Generation of tumor-initiating cells by exogenous delivery of OCT4 transcription factor - PubMed (original) (raw)
Generation of tumor-initiating cells by exogenous delivery of OCT4 transcription factor
Adriana S Beltran et al. Breast Cancer Res. 2011.
Abstract
Introduction: Tumor-initiating cells (TIC) are being extensively studied for their role in tumor etiology, maintenance and resistance to treatment. The isolation of TICs has been limited by the scarcity of this population in the tissue of origin and because the molecular signatures that characterize these cells are not well understood. Herein, we describe the generation of TIC-like cell lines by ectopic expression of the OCT4 transcription factor (TF) in primary breast cell preparations.
Methods: OCT4 cDNA was over-expressed in four different primary human mammary epithelial (HMEC) breast cell preparations from reduction mammoplasty donors. OCT4-transduced breast cells (OTBCs) generated colonies (frequency ~0.01%) in self-renewal conditions (feeder cultures in human embryonic stem cell media). Differentiation assays, immunofluorescence, immunohistochemistry, and flow cytometry were performed to investigate the cell of origin of OTBCs. Serial dilutions of OTBCs were injected in nude mice to address their tumorigenic capabilities. Gene expression microarrays were performed in OTBCs, and the role of downstream targets of OCT4 in maintaining self-renewal was investigated by knock-down experiments.
Results: OTBCs overcame senescence, overexpressed telomerase, and down-regulated p16INK4A. In differentiation conditions, OTBCs generated populations of both myoepithelial and luminal cells at low frequency, suggesting that the cell of origin of some OTBCs was a bi-potent stem cell. Injection of OTBCs in nude mice generated poorly differentiated breast carcinomas with colonization capabilities. Gene expression microarrays of OTBC lines revealed a gene signature that was over-represented in the claudin-low molecular subtype of breast cancer. Lastly, siRNA-mediated knockdown of OCT4 or downstream embryonic targets of OCT4, such as NANOG and ZIC1, suppressed the ability of OTBCs to self-renew.
Conclusions: Transduction of OCT4 in normal breast preparations led to the generation of cell lines possessing tumor-initiating and colonization capabilities. These cells developed high-grade, poorly differentiated breast carcinomas in nude mice. Genome-wide analysis of OTBCs outlined an embryonic TF circuitry that could be operative in TICs, resulting in up-regulation of oncogenes and loss of tumor suppressive functions. These OTBCs represent a patient-specific model system for the discovery of novel oncogenic targets in claudin-low tumors.
Figures
Figure 1
Generation of _OCT4_-transduced breast cells (OTBCs) from primary cultures of mammary cells. Graphical representation of the method used to generate OTBCs from normal breast tissue. Representative images of control cells (empty lentiviral vector) and OTBCs are shown 2 weeks after transduction of OCT4 and seeding in feeder cultures of mouse embryonic fibroblasts in human embryonic stem cell (hESC) media. The OTBCs-86L1 image is a representative example of cells (passage 25) grown in suspension in mammosphere media after cell expansion.
Figure 2
_OCT4_-transduced breast cells (OTBCs) underwent an immortalization process. (a) OTBCs overcame cellular senescence. The histogram quantifies β-galactosidase-positive cells in the OTBCs-86L1 and OTBCs-48L1 lines. Data were normalized to the parental cell lines p86 and p48 and represent the mean ± standard deviation (SD) of three independent experiments (*P ≤0.01). (b) Telomerase mRNA levels are upregulated in OTBCs relative to the parental p86 and p48 lines. The SUM159PT breast cancer cell line was used as a positive telomerase gene expression control. Samples were normalized to the parental p86 and p48 lines. Bar graphs represent the mean ± SD of three independent experiments. (c) OTBCs downregulate p16Ink4a mRNA levels as assessed by quantitative real-time polymerase chain reaction. The p16Ink4a levels in OTBCs were normalized to the parental lines. Bar graphs represent the mean ± SD of three independent experiments. hTERT, human telomerase reverse transcriptase.
