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WNT10B/β-catenin signalling induces HMGA2 and proliferation in metastatic triple-negative breast cancer.

Wend P, Runke S, Wend K, Anchondo B, Yesayan M, Jardon M, Hardie N, Loddenkemper C, Ulasov I, Lesniak MS, Wolsky R, Bentolila LA, Grant SG, Elashoff D, Lehr S, Latimer JJ, Bose S, Sattar H, Krum SA, Miranda-Carboni GA - EMBO Mol Med (2013)

Bottom Line: Treatment of mouse and human triple-negative tumour cells with two Wnt/β-catenin pathway modulators or siRNA to HMGA2 decreases HMGA2 levels and proliferation.We demonstrate that WNT10B has epistatic activity on HMGA2, which is necessary and sufficient for proliferation of TNBC cells.Furthermore, HMGA2 expression predicts relapse-free-survival and metastasis in TNBC patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, David Geffen School of Medicine at UCLA, Jonsson Comprehensive Cancer Center, Los Angeles, CA, USA.

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Triple-negative breast cancer (TNBC) cells in human express a specific gene signature and their self-renewal depends on canonical Wnt/β-catenin signallingA. Human TNBC cell lines (MDA-231, BTL10, MA-11) and other non-triple negative (TN) mammary cell lines tested by qt-PCR for expression of WNT10B and self-renewal markers. Relative mRNA levels are normalized to human MCF7 cells. Error bars represent the means and the standard deviations from three independent experiments.B–E. Proliferation of human TNBC cells (B, D) or human non-TN breast cancer cells (C, E) upon treatment with the Wnt/β-catenin inhibitor ICG-001 (10 µM in 1% DMSO). Error bars represent the means and the standard deviations from three independent experiments. In B, ***p value = 0.0005 versus control-treated cells (Student's t-test). In D, ***p value = 0.0007 versus control-treated cells (Student's t-test).F. Quantification of proliferation of human MDA-MB 231 and MCF7 cells upon treatment with the Wnt pathway inhibitors ICG-001 or Wnt-C59 compared to vehicle control (DMSO). Error bars represent the means and the standard deviations from three independent experiments; **p-value = 0.004 (ANOVA).G. Phase contrast images of BTL10 cells in adherent (upper panel) and mammosphere cell culture (lower panel) upon treatment with the Wnt/β-catenin inhibitor ICG-001 (10 µM in 1% DMSO, n = 3). p Values of <0.05 were considered to be statistically significant (B, D, F).
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fig04: Triple-negative breast cancer (TNBC) cells in human express a specific gene signature and their self-renewal depends on canonical Wnt/β-catenin signallingA. Human TNBC cell lines (MDA-231, BTL10, MA-11) and other non-triple negative (TN) mammary cell lines tested by qt-PCR for expression of WNT10B and self-renewal markers. Relative mRNA levels are normalized to human MCF7 cells. Error bars represent the means and the standard deviations from three independent experiments.B–E. Proliferation of human TNBC cells (B, D) or human non-TN breast cancer cells (C, E) upon treatment with the Wnt/β-catenin inhibitor ICG-001 (10 µM in 1% DMSO). Error bars represent the means and the standard deviations from three independent experiments. In B, ***p value = 0.0005 versus control-treated cells (Student's t-test). In D, ***p value = 0.0007 versus control-treated cells (Student's t-test).F. Quantification of proliferation of human MDA-MB 231 and MCF7 cells upon treatment with the Wnt pathway inhibitors ICG-001 or Wnt-C59 compared to vehicle control (DMSO). Error bars represent the means and the standard deviations from three independent experiments; **p-value = 0.004 (ANOVA).G. Phase contrast images of BTL10 cells in adherent (upper panel) and mammosphere cell culture (lower panel) upon treatment with the Wnt/β-catenin inhibitor ICG-001 (10 µM in 1% DMSO, n = 3). p Values of <0.05 were considered to be statistically significant (B, D, F).

