Research ArticleCancer

Chronic TGF-β exposure drives stabilized EMT, tumor stemness, and cancer drug resistance with vulnerability to bitopic mTOR inhibition

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Science Signaling  26 Feb 2019:
Vol. 12, Issue 570, eaau8544
DOI: 10.1126/scisignal.aau8544
  • Fig. 1 Prolonged TGF-β treatment stabilizes a mesenchymal state in HMLE cells.

    (A) HMLE cells were treated with TGF-β for 12 or 24 days (d), followed by its removal, and then cultured with or without SB431542 for 12 days. The cells were observed by phase-contrast microscopy or subjected to staining for E-cadherin, fibronectin, F-actin, or 4′,6-diamidino-2-phenylindole (DAPI). Scale bars, 50 μm. Images are representative of three independent experiments. (B and C) HMLE cells were treated with TGF-β, followed by its removal as in (A), and were then cultured without TGF-β (Rem) or with SB431542 (SB) for 12 days. The expression of epithelial and mesenchymal markers was analyzed by immunoblotting (B) or quantified by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and normalized to rPL19 mRNA (C). Data are means ± SE from n = 3 independent experiments. *P < 0.05 and **P < 0.01, by a Dunnett’s test. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

  • Fig. 2 Prolonged TGF-β treatment stabilizes a stem cell–like state.

    (A) HMLE cells were treated with TGF-β for 12 or 24 days. TGF-β was removed and cells were cultured without TGF-β (Rem) or in the presence of SB431542 (SB) for 12 days. The expression of the CD44 stem cell marker gene and the stemness-associated NANOG, POU5F1, and SOX2 genes was assessed by qRT-PCR and normalized to rPL19 mRNA. Data are means ± SE from n = 3 independent experiments. *P < 0.05 and **P < 0.01, by a Dunnett’s test. (B and C) HMLE cells were treated with TGF-β for 12 or 24 days, followed by removal of TGF-β, and the cells were cultured with or without SB431542 for 12 days. The expression of the cell surface markers CD44 and CD24 was analyzed by flow cytometry (B). The graph (C) shows the percentages of CD24lowCD44high stem cells in the entire cell populations. Data are means ± SE from n = 3 independent experiments. **P < 0.01 versus untreated cells and ##P < 0.01 versus cells treated with TGF-β for 12 or 24 days, by a Tukey’s test. (D and E) HMLE cells were treated with TGF-β for 12 or 24 days. TGF-β was removed and cells were cultured in the presence (SB) or absence (Rem) of SB431542 for 12 days. Cells were assessed for primary and secondary mammosphere formation. The mammospheres were observed by phase-contrast microscopy (D) and counted (E). Scale bar, 100 μm. Data are means ± SE from n = 3 independent experiments. **P < 0.01, by a Dunnett’s test.

  • Fig. 3 Prolonged TGF-β treatment stabilizes a mesenchymal state in breast cancer cells.

    (A) HMLE cells and Ras-transformed HMLER cells were grown in a monolayer culture to confluence, and the cells were observed by phase-contrast microscopy. Scale bar, 100 μm. Images are representative of three independent cultures. (B) HMLER cells were treated with TGF-β for 2 or 12 days, followed by its removal, and then cultured with or without SB431542 for 6 days. The cells were observed by phase-contrast microscopy or subjected to staining for E-cadherin, fibronectin, F-actin, or DAPI. Scale bars, 50 μm. Images are representative of three independent experiments. (C and D) HMLER cells were treated with TGF-β, followed by its removal, as in (B), and were then cultured without added TGF-β (Rem) or with SB431542 (SB) for 6 days. Epithelial and mesenchymal gene expression were analyzed at the protein level by immunoblot (C) or quantified at the transcript level by qRT-PCR normalized to rPL19 mRNA (D). Data are means ± SE from n = 3 independent experiments.

  • Fig. 4 Prolonged TGF-β treatment, resulting in stabilized EMT, increases drug resistance in breast cancer cells.

    (A to C) HMLER cells treated with TGF-β for 0, 2, or 12 days were trypsinized and reseeded. Left: The cells were then incubated with doxorubicin (A), cisplatin (B), or cyclophosphamide (C) for 48 hours at the concentrations indicated, and the cell viability was quantified and shown relative to the cells not treated with anticancer drugs. Right: The cells were cultured for 48 hours with 250 nM doxorubicin (A), 250 μM cisplatin (B), or 10 μM cyclophosphamide (C), and the cell number was quantified and shown relative to the cells not treated with anticancer drugs. Data are means ± SE from n = 3 independent experiments. *P < 0.05 and **P < 0.01, by a Tukey’s test. (D to F) HMLER cells were treated with or without TGF-β for 12 days and then cultured for 6 days with SB431542 or dimethyl sulfoxide (DMSO) solvent as a control. The cells were then cultured for 48 hours with 250 nM doxorubicin (D), 250 μM cisplatin (E), or 10 μM cyclophosphamide (F), and the cell number was quantified and shown relative to the cells not treated with anticancer drugs. Data are means ± SE from n = 3 independent experiments. *P < 0.05 and **P < 0.01, by a Tukey’s test.

