• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Cancer Res. Author manuscript; available in PMC Jul 1, 2012.
Published in final edited form as:
PMCID: PMC3129359
NIHMSID: NIHMS295475

TGFβ/TNFα-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 sub-population 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 (BCSCs) by epithelial to mesenchymal transition (EMT), although the physiological 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 demonstrated 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 down regulated 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.

Introduction

Breast cancer recurrence is believed to be caused by a sub-population of cancer cells possessing stem cell attributes of tumor initiation, resistance to chemotherapy, radiation and other forms of treatment (13). Breast cancer stem cell (BCSC) characteristics can be induced through genetic and epigenetic mechanisms or extrinsically by micro environmental stimuli, conferring many unique properties such as ability to seed tumors at sites distant from the primary tumor, resistance to apoptosis-inducing drugs and enhanced migratory and metastatic potential (36). Current challenges in BCSC research include identifying unique and reliable molecular markers for BCSC isolation and generating stable, homogeneous BCSCs for propagation in culture for drug screening and to identify therapeutic targets. Several techniques have been used to enrich BCSCs including sorting for CD24−/lowCD44+cells, selecting for side-population (SP) cells that exclude Hoechst dyes, isolating spheroids (mammospheres) from suspension cultures or isolating ALDH1 positive cells (710). Recently, we and others (1113)have demonstrated the generation of CSCs through induction of epithelial-mesenchymal transition (EMT), a biological process involving coordinated molecular, biochemical and cellular changes resulting in the loss of cell-cell adhesion, apical-basolateral polarity, and epithelial markers, but acquisition of motility, spindle-cell shape, and mesenchymal markers (1416).

Defining how EMT processes contribute to BCSC phenotypic characteristics in vivo has been limited by the lack of experimental models that recapitulate the processes known to induce EMT in vivo. Prior studies have shown that stable BCSC populations can be produced by forcing expression of key transcription factors (12). By contrast, induction with TGFβ, a major inducer of EMT during tumor progression (1418) has previously led to only transient activation of BCSC characteristics (12). In our previous report, we showed that in vivo generation of stable BCSCs required CD8 T cells suggesting multiple pathways must be activated in addition to TGF-β (11) Given that prior studies have shown that TNFα, an inflammatory mediator associated with cell-mediated immunity, results in a stable EMT phenotype when used with TGFβ (17), we speculated this combination of cytokines also results in stable generation of cells with the BCSC phenotype. To test this, we exposed breast cancer cells derived from an epithelial breast tumor to the cytokine combination and derived stable cell populations with BCSC characteristics. Furthermore, the ex vivo generated BCSCs had characteristics of the claudin-low breast cancer subtype. These findings provide key insight into BCSC development in vivo and establish a new in vitro experimental model for generating mesenchymal BCSCs for evaluation of characteristics and methods of therapeutic targeting.

Materials and Methods

Cell culture and reagents

Mouse mammary carcinoma cell line (MMC) is an epithelial tumor cell line established from a spontaneous tumor of a neu-tg mouse as previously described (11). Both MMCTT and ETTM were each generated twice by treatment of MMCs with 100 ng/ml TGFβ and 50 ng/ml TNFα for 30 and 60 days, respectively. MMCTTE cells were epithelial cells derived from MMCTT cells upon withdrawal of TGFβ and TNFα treatment and subsequent culture for 30 days. ANV cells are mesenchymal breast cancer stem like tumor cell lines produced in vivo by injection of MMC cells into non-transgenic parental FVB/N mice (11). Derivation of ANV5 was confirmed using fluorescent in situ hybridization of the rat neu gene and karyotyping analysis (11). MMC and ANV5cells were tested for mycoplasmausing PCR-based IMPACT Profile III test (RADIL, Colombia, MO) and together with the in vitro derived MMCTT and ETTM cell lines maintained in RPMI supplemented with 10% FBS, Glutamate, and antibiotics. MCF10A cells were obtained from ATCC(Manassas, VA)and immediately resuscitated and expanded in DMEM/F12 media containing 5% horse serum, EGF, insulin, hydrocortisone and antibiotics. ATCC performed authentication on MCF10A cell lines through short tandem repeat profiling, karyotyping, and cytochrome c oxidase I testing. Test for bacterial and fungal contamination was performed by ATCC using current USP methods for viral testing adhering to United States Code of Federal Regulation (9 CFR 113.53)guidelines, mycoplasma testing via direct culture and Hoechst DNA staining and Limulus Amoebocyte Lysate (LAL) assay to measure Endotoxin values. The cells were then split when enough cells were obtained and then immediately treated with the cytokines or frozen as seeding stocks. All cell lines were treated with Placmocin (InvivoGen, San Diego, CA) every two-weeksto prevent mycoplasma contamination. MCF10ATT cells were generated in duplicate by treatment of MCF10A cells with same doses of TGFβ and TNFα for 40 days. TGFβ and TNFα were purchased from R&D System (St Paul, MN).

