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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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Wnt10b induces Hmga2-dependent proliferation via β-catenin in mouse mammary epithelial and tumour cellsTime course experiment to analyse Hmga2 expression in parental NMuMG (NMG) cells and NMuMG cells overexpressing Wnt10b (NMG-10b) after release from growth arrest (determined by qt-PCR). Error bars represent the means and the standard deviations from three independent experiments.Assessment of enrichment of β-catenin at the Hmga2, c-Myc and Gapdh promoters by chromatin immunoprecipitation (ChIP) 8 h after release from growth arrest. An upstream −6 Kb element of the Hmga2 chromosomal ORF-loci devoid of WRE sites was used as negative control.Forty-eight hours treatment of NMG and NMG-10b cells with the Wnt/β-catenin inhibitor ICG-001 (10 µM in 1% DMSO) leads to decreased Hmga2 expression, as determined by qt-PCR. Error bars represent the means and the standard deviations from three independent experiments; *p-value = 0.04 versus untreated sample (Student's t-test).shRNA-Mediated knockdown of Hmga2 leads to attenuated growth of WZALacZ tumour cells. Shown are two different shHmga2 clones compared to a control shGFP clone. Error bars represent the means and the standard deviations from three independent experiments.Attenuated proliferation of WZALacZ tumour cells upon treatment with ICG-001 (10 µM in 1% DMSO). Error bars represent the means and the standard deviations from three independent experiments.Immunoprecipitation and Western blot analysis verifying decreased interaction of β-catenin and CBP after ICG-001 treatment of WZALacZ tumour cells.Decreased expression of HMGA2 and PCNA in ICG-001-treated WZALacZ tumour cells, as determined by immunoblotting analysis.Quantification of proliferation of WZALacZ 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 = 0.0008 (ANOVA).Images of MSA from primary Wnt10bLacZ mammary tumour cells after control (DMSO, upper panel) or ICG-001 (lower panel) treatment.MSA formation efficiency of primary Wnt10bLacZ mammary tumour cells after control (DMSO), ICG-001, and Wnt-C59 treatment. Error bars represent the means and the standard deviations from 12 independent experiments; ***p value = 0.0006 (ANOVA). p Values of <0.05 were considered to be statistically significant (C, H, J).
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fig03: Wnt10b induces Hmga2-dependent proliferation via β-catenin in mouse mammary epithelial and tumour cellsTime course experiment to analyse Hmga2 expression in parental NMuMG (NMG) cells and NMuMG cells overexpressing Wnt10b (NMG-10b) after release from growth arrest (determined by qt-PCR). Error bars represent the means and the standard deviations from three independent experiments.Assessment of enrichment of β-catenin at the Hmga2, c-Myc and Gapdh promoters by chromatin immunoprecipitation (ChIP) 8 h after release from growth arrest. An upstream −6 Kb element of the Hmga2 chromosomal ORF-loci devoid of WRE sites was used as negative control.Forty-eight hours treatment of NMG and NMG-10b cells with the Wnt/β-catenin inhibitor ICG-001 (10 µM in 1% DMSO) leads to decreased Hmga2 expression, as determined by qt-PCR. Error bars represent the means and the standard deviations from three independent experiments; *p-value = 0.04 versus untreated sample (Student's t-test).shRNA-Mediated knockdown of Hmga2 leads to attenuated growth of WZALacZ tumour cells. Shown are two different shHmga2 clones compared to a control shGFP clone. Error bars represent the means and the standard deviations from three independent experiments.Attenuated proliferation of WZALacZ tumour cells upon treatment with ICG-001 (10 µM in 1% DMSO). Error bars represent the means and the standard deviations from three independent experiments.Immunoprecipitation and Western blot analysis verifying decreased interaction of β-catenin and CBP after ICG-001 treatment of WZALacZ tumour cells.Decreased expression of HMGA2 and PCNA in ICG-001-treated WZALacZ tumour cells, as determined by immunoblotting analysis.Quantification of proliferation of WZALacZ 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 = 0.0008 (ANOVA).Images of MSA from primary Wnt10bLacZ mammary tumour cells after control (DMSO, upper panel) or ICG-001 (lower panel) treatment.MSA formation efficiency of primary Wnt10bLacZ mammary tumour cells after control (DMSO), ICG-001, and Wnt-C59 treatment. Error bars represent the means and the standard deviations from 12 independent experiments; ***p value = 0.0006 (ANOVA). p Values of <0.05 were considered to be statistically significant (C, H, J).

Mentions: To gain mechanistic insights on how Wnt10b regulates HMGA2 expression we turned to our previously published mouse cell line system NMuMG (NMG) and NMuMG-Wnt10b (NMG-10b; Miranda-Carboni et al, 2008). NMG and NMG-10b cells were synchronized in early G1 by maintenance at 100% confluency for 2–3 days. Cells were then released and harvested at various times for analysis by qt-PCR. NMG-10b cell lines induce five- to eightfold greater Hmga2 expression, at all-time points, than the control parental cell line NMG (Fig 3A). We also silenced Hmga2 in the NMG-10b cell line utilizing a Lentiviral-shHmga2 system to verify that the increased proliferation was due to HMGA2 expression. We observed decreased proliferation (>35%) after Hmga2 silencing, but not restored to NMG parental proliferation levels (Supporting Information Fig S3C).