  • Fig. 5 Prolonged TGF-β treatment stabilizes a stem cell state and induces latency in breast cancer cells.

    (A and B) HMLER cells were treated with TGF-β for 2 or 12 days, followed by its removal, as indicated, and then cultured with (SB) or without (Rem) SB431542 for 6 days. The expression of cell surface markers CD44 and CD24 was analyzed by flow cytometry (A). The graph (B) shows the percentages of CD24lowCD44high stem cells in the entire cell population. Data are means ± SE from n = 3 independent experiments. **P < 0.01, by a Dunnett’s test. (C and D) HMLER cells were treated as in (A) and were assessed for mammosphere formation. The mammospheres were observed by phase-contrast microscopy (C) and counted (D). Scale bar, 100 μm. Data are means ± SE from n = 3 independent experiments. *P < 0.05 and **P < 0.01, by a Dunnett’s test. (E) HMLER cells were treated as in (A) and assessed for colony formation in soft agar. Data are means ± SE from n = 3 independent experiments. *P < 0.05 and **P < 0.01, by a Dunnett’s test. (F) HMLER cells were treated with TGF-β for 0, 2, or 12 days and then suspended in phosphate-buffered saline (PBS) with 50% Matrigel, and 10,000 cells were injected orthotopically into a mammary fat pad of NSG mice. The number of mice with a palpable tumor is indicated in the table. (G) HMLER cells were treated with TGF-β for 0, 2, or 12 days and then suspended in PBS, and 500,000 cells were injected into the tail vein of NSG mice. Six weeks after injection, the mice were euthanized, and lungs were harvested. Cancer dissemination into the lungs was measured by counting the tumor nodules in the lungs. Data are means ± SE from n = 3 independent experiments with n = 7 mice. **P < 0.01, by a Dunnett’s test.

  • Fig. 6 Prolonged TGF-β treatment enhances and stabilizes AKT-mTOR signaling.

    (A) HMLER cells were treated with TGF-β for 2 or 12 days, followed by its removal, as indicated, and were cultured in the media without added TGF-β (Rem) or with SB431542 (SB) for 6 days. C-terminal phosphorylation of Smad3 and Smad2 was analyzed by immunoblotting. Blots are representative of n = 3 independent experiments. (B) HMLER cells were treated as in (A), and the expression of TGF-β target genes encoding PAI1, Smad7, p21Cip1, p15Ink4B, or c-Myc was assessed by qRT-PCR and normalized to rPL19 mRNA. Data are means ± SE from n = 3 independent experiments. (C) HMLER cells were treated as in (A), and phosphorylation of AKT, S6K1, S6, 4EBP1, mTOR, and NDRG1 was analyzed by immunoblotting. Blots are representative of n = 3 independent experiments. (D) HMLER cells were treated with TGF-β for 0, 2, or 12 days. The cells were trypsinized and counted. The total protein content normalized for cell number is shown relative to untreated cells. Data are means ± SE from n = 3 independent experiments. *P < 0.05, by a Dunnett’s test.

  • Fig. 7 Inhibition of mTOR signaling suppresses the CSC phenotype.

    (A) HMLER cells were treated with or without TGF-β for 12 days and then cultured for 6 days with MK2206 (500 nM), MLN0128 (100 nM), RapaLink-1 (5 nM), or DMSO solvent as a control. Activation of the AKT-mTOR signaling pathway and expression of epithelial and mesenchymal markers at the protein level were analyzed by immunoblotting. Blots are representative of n = 3 independent experiments. (B) HMLER cells were treated as in (A), and the cells were observed by phase-contrast microscopy. Images are representative of n = 3 independent experiments. Scale bar, 100 μm. (C) HMLER cells were treated as in (A), and the expression of epithelial and mesenchymal markers was analyzed by qRT-PCR and normalized to rPL19 mRNA. Data are means ± SE from n = 3 independent experiments. *P < 0.05 and **P < 0.01 versus the 12 d TGF-β–treated + 6 d control, by a Dunnett’s test. (D) HMLER cells were treated as in (A), and the percentage of CD24lowCD44high stem cells in the entire cell population was determined by flow cytometry. Data are means ± SE from n = 3 independent experiments. **P < 0.01 versus the 12 d TGF-β–treated + 6 d control, by a Dunnett’s test. (E) HMLER cells were treated as in (A) and then assessed for formation of secondary mammospheres. Data are means ± SE from n = 3 independent experiments. **P < 0.01 versus the 12 d TGF-β–treated + 6 d control, by a Dunnett’s test.

