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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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The self-renewal marker HMGA2 is highly expressed in Wnt10bTG mammary tumours and mammary placodesA. IHC for oestrogen receptor α (ERα), progesterone receptor (PR) and Her2 to validate the triple-negative phenotype of Wnt10bLacZ tumours. Wild-type (WT) uterus and Her2 tumour samples were used as positive controls. Highlighted are myoepithelial (myo), glandular epithelial (ge), luminal epithelial (le) and stromal cells (s).B. Hierarchical clustering of microarray data of Hmga2 and Hmga1 expression in Wnt10b-driven tumours.C. Validation of Hmga2 and Hmga1 expression in tumour cells (Tu), Lin−LacZ+ (LacZ+) tumour cells compared to wild-type virgin (Virg) and ErbB2TG tumour cells, as determined by qt-PCR. Error bars represent the means and the standard deviations from three independent experiments; **p-value = 0.007 versus Virg or ErbB2TG Tu samples (Student's t-test).D. IHC of HMGA2 in WT mammary glands, and ErbB2TG and Wnt10bLacZ tumours. Red arrowheads highlight cells positive for HMGA2 and black arrowheads negative cells, respectively.E. IHC of HMGA2 in WT and Wnt10b knockout (Wnt10b−/−) mammary placodes (mp) at embryonic Day 14.5 reveals loss of HMGA2 expression in Wnt10b−/− mp. Dashed black lines indicate Wnt10b−/− mp. Mesenchymal (M: red arrow head), epidermis layer (EL: short black arrow) and mammary anlagen (MA: long black arrow).F, G. Mammosphere assays (MSA) of WT and Wnt10b−/− mammary gland cells and MMTV-Wnt10bLacZ primary tumour cells that were serially passaged (at Day 10 and 20) and analysed at Day 30. Secondary spheres were counted in sequential dilutions and imaged using a phase contrast microscope. Biological replicates (n = 3) and six technical replicates are shown. Wnt10bLacZ-derived MSAs analysed by IHC for HMGA2 is shown (10×) in lower right panel of G (bar, 50 µm). In F error bars represent the means and the standard deviations from three independent experiments; p values: a = 0.04, b = 0.03 versus wild-type or Wnt10b−/− samples (Student's t-test). p Values of <0.05 were considered to be statistically significant (C, F).
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fig02: The self-renewal marker HMGA2 is highly expressed in Wnt10bTG mammary tumours and mammary placodesA. IHC for oestrogen receptor α (ERα), progesterone receptor (PR) and Her2 to validate the triple-negative phenotype of Wnt10bLacZ tumours. Wild-type (WT) uterus and Her2 tumour samples were used as positive controls. Highlighted are myoepithelial (myo), glandular epithelial (ge), luminal epithelial (le) and stromal cells (s).B. Hierarchical clustering of microarray data of Hmga2 and Hmga1 expression in Wnt10b-driven tumours.C. Validation of Hmga2 and Hmga1 expression in tumour cells (Tu), Lin−LacZ+ (LacZ+) tumour cells compared to wild-type virgin (Virg) and ErbB2TG tumour cells, as determined by qt-PCR. Error bars represent the means and the standard deviations from three independent experiments; **p-value = 0.007 versus Virg or ErbB2TG Tu samples (Student's t-test).D. IHC of HMGA2 in WT mammary glands, and ErbB2TG and Wnt10bLacZ tumours. Red arrowheads highlight cells positive for HMGA2 and black arrowheads negative cells, respectively.E. IHC of HMGA2 in WT and Wnt10b knockout (Wnt10b−/−) mammary placodes (mp) at embryonic Day 14.5 reveals loss of HMGA2 expression in Wnt10b−/− mp. Dashed black lines indicate Wnt10b−/− mp. Mesenchymal (M: red arrow head), epidermis layer (EL: short black arrow) and mammary anlagen (MA: long black arrow).F, G. Mammosphere assays (MSA) of WT and Wnt10b−/− mammary gland cells and MMTV-Wnt10bLacZ primary tumour cells that were serially passaged (at Day 10 and 20) and analysed at Day 30. Secondary spheres were counted in sequential dilutions and imaged using a phase contrast microscope. Biological replicates (n = 3) and six technical replicates are shown. Wnt10bLacZ-derived MSAs analysed by IHC for HMGA2 is shown (10×) in lower right panel of G (bar, 50 µm). In F error bars represent the means and the standard deviations from three independent experiments; p values: a = 0.04, b = 0.03 versus wild-type or Wnt10b−/− samples (Student's t-test). p Values of <0.05 were considered to be statistically significant (C, F).

