Twist modulates breast cancer stem cells by transcriptional regulation of CD24 expression

Abstract

The cancer stem cell paradigm postulates that dysregulated tissue-specific stem cells or progenitor cells are precursors for cancer biogenesis. Consequently, identifying cancer stem cells is crucial to our understanding of cancer progression and for the development of novel therapeutic agents. In this study, we demonstrate that the overexpression of Twist in breast cells can promote the generation of a breast cancer stem cell phenotype characterized by the high expression of CD44, little or no expression of CD24, and increased aldehyde dehydrogenase 1 activity, independent of the epithelial-mesenchymal transition. In addition, Twist-overexpressing cells exhibit high efflux of Hoechst 33342 and Rhodamine 123 as a result of increased expression of ABCC1 (MRP1) transporters, a property of cancer stem cells. Moreover, we show that transient expression of Twist can induce the stem cell phenotype in multiple breast cell lines and that decreasing Twist expression by short hairpin RNA in Twist-overexpressing transgenic cell lines MCF-10A/Twist and MCF-7/Twist as well as in MDA-MB-231 partially reverses the stem cell molecular signature. Importantly, we show that inoculums of only 20 cells of the Twist-overexpressing CD44(+)/CD24(-/low) subpopulation are capable of forming tumors in the mammary fat pad of severe combined immunodeficient mice. Finally, with respect to mechanism, we provide data to indicate that Twist transcriptionally regulates CD24 expression in breast cancer cells. Taken together, our data demonstrate the direct involvement of Twist in generating a breast cancer stem cell phenotype through down-regulation of CD24 expression and independent of an epithelial-mesenchymal transition.

Figure 1

Cell surface expression of CD44 and CD24 in Twist-dysregulated breast cells. (A) Immunoblots showing the downregulation of Twist in MCF-10A/Twist, MCF-7/Twist, and MDA-MB-231 breast cancer cell lines by short hairpin RNA against Twist delivered using lentiviral vectors. Lower panels show Twist transient expression levels in MCF-10A and MCF-7 cell lines expressing Twist by retroviral delivery. The antibody against Twist was generated in-house and was validated. Actin was scored as loading control. (B) Flow cytometry analysis of various cell lines for CD44 and CD24 expression. Unstained and single-antibody stained cells were used as controls for setting the quadrants. The events in the lower right quadrant represent the CD44⁺/CD24^-/low subpopulation. The analyzed data are represented as dot plots of (first row) immortalized normal mammary epithelial cell line MCF-10A and breast cancer cell lines MCF-7 and MDA-MB-231, (second row) stable Twistoverexpressing cell lines MCF-10A/Twist and MCF-7/Twist, (third row) transiently transduced Twist-expressing MCF-10A and MCF-7, and (fourth row) Twist knockdown in MCF-10A/Twist, MCF-7/Twist, and MDA-MB-231 breast cancer cells. Ten thousand viable cells were gated for each dot plot acquisition. Results are representative of five separate experiments.

Figure 2

Epithelial-mesenchymal marker expression in Twist cells. (A) Immunoblots showing epithelial marker E-cadherin and mesenchymal marker vimentin in transient knockdown of Twist in stable Twist-expressing cell lines MCF-10A/Twist and MCF-7/ Twist. (B) Immunoblots showing E-cadherin and vimentin in MCF-7 and MCF-7/Twist cells transiently expressing Twist. Transduced cells were lysed and immunoblotted with antibodies against E-cadherin, vimentin (Santa Cruz Biotechnology, Santa Cruz, CA), and Twist (in-house generated). Actin (Sigma-Aldrich) was used as loading control.

Figure 3

Efflux studies in MCF-7 and MCF-7/Twist cells. (A) Representative photomicrographs of Hoechst efflux staining in MCF-7/Twist cells compared with parental MCF-7 cells. The cells, after efflux, were photographed using a Nikon Eclipse 80i fluorescence microscope. (B) Histogram showing quantification of fluorescence intensity per cell. Cell fluorescence (n = 6) in the blue channel was analyzed using dedicated software developed in IDL programming environment that provides an operator-free segmentation of images and determines the average relative fluorescence per cell. Three images were analyzed per sample. (C) Histogram showing expression of drug transporters ABCG2, ABCC1, and ABCA1 in MCF-7/Twist and parental MCF-7 cells. (D) Histogram showing efflux of Rhodamine 123 stain in MCF-7/Twist and in MCF-7 cells. The amount of Rhodamine 123 dye in the cells was determined by flow cytometry. Results are representative of three separate experiments.