Figure 3
Breast cancer stem cell-like properties of OCT4-transduced breast cells (OTBCs) in vitro. (a) Left: Formation of terminal ductal lobular units in three-dimensional (3D) cultures in the presence of Matrigel™ and prolactin. OTBCs were seeded in a 3D culture; 3 weeks later, primary, secondary, and tertiary branching structures were visible. Right: Immunostaining of 3D structures with anti-CK19 (luminal, green) and anti-CK14 (myoepithelial, red) antibodies. (b) Immunostaining of myoepithelial and luminal markers in OTBCs86-L1 cells. Cells were stained for OCT4 (green) and CK14, SMA, Maspin, or CK19 (red) markers. (c) Flow cytometry analysis of antigenic phenotypes characteristic of bi-potent mammary stem cells (CD133lowCD49f+EpCAM-) and cancer stem cells (CD44+CD24-) in the parental line p86 and the clone OTBCs86-L1. The forward scatter channel was plotted in the y-axis, and the fluorescence of the cell surface antigens was plotted in the x-axis. Virtually identical flow cytometry results were obtained from all analyzed clones (Figure S2 in Additional file 5). Experiments were repeated at least three times with similar results. Data shown are representative of one experiment. EpCAM, epithelial cell adhesion molecule.
Figure 4
_OCT4_-transduced breast cells (OTBCs) generate primary tumors and experimental metastases upon injection in immunodeficient mice. (a) Representative image of a prototype subcutaneous primary tumor generated by OTBCs86-L1-DsRed cells. Control mice were injected with the parental p86 line. Animals were injected with 1 × 106 cells; for simplicity, only one animal is shown. The OTBCs86-L1-DsRed was engineered with a lentiviral vector expressing the DsRed gene, which facilitated tumor measurement by fluorescence imaging (IVIS-kinetic; Xenogen). Shown is a representative paraffin-embedded tumor section stained with (b) OCT4 (40×), (c) hematoxylin and eosin (H&E) (40×), (d) H&E magnification from (c), (e) vimentin (VIM) (20×), (f) pan-keratin (AE1/AE3, 20×), (g) keratin 19 (20×), and (h) keratin 8/18 (20×). (i) Representative image of spontaneous metastatic lesions. Immunodeficient mice were injected in the left ventricle of the heart with OTBCs86-L1-DsRed cells, and imaging was performed at day 90 after injection. Shown is a representative paraffin-embedded tumor section stained with (j) OCT4 (40×), (k) H&E (40×), (l) H&E magnification from (k), and (m) VIM (40×). Immunofluorescence detection of (n) DsRed-labeled OTBCs (10×), (o) OCT4 (20×), and (p) merged image of DsRed and OCT4 (10×). The square on the left corner shows a detail of DsRed-positive tumor cells expressing OCT4.
Figure 5
_OCT4_-transduced breast cells (OTBCs) exhibit an epithelial-to-mesenchymal transition (EMT) gene signature. (a) Microarray expression analysis of genes selected across parental (p48 and p86), OTBC lines (OTBCs48-L1 and L2, OTBCs86-L1, L4, and L6), and a subcutaneous tumor derived from OTBCs86-L1 (tumor 86-L1). Array trees were derived by unsupervised hierarchical clustering using markers of EMT, basal, luminal stem cell, cancer stem cell, and epithelial differentiation. Each colored square on the upper right represents the relative mean transcript abundance (in log2 space); highest expression is red, average expression is black, and lowest expression is green. (b) Western blot detection of molecular markers in the parental lines (p86 and p48), OTBCs86-L1 through L6, and OTBCs48-L1. The following markers were analyzed: OCT4, NESTIN, CDH1, Maspin, vimentin, and N-CAM. Tubulin was used as a loading control. Results are representative examples of three independent experiments. (c) Detection of EMT transcription factor expression by quantitative real-time polymerase chain reaction (qRT-PCR). Gene expression levels were normalized to those of the parental cell lines (p86 and p48). Control cell lines used for gene expression analyses include human dermal fibroblasts and the human teratoma cell line NT2. (d) MicroRNAs (miRNAs) known to promote epithelial differentiation are detected by qRT-PCR. Levels of miRNA expression in OTBCs of miR-141, miR-200a, miR-200b, miR-200c, and miR-205 were normalized to those of the parental lines (p86 and p48). Analysis was performed by using miR-U6 as an internal control. Bar graphs represent the mean ± standard deviation of three independent experiments. CDH1, E-cadherin; N-CAM, neural cell adhesion molecule.