Mentions: We next wanted to determine whether our identified gene signature is also expressed in human TNBC cell lines. We analysed by qt-PCR for the presence of WNT10B, HMGA2, HMGA1 and BMI-1 (as a control for self-renewal markers; amongst others) in a subset of human breast cancer cell lines: HMEC (human mammary epithelial cells; normal control), MCF7 (ER+ tumour-derived), MCF-10A (non-tumorigenic mammary epithelial), SK-BR-3 (HER2+ tumour-derived), MDA-MB-231 (mesenchymal stem-cell like (MSL)/TN), JL-BTL10 (BTL10 derived from TNBC stage 2B and grade 3 from a 38-year-old African-American women established by Jean Latimer), MA-11 (TNBC metastasising to bone), and MCF7-Wnt10b (MCF7-10b, a stable cell line expressing mouse Wnt10b in human ER+ tumour-derived cells (Miranda-Carboni et al, 2008; Fig 4A). WNT10B is most highly expressed in TN cell lines when compared to non-TN cell lines. Concurrently, both HMGA2 and HMGA1 are also most highly expressed in the TNBC cell lines and MCF7-10b cells have a high level of induction for HMGA2 (>30-fold) and BMI-1 (>5-fold) but not HMGA1. This may suggest that HMGA2 and BMI-1 are direct downstream targets of the WNT10B ligand. Moreover, HMGA2 is not responsive to 17β-estradiol (E2) treatment of parental MCF7 cells or in MCF7-10b cells; MCF7-10b cells are still responsive to E2 treatment by upregulation of XBP1 and pS2 (Krum et al, 2008b), and express known canonical Wnt-signalling target genes, such as c-myc, CCND1 and DKK1 (Supporting Information Fig S4A; The Wnt homepage: Wnt targets, http://www.stanford.edu/group/nusselab/cgi-bin/wnt/). Importantly, treatment of MCF7-10b cells with ICG-001 blocks proliferation and down regulates HMGA2 expression (Supporting Information Fig S4B and C). MA-11 cells were originally isolated from a malignant bone marrow metastasis of a breast cancer patient and express high levels of both WNT10B and HMGA2 (Micci et al, 2001). BTL10 cells are a novel TNBC cell line from an African-American patient that has been engineered to retain an undifferentiated state while in culture (Latimer et al, 2010). BTL10 cells show a high level of HMGA2 induction (>400-fold), express WNT10B (threefold vs. HMEC control) and are Wnt/β-catenin responsive (Supporting Information Fig S4D).


WNT10B/β-catenin signalling induces HMGA2 and proliferation in metastatic triple-negative breast cancer.

Wend P, Runke S, Wend K, Anchondo B, Yesayan M, Jardon M, Hardie N, Loddenkemper C, Ulasov I, Lesniak MS, Wolsky R, Bentolila LA, Grant SG, Elashoff D, Lehr S, Latimer JJ, Bose S, Sattar H, Krum SA, Miranda-Carboni GA - EMBO Mol Med (2013)