Figure 4

ALDH activity in Twist-dysregulated breast cells. Dot plots of various cell lines analyzed by flow cytometry for ALDH activity. Cells were treated with ALDEFLUOR in the presence or absence of ALDH inhibitor DEAB. After treatment, the samples were analyzed by flow cytometry for the presence of ALDH bright cells. The ALDH bright region was based on the control DEAB sample that was gated to have less than five events. The cell percentage numbers for ALDH positivity are indicated in the bottom right of each histogram. The values presented are the averages of three independent experiments. The analyzed data are represented as dot plots of (first row) parental MCF-10A and MCF-7 breast cancer cell lines, (second row) stable Twist-overexpressing MCF-10A/Twist and MCF-7/Twist breast cancer cell lines, (third row) breast cell lines MCF-10A and MCF-7 transiently transduced with Twist-expressing retroviral constructs, and (fourth row) Twist knockdown breast cancer cell lines MCF-10A/Twist and MCF-7/Twist. Results are representative of five separate experiments.

Figure 5

Self-renewal and mammosphere formation of Twist-expressing MCF-7 cells. MCF-7/Twist cells were stained with CD44 and CD24 antibodies and flow sorted to purify the CD44⁺/CD24^-/low subpopulation. The purified MCF-7/Twist CD44⁺/CD24^-/low subpopulation was propagated in culture, and the percentage of CD44 and CD24 cells was estimated by flow cytometry. (A) Histograms of chronological changes in the CD44⁺/CD24^-/low subpopulation during a period of 13 generations. (B) qRT-PCR analysis of Twist, CD44, and CD24 transcript levels in the purified CD44⁺/CD24^-/low versus CD44⁺/CD24⁺ subpopulations of cells. (C and D) Mammosphere formation by stable Twist-overexpressing cell lines, MCF-7/Twist, and MCF-10A/Twist. Top rows show phase-contrast photomicrographs of mammospheres at a magnification of x20. The lower rows show viability of the cells as indicated by the fluorescence of Calcein-AM stain. Data are represented as histograms on the right. Experiments were performed in quadruplicates and an average of five fields per well were counted.

Figure 6

Twist transcriptional regulation of CD24 expression. (A) Schematic representation of the CD24 promoter sequence showing the location of putative TWIST binding sites relative to the transcription start site (+1). Tick marks denote the canonical E-box sequences (CANNTG) to which Twist can potentially bind. The numbers above the tick marks indicate the relative position of E-box sequences within the promoter region. (B) Reporter assays were performed using CD24 promoter-reporter constructs in MCF-7 with exogenous Twist expression plasmid added and estimated for 2 days. The results of the promoter assays are represented as a histogram showing normalized luciferase readings in CD24 promoter activity. (C) Immunoblot analysis of cell extracts from MCF-7 and MCF-7/Twist cells scored for CD24 (Santa Cruz Biotechnology) and Twist protein expression. Actin was used as a loading control. (D and E) In vivo binding of Twist protein to the CD24 promoter sequence. ChIP was carried out using MCF-7/Twist cells and analyzed using CD24 promoter-specific primers by PCR. Identical volumes from the final precipitate were used for the PCRs except for the input, which was diluted 10-fold.

Figure 7

Growth of low inoculums of purified CD44⁺/CD24^-/low and CD44⁺/CD24⁺ subpopulations in SCID mice. (A) Graphical representation of growth rates of orthotopic xenograft tumors using flow-sorted cells from the Twist⁺/CD44⁺/CD24^-/low and CD44⁺/CD24⁺ subpopulations. We transplanted 100, 50, and 20 cells of the Twist⁺/CD44⁺/CD24^-/low subpopulation, and 100 cells from the Twist⁺/CD44⁺/CD24⁺ subpopulation. Four mice per inoculum were used for this study. Significance was analyzed by a two-sided t-test (**P<.05, ***P <.005). (B) Twist immunohistochemical staining of representative tumors from the CD44⁺/CD24^-/low and CD44+/CD24+ subpopulations. Photomicrographs were taken in bright field settings at a magnification of x40 using Nikon Eclipse 80i fluorescence microscope.