RNA Isolation, RT-PCR and qPCR

Total RNA was purified from cells using RNeasy Plus kit (Qiagen, Valencia, CA). RNA quantity and purity were determined using a NanoDrop ND-1000, and RNA integrity was assessed by determining the RNA integrity number and 28S/18S ratio using a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). PCR primers were designed with Primer Quest (IDT, Inc., Coralville, IA). Reverse transcription-PCR (RT-PCR) was performed using SuperScript One Step RT-PCR with Platinum Taq (Invitrogen, Carlsbad, CA) using 200 ng of RNA in a Bio-Rad MyCycler. PCR samples were analyzed on 1.5% agarose gels and imaged on a Gel Doc XR (Bio-Rad, Hercules, Ca). First strand cDNA for qPCR was synthesized using the RT2 First Strand cDNA Kit (SABiosciences, Frederick, MD). Gene expression and signaling pathway analyses were done using RT2 Profiler PCR Array qPCR kit and detected with the RT2 SYBR Green qPCR Master Mix (SABioscience) according to the manufacturer’s protocol and run on ABI 7900HT with standard 96 block (Applied Biosystems, Carlsbad, Ca). Expression analysis was performed using the manufacturer’s online analysis tool and gene expression was normalized to housekeeping genes. Differential expression is measured as fold expression relative to the MMC cell line.

Immunoblot analysis

Cell lysates were prepared with standard RIPA buffer and after determining protein concentration, equal protein amounts of samples were resolved by SDS-PAGE gel, transferred onto PVDF membranes, blocked with 5% nonfat milk in TBST and incubated with primary antibody at room temperature for 4hrs or overnight at 4°C. After incubation with appropriate horseradish peroxidase-conjugated secondary antibodies in blocking buffer, protein expression was detected using Super Signal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL). Primary polyclonal antibody to E-cadherin, N-Cadherin and β-actin-HRP, and secondary antibodies were purchased from Santa Cruz (Santa Cruz, CA). Anti–β-actin was used as loading control.

Mammosphere formation assay and cell imaging

For mammosphere formation, MMC, MMCTT, ETTM, MCF10A and MCF10ATT cell lines were grown in 4 mls DMEM/F12 media with 1:50 B27 (Invitrogen), 20 ng/mL EGF, 20 ng/mL, 10 μg/mL insulin, penicillin, streptomycin, and amphotericin B in 12-well plates at a density of 5,000 cells/mL as described previously (19). Additional 0.5mLs of media were added every 3 days for 15 days. The number of mammospheres formed were observed and counted under a Leica DC 200 microscope (Leica Microsystems, Bannockburn, IL). Images of spheroids and adherent cells were obtained with a Leica DC 200 microscope (Leica Microsystems) and Fujifilm FinePix 6800 Zoom camera (Fujifilm, Valhalla, NY).

Flow cytometry and cell sorting

Cell surface expression of E-Cadherin, N-Cadherin, CD24 and CD44 on MMC, MMCTT ETTM, MCT10A and MCF10ATT cells was determined by flow cytometry analysis on a FACScan (Becton Dickinson, Franklin Lakes, NJ). Cells were incubated with conjugated or non-conjugated primary antibodies at 4°C for 30 min, washed and then stained with secondary antibody for 30 min. The cells were then fixed with washing buffer containing 0.5% formaldehyde and run on a BD FACScan flow cytometer (Becton Dickinson). Sorting of CD24/CD44+ cells was done on a BD FACS Vantage Cell Sorter and data were analyzed using Win MDI ver 2.8 software (http://en.bio-soft.net/other/WinMDI.html). Antibodies used included anti-CD24 PE (eBioscience, San Diego CA), anti-E-cadherin PE, anti-E-Cadherin FITC and anti-CD44 FITC (BD Pharmingen, San Diego, CA), and anti-N-Cadherin (Santa Cruz Biotechnology). Secondary antibody FITC goat anti-rabbit IgG were from Jackson Immuno Research Laboratories. Isotype antibodies were PE Rat IgG2b (eBioscience) and FITC Rat IgG2b (BD Pharmingen).

In vivo tumorigenicity

Female neu-tg mice on the FVB/N background were maintained as a colony and in vivo tumorigenicity assays were performed according to institutional animal care and use (IACUC) policy. Appropriate numbers of MMC, MMCTT and ETTM cells were injected subcutaneously into the mice and tumor size measured until the mice were sacrificed. Tumors were measured every other day with vernier calipers, and volumes were calculated as the product of length × width × height × 0.5236.

