DOI: http://doi.org/10.15283/ijsc19067

Abstract

BACKGROUND AND OBJECTIVES
Several recent studies have claimed that cancer cells can be reprogrammed into induced pluripotent stem cells (iPSCs). However, in most cases, cancer cells seem to be resistant to cellular reprogramming. Furthermore, the underlying mechanisms of limited reprogramming in cancer cells are largely unknown. Here, we identified the candidate barrier genes and their target genes at the early stage of reprogramming for investigating cancer reprogramming.
METHODS
We tried induction of pluripotency in normal human fibroblasts (BJ) and both human benign (MCF10A) and malignant (MCF7) breast cancer cell lines using a classical retroviral reprogramming method. We conducted RNA-sequencing analysis to compare the transcriptome of the three cell lines at early stage of reprogramming.
RESULTS
We could generate iPSCs from BJ, whereas we were unable to obtain iPSCs from cancer cell lines. To address the underlying mechanism of limited reprogramming in cancer cells, we identified 29 the candidate barrier genes based on RNA-sequencing data. In addition, we found 40 their target genes using Cytoscape software.
CONCLUSIONS
Our data suggest that these genes might one of the roadblock for cancer cell reprogramming. Furthermore, we provide new insights into application of iPSCs technology in cancer cell field for therapeutic purposes.

Keyword

Induced pluripotent stem cells; iPSC generation; RNA-sequencing analysis; Pluripotency; Cancer cell reprogramming

MeSH Terms

Breast Neoplasms
Cell Line
Cellular Reprogramming
Fibroblasts
Humans
Induced Pluripotent Stem Cells*
Methods
Transcriptome
Zidovudine
Zidovudine

Figure

• 

  Fig. 1 Generation of iPSCs from BJ, MCF7, and MCF10A cells. (A) Procedure used for generation of iPSCs. (B) The morphology of iPSC-like colonies formed from BJ cultured on MEFs, but no iPSC-like colonies were formed from MCF7 and MCF10A (OSKM-transduced BJ; 4F-BJ, 4F-MCF7, and 4F-MCF10A). Scale bars: 100 μm. (C) After 25 days, an individual colony of each group was picked up onto Matrigel-coated dishes. Scale bars: 100 μm. (D) Endogenous expression of the OCT4, SOX2, and NANOG in BJ D0, BJ colony, MCF7 D0, MCF7 colony, MCF10A D0, MCF10A colony, and BJ-iPSCs. Expression levels were normalized to those in BJ-iPSCs. Data are shown as mean±SEM of triplicate experiments. Significance was analyzed using one-way ANOVA (analysis of variance) (***p<0.001, ↓=expression undetectable). (E) Immunofluorescence microscopy images of pluripotency markers (OCT4, SOX2, SSEA4, and TRA-1-60) in 4F-BJ, 4F-MCF7, and 4F-MCF10A. Cell nuclei were stained with DAPI. Scale bars: 20 μm.

• 

  Fig. 2 Analysis of exogenous expression of reprogramming factors (OCT4, SOX2, KLF4, and C-MYC) and endogenous expression of OCT4, SOX2, and NANOG. (A) Transgene expression levels were normalized to those of BJ D5. (B) qPCR analysis of the endogenous expression of pluripotency marker genes, OCT4, SOX2, and NANOG. Expression levels were normalized to those in BJ-iPSCs. Data are shown as mean±SEM of triplicate experiments. Significance was analyzed using one-way ANOVA (analysis of variance) (**p<0.001, ***p<0.001, ↓=expression undetectable).

• 

  Fig. 3 Transcriptome profiling analysis by RNA-seq. (A) Heatmap representing the expression patterns of 17 candidate barrier genes up-regulated in BJ D5 but not in MCF7 D5 and MCF10A D5. (B) Heatmap representing the expression patterns of 12 candidate barrier genes down-regulated in BJ D5 but not in MCF7 D5 and MCF10A D5. The heatmaps show the log10 scale fold changes (FPKM values). Red and green colors represent higher and lower gene expression levels, respectively.

• 

  Fig. 4 Genetic interaction network (GIN) between candidate barrier genes and target genes during reprogramming. (A) Construction of GIN based on up-regulated candidate barrier genes, target genes, and core pluripotency genes. (B) Construction of GIN down-regulated candidate barrier genes, target genes, and core pluripotency genes. Black circles, candidate barrier genes; gray circles, target genes.

Reference

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