  • Fig. 8 Inhibition of mTOR signaling suppresses cancer drug resistance and tumorigenesis.

    (A) HMLER cells were treated with or without TGF-β for 12 days and then cultured for 6 days with MK2206 (500 nM), RapaLink-1 (5 nM), or DMSO solvent as a control. The cells were assessed for colony formation in soft-agar. Data are means ± SE from n = 3 independent experiments. **P < 0.01 versus (−) TGF-β; #P < 0.05 and ##P < 0.01 versus control, by a Tukey’s test. (B) HMLER cells were treated with TGF-β for 0, 2, or 12 days and then reseeded and cultured with MK2206 (500 nM) or RapaLink-1 (5 nM) for 2 days. The live cells were counted, and numbers of viable cells relative to cells cultured without the inhibitors were shown as a graph. Data are means ± SE from n = 3 independent experiments. *P < 0.05 versus (−) TGF-β + RapaLink-1, by a Dunnett’s test. (C) HMLER cells were treated with TGF-β for 12 days and subsequently for 48 hours with doxorubicin (250 nM), cisplatin (250 μM), or cyclophosphamide (10 μM) in the presence of MK2206 (500 nM), RapaLink-1 (5 nM), or DMSO solvent. The live cells were counted, and their numbers were normalized to the initial total cell number. Data are means ± SE from n = 3 independent experiments. *P < 0.05 and **P < 0.01 versus 12 d TGF-β–treated + 6 d control, by a Dunnett’s test. (D) HMLER cells were treated with TGF-β for 0, 2, or 12 days and then suspended in 1:1 PBS/Matrigel, and 10,000 cells were injected orthotopically into a mammary fat pad. Primary tumors were stained with antibodies to pSmad3, AKT-pSer473, or 4EBP1-pThr37/46, or with H&E. Images are representative of eight independent tumors. Scale bars, 50 μm. (E to H) HMLER cells were treated with TGF-β for 12 days, and 2 × 106 cells were injected orthotopically into a mammary fat pad. Mice were treated by intraperitoneal injection of vehicle or RapaLink-1 (1.5 mg/kg) every 5 or 7 days starting at day 8. Tumor sizes were measured by caliper twice per week (E). (F) Mice were euthanized at 6 weeks after injection, and tumors or residual stroma at the site of injection were harvested and photographed (F), and sections were stained by H&E (G) or with antibodies to 4EBP1-pThr37/46 and AKT-pSer473 (H). Scale bars, 500 μm (G) and 50 μm (H). Images are representative of the cohort [in (G) and (H), the RapaLink-1 sections were from normal tissue and remaining stroma at the site of injection], and data are means ± SE from n = 6 mice. **P < 0.01 versus vehicle, by a Student’s t test.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/12/570/eaau8544/DC1

    Fig. S1. Neutralizing anti–TGF-β monoclonal antibody and SB431542 partially reverse the stabilized mesenchymal phenotype.

    Fig. S2. Prolonged TGF-β treatment does not maintain the expression of TGF-β ligand.

    Fig. S3. Prolonged TGF-β treatment stabilizes cell migration and invasion.

    Fig. S4. Prolonged TGF-β treatment increases the resistance of breast cancer cells against 5-fluorouracil and paclitaxel.

    Fig. S5. Histological evaluation of tumors from mice injected with HMLER cells.

    Fig. S6. HMLER cells treated with TGF-β for 2 or 12 days maintain their TGF-β responsiveness.

    Fig. S7. Quantification of the phosphorylation of mTOR targets and EMT marker expression.

    Fig. S8. RapaLink-1 partially reduces the colony formation of untreated or short-term TGF-β–treated HMLER cells.

    Table S1. Primer sequences used for quantitative real-time PCR.

  • This PDF file includes:

    • Fig. S1. Neutralizing anti–TGF-β monoclonal antibody and SB431542 partially reverse the stabilized mesenchymal phenotype.
    • Fig. S2. Prolonged TGF-β treatment does not maintain the expression of TGF-β ligand.
    • Fig. S3. Prolonged TGF-β treatment stabilizes cell migration and invasion.
    • Fig. S4. Prolonged TGF-β treatment increases the resistance of breast cancer cells against 5-fluorouracil and paclitaxel.
    • Fig. S5. Histological evaluation of tumors from mice injected with HMLER cells.
    • Fig. S6. HMLER cells treated with TGF-β for 2 or 12 days maintain their TGF-β responsiveness.
    • Fig. S7. Quantification of the phosphorylation of mTOR targets and EMT marker expression.
    • Fig. S8. RapaLink-1 partially reduces the colony formation of untreated or short-term TGF-β–treated HMLER cells.
    • Table S1. Primer sequences used for quantitative real-time PCR.

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