In vitro cell migration and invasion assay

Migration of MMC and ETTM cells were assessed using non-coated membrane transwells (24-well inserts; pore size, 8 μm; BD Biosciences). About 5 × 104 cells suspended in serum free medium were plated in the top chamber and medium supplemented with serum was used as a chemo attractant in the lower chamber. The invasion assay was conducted as described for migration assay using 1.5 × 105 cells and matrigel-coated membrane (24-well insert, pore size, 8 μm; BD Biosciences). After 24 hrs of incubation, cells remaining on top of the membrane and not migrating were removed using a cotton swab. Migrated cells on the lower surface of the membrane were stained with Hema 3 Stain (Fisher Scientific, Pittsburgh, PA), photographed, and counted.

Chemotherapy and cytotoxicity assays

Etoposide and Paclitaxel were from Selleck Chemical (Houston, TX); Oxaliplatin was from purchased from Sigma Aldrich (St. Louis, MO). Chemoresistance of MMC and ETTM cells was determined by measuring cell viability using the Xcelligence system (Roche Applied Science, Indianapolis, IN). The instrument measures cell status (given as cell Index) in a form of electrical impedance which is determined by cell morphology, cell adhesion and cell viability. As cells attach to the bottom of the plate coated with electrodes, a change in local ionic environment occurs resulting in increased impedance. Measurements were performed according to the instructions of the supplier. After seeding 100μL of suspension of 20,000 MMC and ETTM cells into the 16-well of the E-plates, the cell index was taken every 5min for about 18 hrs for the cells to reach log phase. The cells were then exposed to increasing doses of oxaliplatin, paclitaxel and etoposide and the cell index read again for additional 60 hrs.

Statistical analysis

Statistical analysis was performed using GraphPad Prism version 4.00 for Windows, GraphPad Software, San Diego California USA. Two-tailed Student’s t test, the Mann-Whitney Test or the Two-way analysis of variance test was performed to determine statistically significant difference. P < 0.05 was considered as significant.

Results

TGFϐ and TNFα generate mesenchymal tumor cells withBCSC characteristics

We previously isolated murine mammary carcinomas cells (MMCs) from tumors developing inneu-transgenic (i.e. neu-Tg)mice (20). We found that MMC cells had epithelial cell characteristics and rapidly formed tumors when reinjected into neu-tgmice, but when injected into syngeneic wild-type mice, showed an immune-mediated initial delay followed by rapid development of tumors which had undergone EMT and had lost expressionof the neuantigen, generating antigen-negative variants(ANVs) (11, 20). We sought to define a culture model of the MMC/ANV conversion process, and focused on TGFβ and TNFα as key immunologic agents. When MMC cells were cultured with TGFβ and TNFα for extended periods, they were converted to stable mesenchymalcells (named ETTM cells), showing the characteristic change in cell morphology from rounded to elongated, consistent with EMT (Fig. 1A), which was accompanied by loss of expression of the epithelial marker, E-Cadherin, and a gain in expression of mesenchymal markers, N-Cadherin, Snail, Twist and Zeb1 (Figs. 1A-1C). At earlier time points of treatment with TGFβ/TNFα, the cells showed an intermediate phenotype (named MMCTT cells) where although the cells had acquired mesenchymal features, gene expression analysis by immunoblot, RT-PCR and flow cytometry showed only a partial loss of E-Cadherin and partial gain of N-Cadherin suggesting incomplete EMT(Figs. 1A and 1B). While removal of TGFβ/TNFα from the culture media led to reversion of MMCTT cells to an epithelial phenotype (named MCTTE cells), the ETTM cells showed a complete and stable conversion. To assess the ability of ETTM cells to re-differentiate in vivo, they were injected into neu-tg mice and after tumor formation, removed and reassessed for expression of EMT markers. We found that the tumors expressed both E-and N-Cadherin indicating that, despite the stable culture phenotype, they were able to re-establish heterogeneous tumors with epithelial characteristics. Interestingly, the cells isolated from these tumors, when replated in culture, readily reverted to a mesenchymalpheno type (i.e. N-Cadherin positive and E-cadherin negative), similar to the injected ETTM cells suggesting that the cells possess both the ability to differentiate into E-cadherin expressing epithelial cells as well as maintaining strong self-renewal capacity (Fig. 1D).

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

TGFβ/TNFα-mediated EMT was further confirmed by evaluating EMT genes by PCR pathway analysis. The results showed differential expression of EMT genes including higher levels of Col1a2, Col3a1, Col5a2, MMP9, and MMP3 while epithelial genes like Ocln, Erbb3, Tspan13 and Bmp7 were reduced in ETTM cells (Fig. 1E). Overall, these results demonstrate exposure of MMC tumor cells to TGFβ and TNFα results in the generation of stable mesenchymal cells capable of reestablishing heterogeneous tumors with epithelial characteristics.

