Transforming growth factor-β regulates the sphere-initiating stem cell-like feature in breast cancer through miRNA-181 and ATM

Abstract

Recent studies indicate that a subset of cancer cells possessing stem cell properties, referred to as cancer-initiating or cancer stem cells (CSCs), have crucial roles in tumor initiation, metastasis and resistance to anticancer therapies. Transforming growth factor (TGF)-β and their family members have been implicated in both normal (embryonic and somatic) stem cells and CSCs. In this study, we observed that exposure to TGF-β increased the population of breast cancer (BC) cells that can form mammospheres in suspension, a feature endowed by stem cells. This was mediated by the micro (mi)RNA family miR-181, which was upregulated by TGF-β at the post-transcriptional level. Levels of the miR-181 family members were elevated in mammospheres grown in undifferentiating conditions, compared with cells grown in two-dimensional conditions. Ataxia telangiectasia mutated (ATM), a target gene of miR-181, exhibited reduced expression in mammospheres and upon TGF-β treatment. Overexpression of miR-181a/b, or depletion of ATM or its substrate CHK2, was sufficient to induce sphere formation in BC cells. Finally, knockdown of ATM enhanced in vivo tumorigenesis of the MDA361 BC cells. Our results elucidate a novel mechanism through which the TGF-β pathway regulates the CSC property by interfering with the tumor suppressor ATM, providing insights into the cellular and environmental factors regulating CSCs, which may guide future studies on therapeutic strategies targeting these cells.

Fig. 1

Identification of the miR-181 family as TGF-β target genes. A. MiRNAs altered by TGF-β treatment. Total RNAs were prepared from MDA231 and MCF10A cells treated with TGF-β (2 ng/ml) or vehicle for 24 h in 3 independent experiments, and subjected to Agilent miRNA microarrays. The differentially expressed miRNAs were selected by statistical criteria of greater than 1.5-fold changes (log base 2 = ±0.58) and p<0.05 by Students T-test (indicated in log 2 value). FC: fold change (in log 2) of TGF-β-treated sample compared to control sample. B. MDA231 and MCF10A cells were treated with TGF-β (2 ng/ml) or vehicle for indicated time. Total RNA was isolated and subjected to quantitative RT-PCR for miR-181a and miR-181b. Data was normalized to the level of U6, and then compared to that in untreated cells, which was set as 1. Each data point represents the mean ± S.D. of 3 wells. C. BC cell lines BT474, MDA361 and MCF7 were treated with TGF-β at the indicated final concentrations or vehicle for 72 h. Total RNA was isolated and subjected to quantitative RT-PCR for miR-181a and miR-181b.

Fig. 2

The context-dependent effect of TGF-β on the sphere-initiating CSC-like feature. A. BC cell lines BT474, MDA361 and MCF7 contained a small cell population that could initiate spheres when cultured in suspension. Single cells were plated in ultralow attachment plates as described in Materials and Methods, so that cells with stem cell properties were allowed to grow as non-adherent spheroids (mammospheres). Images of the mammospheres were captured on day 7. Numbers of the mammospheres (diameter ≥ 70 μm) were counted, and the sphere forming efficiency (SFE) was calculated based on the numbers of cells that were initially seeded. Each data represents the mean ± S.D. of 3 wells. Bars equal 25 μm. B. Protein extracts were prepared from the parental (P) cells growing in 2D, 7-day sphere (S) cells, and cells that were dissociated from 7-day spheres and subsequently cultured in 2D differentiating condition (SD). Western blot was performed using indicated antibodies. GAPDH was used as a loading control. C. IFA images of MDA361 spheres and 2D cultured cells stained with antibodies against CK5 (green) and CK8/18 (red). DAPI: nuclei staining. The bar equals 25 μm. D. Cells grown in 2D were treated with TGF-β at the indicated concentrations for 72 h, before harvested for sphere formation assay in the absence of TGF-β. Spheres were counted on day 7 and SFE was calculated.

Fig. 3

MiR-181 regulates sphere formation. A. Total RNA was isolated from parental cells, sphere cells and sphere-to-differentiation cells as described in Fig. 2B, and subjected to qRT-PCR for miR-181a, miR-181a*, miR-181b and miR-181d. Data was normalized to the level of U6, and then compared to that in parental cells, which was set as 1. B. Cells grown in 2D were transiently transfected with miR-181a hairpin inhibitor or a control reagent. TGF-β (3.5 ng/ml) or vehicle was added at 6 h after transfection. After 72 h, cells were harvested; half were used for RNA extraction and qRT-PCR of miR-181a/b (bottom), and half for sphere formation assay (top). SFE was assessed on day 7. C. Cells grown in 2D were transiently transfected with a miR-181a/b expression plasmid or the vector control. At 72 h after transfection, cells were analyzed for the levels of miR-181a/b by qRT-PCR (bottom) and for SFE (top). * p<0.005. D. BT474 cells grown in 2D were transiently transfected with the hairpin inhibitors of miR-181a or miR-21, either alone or in combination at equal amount. At 72 h after transfection, cells were analyzed for the levels of miR-181a and miR-21 by qRT-PCR (bottom) and for SFE (top).