Triple-negative breast cancer (TNBC) cells in human express a specific gene signature and their self-renewal depends on canonical Wnt/β-catenin signallingA. Human TNBC cell lines (MDA-231, BTL10, MA-11) and other non-triple negative (TN) mammary cell lines tested by qt-PCR for expression of WNT10B and self-renewal markers. Relative mRNA levels are normalized to human MCF7 cells. Error bars represent the means and the standard deviations from three independent experiments.B–E. Proliferation of human TNBC cells (B, D) or human non-TN breast cancer cells (C, E) upon treatment with the Wnt/β-catenin inhibitor ICG-001 (10 µM in 1% DMSO). Error bars represent the means and the standard deviations from three independent experiments. In B, ***p value = 0.0005 versus control-treated cells (Student's t-test). In D, ***p value = 0.0007 versus control-treated cells (Student's t-test).F. Quantification of proliferation of human MDA-MB 231 and MCF7 cells upon treatment with the Wnt pathway inhibitors ICG-001 or Wnt-C59 compared to vehicle control (DMSO). Error bars represent the means and the standard deviations from three independent experiments; **p-value = 0.004 (ANOVA).G. Phase contrast images of BTL10 cells in adherent (upper panel) and mammosphere cell culture (lower panel) upon treatment with the Wnt/β-catenin inhibitor ICG-001 (10 µM in 1% DMSO, n = 3). p Values of <0.05 were considered to be statistically significant (B, D, F).
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fig04: Triple-negative breast cancer (TNBC) cells in human express a specific gene signature and their self-renewal depends on canonical Wnt/β-catenin signallingA. Human TNBC cell lines (MDA-231, BTL10, MA-11) and other non-triple negative (TN) mammary cell lines tested by qt-PCR for expression of WNT10B and self-renewal markers. Relative mRNA levels are normalized to human MCF7 cells. Error bars represent the means and the standard deviations from three independent experiments.B–E. Proliferation of human TNBC cells (B, D) or human non-TN breast cancer cells (C, E) upon treatment with the Wnt/β-catenin inhibitor ICG-001 (10 µM in 1% DMSO). Error bars represent the means and the standard deviations from three independent experiments. In B, ***p value = 0.0005 versus control-treated cells (Student's t-test). In D, ***p value = 0.0007 versus control-treated cells (Student's t-test).F. Quantification of proliferation of human MDA-MB 231 and MCF7 cells upon treatment with the Wnt pathway inhibitors ICG-001 or Wnt-C59 compared to vehicle control (DMSO). Error bars represent the means and the standard deviations from three independent experiments; **p-value = 0.004 (ANOVA).G. Phase contrast images of BTL10 cells in adherent (upper panel) and mammosphere cell culture (lower panel) upon treatment with the Wnt/β-catenin inhibitor ICG-001 (10 µM in 1% DMSO, n = 3). p Values of <0.05 were considered to be statistically significant (B, D, F).
Mentions: We next wanted to determine whether our identified gene signature is also expressed in human TNBC cell lines. We analysed by qt-PCR for the presence of WNT10B, HMGA2, HMGA1 and BMI-1 (as a control for self-renewal markers; amongst others) in a subset of human breast cancer cell lines: HMEC (human mammary epithelial cells; normal control), MCF7 (ER+ tumour-derived), MCF-10A (non-tumorigenic mammary epithelial), SK-BR-3 (HER2+ tumour-derived), MDA-MB-231 (mesenchymal stem-cell like (MSL)/TN), JL-BTL10 (BTL10 derived from TNBC stage 2B and grade 3 from a 38-year-old African-American women established by Jean Latimer), MA-11 (TNBC metastasising to bone), and MCF7-Wnt10b (MCF7-10b, a stable cell line expressing mouse Wnt10b in human ER+ tumour-derived cells (Miranda-Carboni et al, 2008; Fig 4A). WNT10B is most highly expressed in TN cell lines when compared to non-TN cell lines. Concurrently, both HMGA2 and HMGA1 are also most highly expressed in the TNBC cell lines and MCF7-10b cells have a high level of induction for HMGA2 (>30-fold) and BMI-1 (>5-fold) but not HMGA1. This may suggest that HMGA2 and BMI-1 are direct downstream targets of the WNT10B ligand. Moreover, HMGA2 is not responsive to 17β-estradiol (E2) treatment of parental MCF7 cells or in MCF7-10b cells; MCF7-10b cells are still responsive to E2 treatment by upregulation of XBP1 and pS2 (Krum et al, 2008b), and express known canonical Wnt-signalling target genes, such as c-myc, CCND1 and DKK1 (Supporting Information Fig S4A; The Wnt homepage: Wnt targets, http://www.stanford.edu/group/nusselab/cgi-bin/wnt/). Importantly, treatment of MCF7-10b cells with ICG-001 blocks proliferation and down regulates HMGA2 expression (Supporting Information Fig S4B and C). MA-11 cells were originally isolated from a malignant bone marrow metastasis of a breast cancer patient and express high levels of both WNT10B and HMGA2 (Micci et al, 2001). BTL10 cells are a novel TNBC cell line from an African-American patient that has been engineered to retain an undifferentiated state while in culture (Latimer et al, 2010). BTL10 cells show a high level of HMGA2 induction (>400-fold), express WNT10B (threefold vs. HMEC control) and are Wnt/β-catenin responsive (Supporting Information Fig S4D).

Bottom Line: Treatment of mouse and human triple-negative tumour cells with two Wnt/β-catenin pathway modulators or siRNA to HMGA2 decreases HMGA2 levels and proliferation.We demonstrate that WNT10B has epistatic activity on HMGA2, which is necessary and sufficient for proliferation of TNBC cells.Furthermore, HMGA2 expression predicts relapse-free-survival and metastasis in TNBC patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, David Geffen School of Medicine at UCLA, Jonsson Comprehensive Cancer Center, Los Angeles, CA, USA.

Show MeSH
Related in: MedlinePlus