Mentions: To determine how well our novel MMTV-Wnt10b-IRES-LacZ (Wnt10bLacZ) mouse model resembles human TNBC, we conducted IHC for estrogen receptor-alpha (ERα), progesterone receptor (PR) and HER2 protein expression levels (Fig 2A). Wnt10b-driven tumours are phenotypically like human TNBC—devoid of ERα, PR and HER2. In contrast, ERα and PR protein expression, in the uterus of wild-type female mice, is present in various subpopulations of myoepithelial, stromal cells and both luminal and glandular epithelial cells (Fig 2A). Interestingly, the Wnt10b tumorigenic mouse model contrasts the MMTV-Wnt1 model, in part, because Wnt1-driven tumours have been reported to express hormone receptors (Teissedre et al, 2009). HER2 protein expression is very high in mouse MMTV-neu2/ErbB2 (ErbB2TG) tumours but not expressed at all in the Wnt10bLacZ tumours. Furthermore, Wnt10b-driven tumours express the basal-epithelial markers CK5 and CK6, in contrast to ErbB2TG tumours (Supporting Information Fig S2A–C). We also show that Wnt10bLacZ-driven tumours express high levels of β-galactosidase activity, have transcriptionally active nuclear β-catenin and correlating with high levels of AXIN2 protein expression (Supporting Information Fig S2D–F).


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)

The self-renewal marker HMGA2 is highly expressed in Wnt10bTG mammary tumours and mammary placodesA. IHC for oestrogen receptor α (ERα), progesterone receptor (PR) and Her2 to validate the triple-negative phenotype of Wnt10bLacZ tumours. Wild-type (WT) uterus and Her2 tumour samples were used as positive controls. Highlighted are myoepithelial (myo), glandular epithelial (ge), luminal epithelial (le) and stromal cells (s).B. Hierarchical clustering of microarray data of Hmga2 and Hmga1 expression in Wnt10b-driven tumours.C. Validation of Hmga2 and Hmga1 expression in tumour cells (Tu), Lin−LacZ+ (LacZ+) tumour cells compared to wild-type virgin (Virg) and ErbB2TG tumour cells, as determined by qt-PCR. Error bars represent the means and the standard deviations from three independent experiments; **p-value = 0.007 versus Virg or ErbB2TG Tu samples (Student's t-test).D. IHC of HMGA2 in WT mammary glands, and ErbB2TG and Wnt10bLacZ tumours. Red arrowheads highlight cells positive for HMGA2 and black arrowheads negative cells, respectively.E. IHC of HMGA2 in WT and Wnt10b knockout (Wnt10b−/−) mammary placodes (mp) at embryonic Day 14.5 reveals loss of HMGA2 expression in Wnt10b−/− mp. Dashed black lines indicate Wnt10b−/− mp. Mesenchymal (M: red arrow head), epidermis layer (EL: short black arrow) and mammary anlagen (MA: long black arrow).F, G. Mammosphere assays (MSA) of WT and Wnt10b−/− mammary gland cells and MMTV-Wnt10bLacZ primary tumour cells that were serially passaged (at Day 10 and 20) and analysed at Day 30. Secondary spheres were counted in sequential dilutions and imaged using a phase contrast microscope. Biological replicates (n = 3) and six technical replicates are shown. Wnt10bLacZ-derived MSAs analysed by IHC for HMGA2 is shown (10×) in lower right panel of G (bar, 50 µm). In F error bars represent the means and the standard deviations from three independent experiments; p values: a = 0.04, b = 0.03 versus wild-type or Wnt10b−/− samples (Student's t-test). p Values of <0.05 were considered to be statistically significant (C, F).