Since EMT induction by over expression of Snail, Twist or K-ras resulted in generation of cell populations with BCSC characteristics in prior studies, we questioned whether TGFβ/TNFα-generated mesenchymal cells also have these BCSC characteristics, such as enhanced self-renewal, a CD24−/lowCD44+ phenotype, ability to potently establish heterogeneous tumor and resistance to chemotherapy (7, 11, 12). Self-renewal was examined by assessing for an increased ability of the MMCTT(incomplete transition)and ETTM cells to form mammospheres compared to the MMCs. The results showed that ETTM cells were the most efficient in mammosphere formation, producing more compact and rounded spheroids, than MMCTT cells. MMCs, in contrast, were the least efficient in forming mammospheres(Fig. 2A). Representative staining for CD24 and CD44 showed that the CD24−/lowCD44+ population within cultures increased with combined TGFβ and TNFα treatment from 33% in the MMCs, to 77% in the MCTTs and 100% in the ETTMs (Fig. 2A). The observation that 33% of MMCs were CD24−/lowCD44+ despite a poorer mammosphere forming efficiency suggests that many of the CD24−/CD44+ cell are not mammosphere-forming cells, which is consistent with Al-Hajj and colleagues work also showing that only a subset of human CD24−/lowCD44+ cells are BCSCs (7).

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

Using the mixed epithelial and mesenchymal tumor cell line, MMCTT, we evaluated the morphology and mammosphere formation efficiency of isolated CD24+/CD44+ and CD24/CD44+ subpopulations to confirm that the mesenchymaland self-renewing phenotype is associated with the CD24/CD44+ subset. Results in Figure 2B show that only CD24/CD44+ cells had a mesenchymal phenotype and effectively formed mammospheres (Fig 2b). Staining of tumor cells isolated from ETTM-induced tumors showed CD24+/CD44+ and CD24/CD44+ populations. Consistent with Figure 1D showing that cultures of ETTM-generated tumor cell reverted to mesenchymal cells, the cultured tumor cells also reverted entirely to the CD24/CD44+ cells phenotype suggesting that the ETTM cells have the ability to differentiate and to self-renew (Fig. 2C).

A recently identified molecular subtype of breast cancer, claudin-low, is associated with increased expression of mesenchymal markers and correlates with poor prognosis (21, 22). Additionally, residual subpopulations of breast cancer cells that evade chemo-and endocrine therapy, in humans, have a claudin-low and BCSC phenotype characterized by reduced levels of claudin 3, 4, and 7, enrichment of CD24−/lowCD44+ cells, and increased expression of stem cell-related genes (23). Parallel to these studies, we observed that ETTM cells have considerably reduced expression of claudin 3, 4, and 7 and the luminal marker KRT18, relative to the parental MMC tumor cells (Fig. 2D). Furthermore, pathway analysis showed enriched expression of stem cell-associated genes, including Col1a1, Fgf2, Ccnd2, Igf1, Notch1, ABCG2 in the ETTM cells (Fig. 2E).

TGFϐ/TNFα mediated EMT in human MCF10A cells generates mesenchymal cells with a stem cell-like phenotype

To determine whether TGFβ/TNFα induced EMT in human epithelial mammary cells can generate stem cell-like cells, we treated normal human mammary epithelial MCF10A cells with the cytokines for 40 days, giving rise to MCF10ATT cells. As expected, exposure to TGF and TNF led to acquisition of mesenchymal phenotype accompanied by upregulation of mesenchymal genes (such as Vimentin, Sparc, Foxc2, CDH2) and downregulation of epithelial markers (CDH1, Krt14, Ocln) as shown by real-time qPCR analysis (Figs. 3A and B). We then evaluated the breast cancer stem cell properties of the MCF10ATTcells by flow cytometry of CD24 and CD44 expression. We observed that all the MCF10ATTcells were CD24−/CD44+ compared to the MCF10A cells which comprised mainly CD24+/CD44+ population (Fig. 3C). Real-time qPCR pathway analysis also showed differential regulation of stem cell gene (Fig. 3D). To assess the self-renewal ability of the EMT generated MCF10ATTcells, we examined their mammosphere-forming capacity compared to the parental MCF10Acells. As shown in Figs. 3E and F, we found that the stem cell–like MCF10ATT cells were more efficient mammosphere forming units generating more than 100 fold mammospheres compared to the MCF10A cells.

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

Regulation of EMT and stem cell associated genes in ETTM cells

To further assess the BCSC characteristics of the ETTM cells, pathway analysis of transcription factors and signaling pathways associated with cancer stem cells such as Home box genes, TGFβ, Notch and Hedgehog signaling pathways were performed (2426). These analyses revealed increased expression, in ETTM relative to MMC, of Homeo box-containing genes such as Dlx1, Hoxc8 and Hoxa9 as well as lower levels of Hoxd13, Pdx1 and Pitx1 (Fig. 4A). TGFβ/BMP signaling genes including TGFB1, Col1a1, Serpine1, TGFB2 and Igfbp3 were differentially expressed(Fig. 4B), whereas analysis of Hedgehog and Notch signaling genes identified differential expression of Wnt6, BMP4, Wnt10a, Gas1, Gli3, Ptchd2, Ptch1Wisp1, Notch4, Fosl1, Fzd1, Notch3 and Hey1 (Figs. 4C and D). These observations indicate widespread regulation of stem-associated genes in ETTMBCSCs. Overall, these observations together shows that TGFβ/TNFα mediate EMT leading to the generation CD24/CD44+ cells BCSCs with a claudin-low phenotype.