Figure 6
_OCT4_-transduced breast cells (OTBCs) up- and downregulate genes clustered with the claudin-low molecular subtype of breast cancer. Box-and-whisker plot for the mean expression of the upregulated (534 genes) and downregulated (1, 144 genes) gene signatures (Tables S4 and S5 in Additional files 7 and 8, respectively) in OTBCs compared with the parental cell lines across the intrinsic molecular subtypes of breast cancer by using the published breast cancer patient database (UNC337) [6]. P values have been calculated by comparing gene expression means across all subtypes. HER2, epidermal growth factor receptor 2.
Figure 7
Differential regulation of the human embryonic targets of NANOG, OCT4, and SOX2 (NOS targets) in _OCT4_-transduced breast cells (OTBCs). (a) Expression of selected NOS targets across the parental cell line p86, the OTBCs86-L1 cell line, and the same line OTBCs86-L1 transfected with a short interfering RNA specific for OCT4. Efficient knockdown of OCT4 was validated by Western blot. Each colored square on the left panels represents the mean relative transcript abundance (in log2 space); highest expression is red, average expression is black, and lowest expression is green. Data represent the average of three independent experiments. (b) Detection of self-renewal transcription factors OCT4, NANOG, SOX2, and ZIC1 expression by quantitative real-time polymerase chain reaction (qRT-PCR). Control cell lines used for gene expression analyses include human dermal fibroblasts and human embryonic stem cells (hESCs). The mRNA levels were normalized to those of the parental cell lines (p86 and p48). Bar graph represents the mean ± standard deviation (SD) of three independent experiments. (c) Detection of tumor suppressor gene expression by qRT-PCR. Genes analyzed include p21WAF1/cip1, Dickkopf-related protein 1 (DKK1), methylguanine-DNA methyltransferase (MGMT), Maspin (SERPINB5), and E-cadherin (CDH1). The mRNA levels were normalized to those of the parental cell lines (p86 and p48). Bar graph represents the mean ± SD of three independent experiments. NOS, NANOG, OCT4, and SOX2 targets.
Figure 8
Knockdown of OCT4, NANOG, and ZIC1 in _OCT4_-transduced breast cells (OTBCs) affects self-renewal. (a) Cell viability was assessed in OTBCs86-L1 cells upon transfection of short interfering RNAs (siRNAs) designed to knock down OCT4, NANOG, and ZIC1. Data were normalized to cells transfected with a mismatch siRNA pool. Mock refers to untransfected OTBCs86-L1 cells. Cells were transfected with the corresponding siRNAs and placed in self-renewal conditions for 72 hours. Data represent the mean ± standard deviation (SD) of three independent experiments and were analyzed with student t test with P values set at *P ≤0.01 and **P ≤0.001. (b) Knockdown of OCT4 in immortalized human mammary epithelial cells (HUMECs). Transfection of siRNAs was performed as described above. (c) Real-time polymerase chain reaction quantification of OCT4, NANOG, and ZIC1 mRNA expression after knockdown in OTBCs86-L1 cells; data were normalized to cells transfected with a mismatch siRNA pool. (d) Protein expression levels of OCT4 and ZIC1 assessed by Western blot in OTBCs86-L1 (MOCK), knockdown OTBCs86-L1 (OCT4 or ZIC1), and mismatch siRNA pool cell population (MISMATCH). One of three independent experiments is shown.
Figure 9
Graphical model of the generation of tumor-initiating cells (TICs) and the OCT4 molecular targets regulated in _OCT4_-transduced breast cells (OTBCs). (a) Illustration of the proposed model for the generation of OTBCs endowed with tumor-initiating ability. (b) Endogenous expression of OCT4 in normal breast tissue results in aberrant self-renewal and expansion of an undifferentiated early stem/progenitor population. OCT4 expression results in an activation of self-renewal transcription factors (TFs) and a concomitant repression of tumor suppressor genes that allow cells to immortalize and gain stem cell-like features. CDH1, E-cadherin; EMT, epithelial-to-mesenchymal transition; hTERT, human telomerase reverse transcriptase; miRNA, microRNA; TSG, tumor suppressor gene.
References
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