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fig02: The self-renewal marker HMGA2 is highly expressed in Wnt10bTG mammary tumours and mammary placodesA. IHC for oestrogen receptor α (ERα), progesterone receptor (PR) and Her2 to validate the triple-negative phenotype of Wnt10bLacZ tumours. Wild-type (WT) uterus and Her2 tumour samples were used as positive controls. Highlighted are myoepithelial (myo), glandular epithelial (ge), luminal epithelial (le) and stromal cells (s).B. Hierarchical clustering of microarray data of Hmga2 and Hmga1 expression in Wnt10b-driven tumours.C. Validation of Hmga2 and Hmga1 expression in tumour cells (Tu), Lin−LacZ+ (LacZ+) tumour cells compared to wild-type virgin (Virg) and ErbB2TG tumour cells, as determined by qt-PCR. Error bars represent the means and the standard deviations from three independent experiments; **p-value = 0.007 versus Virg or ErbB2TG Tu samples (Student's t-test).D. IHC of HMGA2 in WT mammary glands, and ErbB2TG and Wnt10bLacZ tumours. Red arrowheads highlight cells positive for HMGA2 and black arrowheads negative cells, respectively.E. IHC of HMGA2 in WT and Wnt10b knockout (Wnt10b−/−) mammary placodes (mp) at embryonic Day 14.5 reveals loss of HMGA2 expression in Wnt10b−/− mp. Dashed black lines indicate Wnt10b−/− mp. Mesenchymal (M: red arrow head), epidermis layer (EL: short black arrow) and mammary anlagen (MA: long black arrow).F, G. Mammosphere assays (MSA) of WT and Wnt10b−/− mammary gland cells and MMTV-Wnt10bLacZ primary tumour cells that were serially passaged (at Day 10 and 20) and analysed at Day 30. Secondary spheres were counted in sequential dilutions and imaged using a phase contrast microscope. Biological replicates (n = 3) and six technical replicates are shown. Wnt10bLacZ-derived MSAs analysed by IHC for HMGA2 is shown (10×) in lower right panel of G (bar, 50 µm). In F error bars represent the means and the standard deviations from three independent experiments; p values: a = 0.04, b = 0.03 versus wild-type or Wnt10b−/− samples (Student's t-test). p Values of <0.05 were considered to be statistically significant (C, F).
Mentions: To determine how well our novel MMTV-Wnt10b-IRES-LacZ (Wnt10bLacZ) mouse model resembles human TNBC, we conducted IHC for estrogen receptor-alpha (ERα), progesterone receptor (PR) and HER2 protein expression levels (Fig 2A). Wnt10b-driven tumours are phenotypically like human TNBC—devoid of ERα, PR and HER2. In contrast, ERα and PR protein expression, in the uterus of wild-type female mice, is present in various subpopulations of myoepithelial, stromal cells and both luminal and glandular epithelial cells (Fig 2A). Interestingly, the Wnt10b tumorigenic mouse model contrasts the MMTV-Wnt1 model, in part, because Wnt1-driven tumours have been reported to express hormone receptors (Teissedre et al, 2009). HER2 protein expression is very high in mouse MMTV-neu2/ErbB2 (ErbB2TG) tumours but not expressed at all in the Wnt10bLacZ tumours. Furthermore, Wnt10b-driven tumours express the basal-epithelial markers CK5 and CK6, in contrast to ErbB2TG tumours (Supporting Information Fig S2A–C). We also show that Wnt10bLacZ-driven tumours express high levels of β-galactosidase activity, have transcriptionally active nuclear β-catenin and correlating with high levels of AXIN2 protein expression (Supporting Information Fig S2D–F).

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