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

TGFβ/TNFα-mediated EMT results in increased tumor aggressiveness

Mesenchymal BCSCs are known to have increased aggressiveness relative to epithelial tumor cells. Important characteristics include high tumorigenicity nature as well as enhanced migration, invasion and chemoresistance (12, 13, 27). Consistent with their mesenchymal phenotype, ETTM cells demonstrated enhanced migration and invasion compared to MMCs (Fig 5A). Relative to MMC and MMCTT, ETTM cells were also much more tumorigenic than the MMCTTs (Fig. 5B). Furthermore, serial dilution of the ETTM cells showed ability to form tumors with as low as 100 cells compared to the MMCs which could only form tumors at high cell numbers (11) (Fig. 5C).

Figure 5
ETTM cells are highly migratory, invasive and tumorigenic

Human CD24−/lowCD44+ BCSCs are known to have a metastatic gene signature and are associated with a poor prognosis (28). Analysis of expression of metastasis-associated genes in ETTM cells revealed higher levels of Ccl7, Mmp13, Mmp9, Mmp3, Cdh11, Hgf, Mmp10, Ctsk and Cdh6 as well as lower levels of Mcam and Tnfsf10, as compared to MMC(Fig 5D). In addition, assessment of oncogenes and tumor suppressor gene expression signatures showed higher expression of S100a4, Tnf, kitlg, P53, Mycn and Est1 as well as lower levels of Myb, Serpinb5, Cdh1 and Kit (Fig. 5E). Overall, these results demonstrated that ETTM cells, like previously reported CSCs, are substantially more aggressive than their epithelial differentiated counterparts.

ETTM BSCSs are chemoresistant

A clinically important attribute of CSCs is their resistance to conventional therapies such as chemotherapy (2, 29). Prior work by us and others has shown that CD24−/lowCD44+ BCSCs have elevated levels of drug pumps and DNA repair proteins thereby conferring profound resistance to chemo-and radio-therapies (11, 30). To assess the resistance of ETTM and MMC cells, they were treated with increasing doses of Oxaliplatin, Paclitaxel and Etoposide (11)and growth measured in real time (31). As shown in Figs.6A–C, BCSCs generated with TGFβ/TNFα were more resistant to chemotherapy as compared to epithelial MMC.

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

Analysis of drug resistance genes showed increased expression of ABC transporter genes, Abcb1b, Abcc3 and Abcc5 as well as resistance genes Hif1a, and Pparg but lower levels of Erbb3 and Tnfrsf11a (Fig. 6D). Analysis of apoptotic genes showed higher expression of Bcl2l2, Birc3 and Traf1 genes but decreased expression of Bcl2, Bcl2l1, Mme5 and Dapk1 (Fig 6E). DNA damage and repair signaling such as P53, Rad9b and Rad51L1 were higher whereas Gadd45a, Rad50 and Rad54l1 were lower (Fig. 6F).

ETTM cells show altered expression of breast cancer and cell survival pathways

A number of signaling pathways including chemokine pathwayshavea role in breast cancer pathogenesis. Studies have shown correlation between chemokine-secreting tumor-infiltrating macrophages and poor prognosis of breast cancer (32, 33). Assessment of chemokine receptor signaling genes in ETTM showed increased expression of Ccl13, Cxcr7, Ccl11, Cxcl12 and Tnf as well as downregulation of Cxcr4 relative to MMC (Fig. 7A). We also observed higher amounts of inflammatory cytokines and receptor genes Ccl2, Ccl5, Ccl6, Ccl7, Ccl8, Ccl11, Ccr1, Ccr7 and Il1r1 but lower Ccr3, Cxcl1, Il1f6 and Ccl20 (Fig. 7B). In breast cancer, expression of estrogen receptors is both a predictive and prognostic marker as well as an effective means of targeting hormone-dependent breast cancers (34). Gene analysis of breast cancer estrogen receptor signaling identified differential expression of Thbs2, Serpine1, Serpineb5, Cldn7, Krt18 and Kit genes (Fig. 7C). Cell cycle regulators are implicated in cancer initiation, progression and resistance to therapy, and were also found to be differentially expressed in ETTM (Fig. 7D) (35). Finally, higher levels of angiogenesis genes were observed with generation of ETTM cells (Fig. 7E). The results of these gene expression analyses of breast cancer and cell survival-associated pathways supports the genotype and phenotype of the ETTM cells as BCSCs.