Fig. 4

TGF-β induces miR-181a/b at the post-transcriptional level. A. Quantitative RT-PCR of the primary (pri-) miR-181a-1 and the precursor (pre-) miR-181a-1 in cells treated with TGF-β at the indicated concentrations for 72 h. Data was normalized to GAPDH in each sample and compared to that of untreated cells, which was set as 1. B. RIP was carried out in BT474 cells treated with TGF-β (3.5 ng/ml) or vehicle for 24 h. Antibodies against Smad2/3, Drosha or IgG (as a negative control) were used in the immunoprecipitation as described in the Materials and Methods. Quantitative RT-PCR of pri-miR-181a-1 and the mature miR-181a were performed, using the pre-RIP RNA sample without reverse transcription reaction (−RT) as a negative control. Data was compared to the control RIP sample with IgG. C. BT474 cells grown in 2D were transiently transfected with siRNA targeting Smad4 or control siRNA. After 72 h, cells were analyzed for the levels of miR-181a/a*/b/d by qRT-PCR (left) and for SFE (right). * p<0.005.

Fig. 5

ATM, a miR-181 target, suppresses sphere formation through phosphorylating CHK2. A. The miR-181a/b/c/d targeting sites in the 3′UTR of ATM mRNA predicted by TargetScanHuman 5.1 (www.targetscan.org) and miRDB (www.mirdb.org). B. The psiCHECK luciferase reporters containing the miR-181 targeting site at 123, the site at 3525 or both sites, or a control reporter containing a scrambled sequence, were used to transfect 293 cells, together with a miR-181a/b expressing plasmid or vector. For the TGF-β treatment experiments, cells were transfected with each reporter; TGF-β (3.5 ng/ml) or vehicle was added at 24 h after transfection. Luciferase activity was analyzed at 48 h post transfection, and compared to the cells transfected with the control plasmids. C. The luciferase reporter containing both miR-181 targeting sites in ATM 3′UTR was transfected into BT474, MDA361 or MCF7 cells, together with the miR-181a/b expressing plasmid or vector. For the TGF-β treatment experiments, cells were transfected with the reporter only; TGF-β (3.5 ng/ml) or vehicle was added at 24 h after transfection. Luciferase activity was analyzed at 48 h post transfection, and compared to the cells transfected with the control plasmids. D. Protein extracts of parental (P) and sphere (S) cells were prepared and subjected to Western blot analysis. GAPDH was used as a loading control. E. BT474 cells grown in 2D were transfected with a miR-181a hairpin inhibitor (or control reagent) or a miR-181a/b expressing plasmid (or vector). TGF-β or vehicle was added at 6 h after transfection. After 72 h, cells were harvested; half were used for RNA extraction and qRT-PCR of miR-181a (bottom), and half for protein extraction and Western blot analysis (top). F. Total RNA isolated from cells treated with TGF-β at the indicated concentrations for 72 h was analyzed for ATM mRNA level by qRT-PCR. Data was normalized to GAPDH in each sample and compared to that of untreated cells. G. Cells grown in 2D were treated with TGF-β or vehicle for 72 h before lysed and subjected to Western blot analysis. H. Cells grown in 2D were transiently transfected with siRNA targeting ATM, CHEK2, BRCA1, p53 or control siRNA. At 72 h after transfection, cells were analyzed for SFE as described in Fig. 3B. * p<0.005. I. Western blot of the indicated proteins in cells transfected with ATM siRNA or control siRNA.

Fig. 6

Knockdown of ATM enhances the in vivo tumorigenesis of BC cells. A. MDA361/tetO-shRNA(ATM) cells were treated in the absence or presence of Dox (1 μg/mL) for 48 h and analyzed for ATM expression by Western blot. B. The same cells were treated in the absence or presence of Dox for 48 h before analyzed for SFE as described. * p<0.005. C. NSG mice were injected in the no. 4 mammary fat pad with 5 × 10⁵ of MDA361/tetO-shRNA(ATM) cells, and divided into 2 groups (5 mice per group) for treatment with Dox or control. Tumor formation was indicated by Tumors/Injections at 3 weeks after injection.