Tumorigenic potential is restored during differentiation in fusion-reprogrammed cancer cells

Abstract

Detailed understanding of the mechanistic steps underlying tumor initiation and malignant progression is critical for insights of potentially novel therapeutic modalities. Cellular reprogramming is an approach of particular interest because it can provide a means to reset the differentiation state of the cancer cells and to revert these cells to a state of non-malignancy. Here, we investigated the relationship between cellular differentiation and malignant progression by the fusion of four independent mouse cancer cell lines from different tissues, each with differing developmental potentials, to pluripotent mouse embryonic stem (ES) cells. Fusion was accompanied by loss of differentiated properties of the four parental cancer cell lines and concomitant emergence of pluripotency, demonstrating the feasibility to reprogram the malignant and differentiative properties of cancer cells. However, the original malignant and differentiative phenotypes re-emerge upon withdrawal of the fused cells from the embryonic environment in which they were maintained. cDNA array analysis of the malignant hepatoma progression implicated a role for Foxa1, and silencing Foxa1 prevented the re-emergence of malignant and differentiation-associated gene expression. Our findings support the hypothesis that tumor progression results from deregulation of stem cells, and our approach provides a strategy to analyze possible mechanisms in the cancer initiation.

Figure 1

The formation of hybrid colonies from ES cell and cancer cell fusions. (a) The strategy was to generate hybrids by mouse ES cell and cancer cell fusions. Cancer cells with distinct differentiation potentials were fused with ES cells to generate stable hybrid cells. Existing cancer cells and ES cells were stably transfected with independent drug-resistant and fluorescent markers. Fused cells were generated in the presence of polyethylene glycol (PEG) and grown under standard conditions in the presence of antibiotics to select for double fluorescence-positive and double resistance-positive cell hybrids. After differentiation, the tumorigenic properties were rebuilt. (b) Plates containing hybrid colonies from PEG fusions of ES cells and cancer cells are shown on the left. The colonies of each plate were counted, and the numbers are compared on the right. (c) Morphological characters of the EP, EF, EHe and EB cell lines. Microscopic images of bright field (top), green fluorescence (middle) and red fluorescence (bottom) are shown. Scale bar: 100 μm

Figure 2

Gene expression analysis in ES cells, cancer cells and ES-cancer cell hybrids. (a) Pluripotent gene expression in ES-embryonic carcinoma cells were analyzed by reverse transcription-PCR (RT-PCR) and real-time PCR. Error bars, S.E. of the average values. (b) Pluripotent gene expression (top) and tissue-specific gene expression (bottom) in EHe and EB lines were analyzed by RT-PCR. (c) RNA-FISH staining for Oct4 in EHe and EB hybrid cells. Yellow arrowheads indicate Oct4 RNA nascent transcripts. The percentage of cells showing the expected allelic expression is indicated. Representative images show sites of Oct4 transcription (green) merged with 4′, 6-diamidino-2-phenylindole (DAPI) in ES, Hepa1-6, B16, EHe and EB hybrid cells. Scale bar, 50 μm. (d) Bisulfite genomic sequencing of the promoter regions of Oct4 in ES-cancer hybrid cells, ES cells and cancer cells. Open circles indicate unmethylated CpG dinucleotides, whereas closed circles indicate methylated CpGs. (e) Cancer-related gene expression in ES cells, cancer cells and ESC-cancer cell hybrids were analyzed by RT-PCR

Figure 3

Induce ES cells and hybrids to differentiate in vitro. (a) Morphology of representative day 5 embryoid bodies derived from ES-cancer cell hybrids. Scale bar, 100 μm. (b) Reverse transcription-PCR (RT-PCR) analysis of tumor-related gene expression in ES-cancer hybrid cells over the indicated number of days. β-Actin was used as a ubiquitously expressed control. (c) RT-PCR analysis of tissue-specific gene and pluripotent gene expression in ES-cancer hybrid cells over the indicated number of days. β-Actin was used as a ubiquitously expressed control

Figure 4

Identification and verification of differentiation-related tumorigenic genes from Foxa1/AR targets. (a) Analysis of gene expression by cDNA microarray assay. Expression profiles were clustered by a Pearson's correlation analysis. Expression levels are depicted in color. (b) Functional pathway analysis of 558 differential gene expressions (fold change >3, P<0.01, t-test) in undifferentiated and differentiated ES-hepatoma hybrids. (c) Intersect analysis of 667 potential cancer-promoting genes that are targeted by Foxa1/AR and 558 differentially expressed genes from the array. (d) Quantitative PCR (qPCR) analysis of transcripts in hepatoma cells, ES and EHe hybrids differentiated at day 14. Genes on the left side of the red dash are the ones being bound at the promoter region, whereas genes on the left side are the ones being bound at the intron enhancer region. Tgfa, Junb and Egfr are the genes being bound at both of the regions. Transcripts of ES cells at day 0 were normalized to 1. Error bars, S.E. of the average values. (e) The decreased expression level of Foxa1 by siRNA was verified by qPCR and western blot. Transcripts of control siRNA (sicon)-transfected ES cells at differentiation day 14 was normalized as 1. Error bars, S.E. of the average values. *P<0.05. (f) Dynamic expression level of oncogenic Foxa1-binding genes (Tgfa, Egfr and c-Jun) and non foxa1-binding gene p16^INK4a from days 0 to 14 in sicon and Foxa1 silencing group (siFoxa1). Transcript levels at day 0 were normalized to 1

Figure 5

Teratoma analysis of hybrid cells and corresponding parental cell lines. ES cells, cancer cell lines and ES-cancer cell hybrids were injected subcutaneously into nude mice. Representative haematoxylin- and eosin-stained sections of tumors from (a) ES cells; (b) ES-lymphocyte cell hybrids established broad range of mature tissue types; (c) P19, (d) EP hybrids can spontaneously differentiated into immature neuroepithelium marked by rosette formation (▴); (e) F9 and (f) EF hybrids established undifferentiated tissue types in the teratoma; (g) Hepa1-6-derived tumor showed a hepatocarcinoma tissue type; and (h) in tumors derived from EHe hybrids, >80% of the areas showed undifferentiated malignant cell types with enlarged nuclei and less cytoplasm; in tumors derived from B16 (i) and 70% area of EB hybrids (j), undifferentiated cells with hyperchromic nuclei and melanin granule (↑) were observed. Scale bar, 1000 μm

Figure 5

Teratoma analysis of hybrid cells and corresponding parental cell lines. ES cells, cancer cell lines and ES-cancer cell hybrids were injected subcutaneously into nude mice. Representative haematoxylin- and eosin-stained sections of tumors from (a) ES cells; (b) ES-lymphocyte cell hybrids established broad range of mature tissue types; (c) P19, (d) EP hybrids can spontaneously differentiated into immature neuroepithelium marked by rosette formation (▴); (e) F9 and (f) EF hybrids established undifferentiated tissue types in the teratoma; (g) Hepa1-6-derived tumor showed a hepatocarcinoma tissue type; and (h) in tumors derived from EHe hybrids, >80% of the areas showed undifferentiated malignant cell types with enlarged nuclei and less cytoplasm; in tumors derived from B16 (i) and 70% area of EB hybrids (j), undifferentiated cells with hyperchromic nuclei and melanin granule (↑) were observed. Scale bar, 1000 μm