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

Discussion

The key BCSC characteristics are increased tumorigenicity, ability to reconstitute a heterogeneous tumor, self-renewal and resistance to therapies. We have shown here that treatment of epithelial MMCs with TGFβ/TNFα generates stable mesenchymal BCSCs, which have increased expression of mesenchymal markers such N-Cadherin, Snail, Slug, Twist, Zeb1, Mmp3 and Mmp9. Different cancer cell lines have different degrees of sensitivity and resistance to TGFβ-induced EMT (3640). The incomplete and reversible EMT observed in MMCTT cells, in contrast to complete and stable ETTMs, generated from the cell epithelial MMC cells present an intriguing observation, suggesting that although EMT can begin within a few days of exposure to cytokine, complete EMTinvolves not only early regulation of actin cytoskeleton as evident in cell morphological changes, but is associated with regulation of a plethora of genes and activation of an array of molecular pathways leading to a transformation of epithelial cells to more migratory and invasive mesenchymal cells. Thus, EMT may not be a rapid but rather a long term process involving regulation of many genes which act in concert to produce the profound observed cellular, molecular, phenotypic and functional changes.

The generation of BCSCs by treatment with TGFβ/TNFα suggests that factors secreted by the immune system play an important role in breast cancer progression, which is consistent with our prior in vivo studies (11). It also implicates inflammation-induced EMT in cancer metastases and recurrence. TGFβ is a potent growth inhibitor which normally functions to regulate aberrant growth of epithelial and hematopoietic cells (41). However, TGFβ can also induce proliferation and invasiveness in cancer cells that have evaded the inhibitory effect of TGFβ signaling through activation of specific biological processes (42). Although the mechanism by which TGFβ can alternatively suppress and promote cancer progression at different stages of tumor development has not been completely elucidated, induction of EMT or at least regulation of mesenchymal genes could be one possible scenario. Furthermore, the simultaneous expression of immune factors and receptors may deregulate immunity and allow the CSCs to subvert immune-mediate delimination, thereby permitting long term survival, which would be consistent with long-term recurrence risk among treated breast cancer patients.

The ability of the BCSCs to interconvert between the CD24−/lowCD44+ BCSCs and CD24+/CD44+ tumor cells, as shown by de-differentiation of epithelial cells or through selection in culture presents a unique model for understanding how to inhibit the growth and regeneration of BCSCs (43). Identification of cell surface markers of BCSCs for accurate identification and isolation is essential if BCSC research is to make an impact in patient care. In addition, identification of functionally relevant biological targets for development of monoclonal antibodies or small molecule inhibitors to target BCSCs in combination with conventional treatments could provide the breakthrough needed to minimize metastatic recurrence of breast cancer.

We observed upregulation of signaling pathways linked to stem cells including Home box, hedgehog, TGFβ/BMP, Notch and chemokine and chemokine receptors. These pathways are subjects of on-going research to identify possible targets. Chemo resistance to cytotoxic drugs such as Oxaliplatin, Etoposide and Paclitaxel is a crucial property of CSCs which is central to their survival and evasion. The robust chemoresistant and regulation of genes and pathways associated with stemness and survival underscores the major problem posed by CSCs, specifically their ability to be eradicated with existing therapeutic modalities.

There is increasing evidence of the importance of EMT in the generation of the claudin-low sub-type (22). The claudin-low subtype is a recent addition to the four major subtypes of breast cancer, namely luminal A, luminal B, basal-like and ERBB2 positive subtypes (44). This new subtype exhibits stem cell characteristics with increased expression of immune response genes, EMT genes, and lower expression of luminal differentiation genes (45). The claudin-low subtype also constitutes the residual subpopulation of breast cancer cells that survived after chemo-and endocrine therapy and are enriched in CD24−/low/CD44+ cells (23). Our results show that TGFβ/TNFα treatment of breast cancer cells with a predominant luminal phenotype results in generation of the claudin-low cells and suggests that targeting proteins involved in the induction or survival of this subtype could be a therapeutic strategy for improving survival of breast cancer patients regardless of the primary subtype.

In conclusion, this study provides new information on the role of the cytokines, TGFβ and TNFα in EMT. It also provides initial evidence linking immunity (i.e. cytokines) to the generation of the BCSC through EMT. The ex vivo generation of BCSCs cells with cytokines may enable a better understanding of the biology of CSC, identification of definitive biomarkers, and the discovery of biological targets and pathways for development of effective therapies. It could also be useful for drug screening to determine the effectiveness and required doses of CSC-targeted therapies.

Acknowledgments

Grant Support

This work was supported by a generous gift from Martha and Bruce Atwater (KLK); Howard Temin Award, K01-CA100764 (KLK); R01-CA122086 (DCR); and the Mayo Clinic Breast Cancer Specialized Program of Research Excellence Award, P50-CA116201 (JI).

The authors acknowledge the strong support of the Mayo Clinic Comprehensive Cancer Center for providing access to core facilities.

