TGFbeta/TNF(alpha)-mediated epithelial-mesenchymal transition generates breast cancer stem cells with a claudin-low phenotype

Abstract

Breast cancer recurrence is believed to be caused by a subpopulation of cancer cells that possess the stem cell attribute of treatment resistance. Recently, we and others have reported the generation of breast cancer stem cells (BCSC) by epithelial-mesenchymal transition (EMT), although the physiologic process by which these cells may arise in vivo remains unclear. We show here that exposure of tumor cells to TGFβ and TNFα induces EMT and, more importantly, generates cells with a stable BCSC phenotype which is shown by increased self-renewing capacity, greatly increased tumorigenicity, and increased resistance to oxaliplatin, etoposide, and paclitaxel. Furthermore, gene expression analyses found that the TGFβ/TNFα-derived BCSCs showed downregulated expression of genes encoding claudin 3, 4, and 7 and the luminal marker, cytokeratin 18. These changes indicate a shift to the claudin-low molecular subtype, a recently identified breast cancer subtype characterized by the expression of mesenchymal and stem cell-associated markers and correlated with a poor prognosis. Taken together, the data show that cytokine exposure can be used to generate stable BCSCs ex vivo, and suggest that these cells may provide a valuable tool in the identification of stem cell-directed biomarkers and therapies in breast cancer.

Figure 1. TGFϐ with TNFα generates tumor cells with a stable EMT phenotype that form heterogenous tumors in vivo

A) Photomicrograph (upper) and flow cytometry analysis (bottom) of epithelial MMC and mesenchymal MMCTT and ETTM cells. B) RT-PCR of mRNA isolated from MMC, MMCTT, MMCTTE, ETTM and ANV5 cells using primers specific for E-cadherin, N-Cadherin, Zeb1, Twist, Snail and GAPDH. ANV5 is a stable BCSC cell line derived in our prior studies (11). C) Immunoblot of cells in (B) showing expression of E-Cadherin, N-Cadherin and β-actin. D) Flow cytometry analysis of N-Cadherin and E-Cadherin on ETTM cells, ETTM primarytumor and cultured cells. E) Expression of EMT-associated genes by qPCR analysis.

Figure 2. Generation of breast cancer stem cells by TGFβ and TNFα

A) top left, Photograph of mammospheres formed by MMC, MMCTT and ETTM cells. A) top right, graph of the mean and s.e.m. (n=6) of mammospheres formed by MMC, MMCTT and ETTM cells. *** = p < 0.05. A) bottom left, flow cytometry analysis of co-expression of CD24 and CD44 on MMC, MMCTT and ETTM cells. B) Photograph of adherent cells and mammospheres formed by CD24⁺/CD44⁺ and CD24⁻/CD44⁺ of MMCTT cells. C) Flow cytometry analysis showing expression of CD24 and CD44 on ETTM cells, freshly isolated ETTM tumor cells and cultured ETTM tumor cells. D) Real-time PCR using gene-specific primers detect gene expression of Keratin 18, Claudin 3, 4, 7 and 9. Error bars represent SD of two independent experiments. E) Gene expression analysis of stem cells genes using qPCR profiler arrays. Errors bars represent SD of two independent experiments.

Figure 3. Human immortalized breast epithelial cells exposed to TGFβ and TNFα undergo EMT and acquire breast cancer stem cell phenotype

A) Photograph of adherent epithelial MCF10A cells and mesenchymal MCF10ATT cells (MCF10A cells grown in TGFβ and TNFα for 40 days). B) Fold regulation plot of EMT-associated genes in MCF10ATT cells assessed by qPCR using MCF10A cells as a reference. Errors bars represent SD of two independent experiments. C) Flow cytometry analysis of cell surface expression of CD24 and CD44 on MCF10A and MCF10ATT cells. D) Fold regulation plot of stem cell genes by qPCR using profiler arrays using MCF10A as reference. Errors bars represent SD of two independent experiments. Photograph (E) and quantification (F) of mammospheres formed by MCF10A and MCF10TT cells. Each bar is the mean (s.e.m.) of 6 replicates.

Figure 4. Regulation of EMT and stem cell associated genes in ETTM cells

a) Gene expression analysis of Home box genes showing upregulation of Dlx1, Hoxc9, Hoxc8, and Hoxa9. b) qPCR pathway array analysis shows regulation of genes involved in TGFβ/BMP signaling pathway. c) Assessment of regulation of Hedgehog signaling genes showing differential regulation of Wnt6, Wnt10A, Gas1, Wnt5a, gli3, Ptch1, PtcgD2 and Wnt5b. d) qPCR gene expression analysis detects upregulation of Wisp1, Fosl1, Notch4 and downregulation of Notch3 and Hey1. Error bars represent SD of two independent experiments.

Figure 5. ETTM cells are highly migratory, invasive and tumorigenic

A) top, photomicrograph of MMC and ETTM cells migrated and invaded through matrigel coated or uncoated trans well inserts. A) bottom, quantification of migrated and invaded cells showing statistically significant differences between MMC and ETTM cells. B,C) Mean (± s.e.m., n=5) tumor sizes over time (days) following implantation of 100,000 MMC, MMCTTand ETTMcells as wellas serial dilution of ETTM cells showing high tumorigenicity of ETTM cells and the ability to form tumors at 100 cells. The inset fraction represents the number of mice with tumors in this experiment. For both the 10,000 and 1000 cell doses, the variability was so minor that the error bars are obscured by the symobols D, E) Gene expression analysis of metastases, oncogenes and tumor suppressor genes showing differential regulation of metal loproteases and adhesion genes such as Mmp13, Mmp9, Mmp3, Cdh1, cdh66 andCdh11. *=p<0.05. Error bars represent SD of two independent experiments.

Figure 6. ETTM stem cells are resistant to oxaliplatin, etoposide and paclitaxel

A) Real-time measurement of MMC and ETTM cell growth (Cell index) following treatment with oxaliplatin over a period of 40 hrs. Lines are the means of 3 replicates. B,C) Treatment of MMC and ETTM with different doses of etoposide and paclitaxel for 45 or60 hrs showing resistance to cell death by ETTM cells as measured by electrical impedence. Bars are the mean (s.e.m.) of 3 replicates D, E) qPCR pathway array analyses of drug resistance and apoptosis genes, F) qPCR pathway array analyses of DNA damage and repair genes. For panels D-F, error bars represent SD of two independent experiments.

Figure 7. Breast cancer and stem cell associated genes are regulated in ETTM cells

qPCR analysis of chemokine receptor genes (A), inflammatory cytokine and receptor genes (B), breast cancer and estrogen signaling genes (C) and cell cycle and angiogenesis genes (D and E). shows differential regulation of these signaling pathways in ETTM BCSCs compared to non-BCSC parental cells. Error bars represent SD of two independent experiments.