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)

Wnt10b induces Hmga2-dependent proliferation via β-catenin in mouse mammary epithelial and tumour cellsTime course experiment to analyse Hmga2 expression in parental NMuMG (NMG) cells and NMuMG cells overexpressing Wnt10b (NMG-10b) after release from growth arrest (determined by qt-PCR). Error bars represent the means and the standard deviations from three independent experiments.Assessment of enrichment of β-catenin at the Hmga2, c-Myc and Gapdh promoters by chromatin immunoprecipitation (ChIP) 8 h after release from growth arrest. An upstream −6 Kb element of the Hmga2 chromosomal ORF-loci devoid of WRE sites was used as negative control.Forty-eight hours treatment of NMG and NMG-10b cells with the Wnt/β-catenin inhibitor ICG-001 (10 µM in 1% DMSO) leads to decreased Hmga2 expression, as determined by qt-PCR. Error bars represent the means and the standard deviations from three independent experiments; *p-value = 0.04 versus untreated sample (Student's t-test).shRNA-Mediated knockdown of Hmga2 leads to attenuated growth of WZALacZ tumour cells. Shown are two different shHmga2 clones compared to a control shGFP clone. Error bars represent the means and the standard deviations from three independent experiments.Attenuated proliferation of WZALacZ tumour cells upon treatment with ICG-001 (10 µM in 1% DMSO). Error bars represent the means and the standard deviations from three independent experiments.Immunoprecipitation and Western blot analysis verifying decreased interaction of β-catenin and CBP after ICG-001 treatment of WZALacZ tumour cells.Decreased expression of HMGA2 and PCNA in ICG-001-treated WZALacZ tumour cells, as determined by immunoblotting analysis.Quantification of proliferation of WZALacZ 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 = 0.0008 (ANOVA).Images of MSA from primary Wnt10bLacZ mammary tumour cells after control (DMSO, upper panel) or ICG-001 (lower panel) treatment.MSA formation efficiency of primary Wnt10bLacZ mammary tumour cells after control (DMSO), ICG-001, and Wnt-C59 treatment. Error bars represent the means and the standard deviations from 12 independent experiments; ***p value = 0.0006 (ANOVA). p Values of <0.05 were considered to be statistically significant (C, H, J).
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fig03: Wnt10b induces Hmga2-dependent proliferation via β-catenin in mouse mammary epithelial and tumour cellsTime course experiment to analyse Hmga2 expression in parental NMuMG (NMG) cells and NMuMG cells overexpressing Wnt10b (NMG-10b) after release from growth arrest (determined by qt-PCR). Error bars represent the means and the standard deviations from three independent experiments.Assessment of enrichment of β-catenin at the Hmga2, c-Myc and Gapdh promoters by chromatin immunoprecipitation (ChIP) 8 h after release from growth arrest. An upstream −6 Kb element of the Hmga2 chromosomal ORF-loci devoid of WRE sites was used as negative control.Forty-eight hours treatment of NMG and NMG-10b cells with the Wnt/β-catenin inhibitor ICG-001 (10 µM in 1% DMSO) leads to decreased Hmga2 expression, as determined by qt-PCR. Error bars represent the means and the standard deviations from three independent experiments; *p-value = 0.04 versus untreated sample (Student's t-test).shRNA-Mediated knockdown of Hmga2 leads to attenuated growth of WZALacZ tumour cells. Shown are two different shHmga2 clones compared to a control shGFP clone. Error bars represent the means and the standard deviations from three independent experiments.Attenuated proliferation of WZALacZ tumour cells upon treatment with ICG-001 (10 µM in 1% DMSO). Error bars represent the means and the standard deviations from three independent experiments.Immunoprecipitation and Western blot analysis verifying decreased interaction of β-catenin and CBP after ICG-001 treatment of WZALacZ tumour cells.Decreased expression of HMGA2 and PCNA in ICG-001-treated WZALacZ tumour cells, as determined by immunoblotting analysis.Quantification of proliferation of WZALacZ 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 = 0.0008 (ANOVA).Images of MSA from primary Wnt10bLacZ mammary tumour cells after control (DMSO, upper panel) or ICG-001 (lower panel) treatment.MSA formation efficiency of primary Wnt10bLacZ mammary tumour cells after control (DMSO), ICG-001, and Wnt-C59 treatment. Error bars represent the means and the standard deviations from 12 independent experiments; ***p value = 0.0006 (ANOVA). p Values of <0.05 were considered to be statistically significant (C, H, J).
Mentions: To gain mechanistic insights on how Wnt10b regulates HMGA2 expression we turned to our previously published mouse cell line system NMuMG (NMG) and NMuMG-Wnt10b (NMG-10b; Miranda-Carboni et al, 2008). NMG and NMG-10b cells were synchronized in early G1 by maintenance at 100% confluency for 2–3 days. Cells were then released and harvested at various times for analysis by qt-PCR. NMG-10b cell lines induce five- to eightfold greater Hmga2 expression, at all-time points, than the control parental cell line NMG (Fig 3A). We also silenced Hmga2 in the NMG-10b cell line utilizing a Lentiviral-shHmga2 system to verify that the increased proliferation was due to HMGA2 expression. We observed decreased proliferation (>35%) after Hmga2 silencing, but not restored to NMG parental proliferation levels (Supporting Information Fig S3C).

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