References

1. Han JS, Crowe DL. Tumor initiating cancer stem cells from human breast cancer cell lines. Int J Oncol. 2009;34:1449–53. [PubMed]
2. Bapat SA. Evolution of cancer stem cells. Semin Cancer Biol. 2007;17:204–13. [PubMed]
3. Miller SJ, Lavker RM, Sun TT. Interpreting epithelial cancer biology in the context of stem cells: tumor properties and therapeutic implications. Biochim Biophys Acta. 2005;1756:25–52. [PubMed]
4. Yoshiura K, Kanai Y, Ochiai A, Shimoyama Y, Sugimura T, Hirohashi S. Silencing of the E-cadherin invasion-suppressor gene by CpG methylation in human carcinomas. Proc Natl Acad Sci U S A. 1995;92:7416–9. [PMC free article] [PubMed]
5. Bissell MJ, Labarge MA. Context, tissue plasticity, and cancer: are tumor stem cells also regulated by the microenvironment? Cancer Cell. 2005;7:17–23. [PMC free article] [PubMed]
6. Ichim CV, Wells RA. First among equals: the cancer cell hierarchy. Leuk Lymphoma. 2006;47:2017–27. [PubMed]
7. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A. 2003;100:3983–8. [PMC free article] [PubMed]
8. Patrawala L, Calhoun T, Schneider-Broussard R, Zhou J, Claypool K, Tang DG. Side population is enriched in tumorigenic, stem-like cancer cells, whereas ABCG2+ and ABCG2-cancer cells are similarly tumorigenic. Cancer Res. 2005;65:6207–19. [PubMed]
9. Ponti D, Costa A, Zaffaroni N, Pratesi G, Petrangolini G, Coradini D, et al. Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties. Cancer Res. 2005;65:5506–11. [PubMed]
10. Ginestier C, Hur MH, Charafe-Jauffret E, Monville F, Dutcher J, Brown M, et al. ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell. 2007;1:555–67. [PMC free article] [PubMed]
11. Santisteban M, Reiman JM, Asiedu MK, Behrens MD, Nassar A, Kalli KR, et al. Immune-induced epithelial to mesenchymal transition in vivo generates breast cancer stem cells. Cancer Res. 2009;69:2887–95. [PMC free article] [PubMed]
12. Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, et al. The epithelial- mesenchymal transition generates cells with properties of stem cells. Cell. 2008;133:704–15. [PMC free article] [PubMed]
13. Morel AP, Lievre M, Thomas C, Hinkal G, Ansieau S, Puisieux A. Generation of breast cancer stem cells through epithelial-mesenchymal transition. PLoS One. 2008;3:e2888. [PMC free article] [PubMed]
14. Lee JM, Dedhar S, Kalluri R, Thompson EW. The epithelial-mesenchymal transition: new insights in signaling, development, and disease. J Cell Biol. 2006;172:973–81. [PMC free article] [PubMed]
15. Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial-mesenchymal transitions in development and disease. Cell. 2009;139:871–90. [PubMed]
16. Cowin P, Welch DR. Breast cancer progression: controversies and consensus in the molecular mechanisms of metastasis and EMT. J Mammary Gland Biol Neoplasia. 2007;12:99–102. [PMC free article] [PubMed]
17. Bates RC, Mercurio AM. Tumor necrosis factor-alpha stimulates the epithelial-to- mesenchymal transition of human colonic or ganoids. Mol Biol Cell. 2003;14:1790–800. [PMC free article] [PubMed]
18. Radisky DC. Epithelial-mesenchymal transition. J Cell Sci. 2005;118:4325–6. [PubMed]
19. Grimshaw MJ, Cooper L, Papazisis K, Coleman JA, Bohnenkamp HR, Chiapero-Stanke L, et al. Mammosphere culture of metastatic breast cancer cells enriches for tumorigenic breast cancer cells. Breast Cancer Res. 2008;10:R52. [PMC free article] [PubMed]
20. Knutson KL, Almand B, Dang Y, Disis ML. Neu antigen-negative variants can be generated after neu-specific antibody therapy in neu transgenic mice. Cancer Res. 2004;64:1146–51. [PubMed]
21. Hennessy BT, Gonzalez-Angulo AM, Stemke-Hale K, Gilcrease MZ, Krishnamurthy S, Lee JS, et al. Characterization of a naturally occurring breast cancer subset enriched in epithelial-to-mesenchymal transition and stem cell characteristics. Cancer Res. 2009;69:4116–24. [PMC free article] [PubMed]
22. Herschkowitz JI, Simin K, Weigman VJ, Mikaelian I, Usary J, Hu Z, et al. Identification of conserved gene expression features between murine mammary carcinoma models and human breast tumors. Genome Biol. 2007;8:R76. [PMC free article] [PubMed]
23. Creighton CJ, Li X, Landis M, Dixon JM, Neumeister VM, Sjolund A, et al. Residual breast cancers after conventional therapy display mesenchymal as well as tumor-initiating features. Proc Natl AcadSci U S A. 2009;106:13820–5. [PMC free article] [PubMed]
24. Pannuti A, Foreman K, Rizzo P, Osipo C, Golde T, Osborne B, et al. Targeting Notch to target cancer stem cells. Clin Cancer Res. 16:3141–52. [PMC free article] [PubMed]
25. Liu J, Sato C, Cerletti M, Wagers A. Notch signaling in the regulation of stem cell self- renewal and differentiation. Curr Top Dev Biol. 92:367–409. [PubMed]
26. Liu S, Dontu G, Mantle ID, Patel S, Ahn NS, Jackson KW, et al. Hedgehog signaling and Bmi-1 regulate self-renewal of normal and malignant human mammary stem cells. Cancer Res. 2006;66:6063–71. [PubMed]
27. Gupta PB, Onder TT, Jiang G, Tao K, Kuperwasser C, Weinberg RA, et al. Identification of selective inhibitors of cancer stem cells by high-throughput screening. Cell. 2009;138:645–59. [PubMed]
28. Liu R, Wang X, Chen GY, Dalerba P, Gurney A, Hoey T, et al. The prognostic role of a gene signature from tumorigenic breast-cancer cells. N Engl J of Med. 2007;356:217–26. [PubMed]
29. Gangemi R, Paleari L, Orengo AM, Cesario A, Chessa L, Ferrini S, et al. Cancer stem cells: a new paradigm for understanding tumor growth and progression and drug resistance. Curr Med Chem. 2009;16:1688–703. [PubMed]
30. Phillips TM, McBride WH, Pajonk F. The response of CD24(−/low)/CD44+ breast cancer- initiating cells to radiation. J Natl Cancer Inst. 2006;98:1777–85. [PubMed]
31. Vistejnova L, Dvorakova J, Hasova M, Muthny T, Velebny V, Soucek K, et al. The comparison of impedance-based method of cell proliferation monitoring with commonly used metabolic-based techniques. Neuro Endocrinol Lett. 2009;30 (Suppl 1):121–7. [PubMed]
32. Leek RD, Harris AL. Tumor-associated macrophages in breast cancer. J Mammary Gland Biol Neoplasia. 2002;7:177–89. [PubMed]
33. Lin EY, Pollard JW. Macrophages: modulators of breast cancer progression. Novartis Found Symp. 2004;256:158–68. discussion 68–72, 259–69. [PubMed]
34. Johnston SR. New strategies in estrogen receptor-positive breast cancer. Clin Cancer Res. 16:1979–87. [PubMed]
35. Helleday T, Petermann E, Lundin C, Hodgson B, Sharma RA. DNA repair pathways as targets for cancer therapy. Nat Rev Cancer. 2008;8:193–204. [PubMed]
36. Illman SA, Lehti K, Keski-Oja J, Lohi J. Epilysin (MMP-28) induces TGF-beta mediated epithelial to mesenchymal transition in lung carcinoma cells. J Cell Sci. 2006;119:3856–65. [PubMed]
37. Tavares AL, Mercado-Pimentel ME, Runyan RB, Kitten GT. TGF beta-mediated RhoA expression is necessary for epithelial-mesenchymal transition in the embryonic chick heart. Dev Dyn. 2006;235:1589–98. [PubMed]
38. Doerner AM, Zuraw BL. TGF-beta1 induced epithelial to mesenchymal transition (EMT) in human bronchial epithelial cells is enhanced by IL-1beta but not abrogated by corticosteroids. Respir Res. 2009;10:100. [PMC free article] [PubMed]
39. Hills CE, Squires PE. TGF-beta1-induced epithelial-to-mesenchymal transition and therapeutic intervention in diabetic nephropathy. Am J Nephrol. 31:68–74. [PubMed]
40. Zavadil J, Bottinger EP. TGF-beta and epithelial-to-mesenchymal transitions. Oncogene. 2005;24:5764–74. [PubMed]
41. Massague J, Blain SW, Lo RS. TGFbeta signaling in growth control, cancer, and heritable disorders. Cell. 2000;103:295–309. [PubMed]
42. Galliher AJ, Neil JR, Schiemann WP. Role of transforming growth factor-beta in cancer progression. Future Oncol. 2006;2:743–63. [PubMed]
43. Gupta PB, Chaffer CL, Weinberg RA. Cancer stem cells: mirage or reality? Nat Med. 2009;15:1010–2. [PubMed]
44. Sorlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H, et al. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci U S A. 2001;98:10869–74. [PMC free article] [PubMed]
45. Horwitz EM, Prather WR. Cytokines as the major mechanism of mesenchymal stem cell clinical activity: expanding the spectrum of cell therapy. Isr Med Assoc J. 2009;11:209–11. [PubMed]
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...