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Effect of glycogen synthase kinase-3 inactivation on mouse mammary gland development and oncogenesis.

Dembowy J, Adissu HA, Liu JC, Zacksenhaus E, Woodgett JR - Oncogene (2014)

Bottom Line: To uncover possible β-catenin-independent activities of GSK-3, we generated mammary-specific knockouts of GSK-3 and β-catenin.At 10 months, adenocarcinomas that developed in glands lacking GSK-3 and β-catenin displayed elevated levels of γ-catenin/plakoglobin as well as activation of the Hedgehog and Notch pathways.Collectively, these results establish the two isoforms of GSK-3 as essential integrators of multiple developmental signals that act to maintain normal mammary gland function and suppress tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: 1] Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada [2] Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT
Many components of the Wnt/β-catenin signaling pathway have critical functions in mammary gland development and tumor formation, yet the contribution of glycogen synthase kinase-3 (GSK-3α and GSK-3β) to mammopoiesis and oncogenesis is unclear. Here, we report that WAP-Cre-mediated deletion of GSK-3 in the mammary epithelium results in activation of Wnt/β-catenin signaling and induces mammary intraepithelial neoplasia that progresses to squamous transdifferentiation and development of adenosquamous carcinomas at 6 months. To uncover possible β-catenin-independent activities of GSK-3, we generated mammary-specific knockouts of GSK-3 and β-catenin. Squamous transdifferentiation of the mammary epithelium was largely attenuated, however, mammary epithelial cells lost the ability to form mammospheres suggesting perturbation of stem cell properties unrelated to loss of β-catenin alone. At 10 months, adenocarcinomas that developed in glands lacking GSK-3 and β-catenin displayed elevated levels of γ-catenin/plakoglobin as well as activation of the Hedgehog and Notch pathways. Collectively, these results establish the two isoforms of GSK-3 as essential integrators of multiple developmental signals that act to maintain normal mammary gland function and suppress tumorigenesis.

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Related in: MedlinePlus

Mammosphere formation in MECs depleted of GSK-3 and β-catenin. β-cateninActive (a) or GSK-3-ablated (b) MECs were cultured under conditions promoting MS formation and imaged on day 12 post infection. MS were then dissociated and analyzed by flow cytometry to assess the CD24:CD49f stem cell profile. Shown are representative of three separate experiments demonstrating efficient MS formation across all genotypes. A CD24hi:CD49flo/− population was consistently observed in β-cateninActive MECs. (c) PCR genotyping of an aliquot of Ad-GFP or Ad-Cre-GFP MECs of indicated genotypes analyzed using primers specific for detection of floxed and deleted alleles of GSK-3β (850 bp, 250 bp) and β-cateninFL-Ex3/FL-Ex3 (650 bp, 400 bp) showing efficient Cre excision at both loci at day 12 post infection.
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fig3: Mammosphere formation in MECs depleted of GSK-3 and β-catenin. β-cateninActive (a) or GSK-3-ablated (b) MECs were cultured under conditions promoting MS formation and imaged on day 12 post infection. MS were then dissociated and analyzed by flow cytometry to assess the CD24:CD49f stem cell profile. Shown are representative of three separate experiments demonstrating efficient MS formation across all genotypes. A CD24hi:CD49flo/− population was consistently observed in β-cateninActive MECs. (c) PCR genotyping of an aliquot of Ad-GFP or Ad-Cre-GFP MECs of indicated genotypes analyzed using primers specific for detection of floxed and deleted alleles of GSK-3β (850 bp, 250 bp) and β-cateninFL-Ex3/FL-Ex3 (650 bp, 400 bp) showing efficient Cre excision at both loci at day 12 post infection.

Mentions: To further investigate GSK-3 functions, we employed a mammosphere (MS) assay to measure in vitro stem/progenitor cell frequency in primary MEC preparations.64 In the absence of attachment to an exogenous substratum or cell–cell adhesion, stem cells are able to survive and proliferate and we found no significant differences in primary or secondary MS formation of GSK-3 DKO MECs generated by infecting Lin− GSK-3α−/−; GSK-3βFL/FL cells with either Ad-GFP or Ad-Cre-GFP. Interestingly, whereas the CD24:CD49f marker profile of MS dissociated on day 12 post infection was not significantly changed upon loss of GSK-3 across replicate experiments (Figures 3b and c), a prominent CD24hi:CD49flo/− population was observed upon stabilization of β-catenin (Figures 3a and c). To further investigate the functional relevance of these populations, β-cateninActive MECs were sorted into CD24hi:CD49flo/− and CD24lo/−:CD49fhi fractions with only CD24lo/−:CD49fhi cells capable of forming secondary MS, although CD24hi:CD49flo/− MEC population was regenerated (data not shown). On the basis of these in vitro data, we conclude that, unlike direct stabilization of β-catenin, GSK-3 loss does not significantly impact the mammary gland stem cell compartment.


Effect of glycogen synthase kinase-3 inactivation on mouse mammary gland development and oncogenesis.

Dembowy J, Adissu HA, Liu JC, Zacksenhaus E, Woodgett JR - Oncogene (2014)

Mammosphere formation in MECs depleted of GSK-3 and β-catenin. β-cateninActive (a) or GSK-3-ablated (b) MECs were cultured under conditions promoting MS formation and imaged on day 12 post infection. MS were then dissociated and analyzed by flow cytometry to assess the CD24:CD49f stem cell profile. Shown are representative of three separate experiments demonstrating efficient MS formation across all genotypes. A CD24hi:CD49flo/− population was consistently observed in β-cateninActive MECs. (c) PCR genotyping of an aliquot of Ad-GFP or Ad-Cre-GFP MECs of indicated genotypes analyzed using primers specific for detection of floxed and deleted alleles of GSK-3β (850 bp, 250 bp) and β-cateninFL-Ex3/FL-Ex3 (650 bp, 400 bp) showing efficient Cre excision at both loci at day 12 post infection.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4490903&req=5

fig3: Mammosphere formation in MECs depleted of GSK-3 and β-catenin. β-cateninActive (a) or GSK-3-ablated (b) MECs were cultured under conditions promoting MS formation and imaged on day 12 post infection. MS were then dissociated and analyzed by flow cytometry to assess the CD24:CD49f stem cell profile. Shown are representative of three separate experiments demonstrating efficient MS formation across all genotypes. A CD24hi:CD49flo/− population was consistently observed in β-cateninActive MECs. (c) PCR genotyping of an aliquot of Ad-GFP or Ad-Cre-GFP MECs of indicated genotypes analyzed using primers specific for detection of floxed and deleted alleles of GSK-3β (850 bp, 250 bp) and β-cateninFL-Ex3/FL-Ex3 (650 bp, 400 bp) showing efficient Cre excision at both loci at day 12 post infection.
Mentions: To further investigate GSK-3 functions, we employed a mammosphere (MS) assay to measure in vitro stem/progenitor cell frequency in primary MEC preparations.64 In the absence of attachment to an exogenous substratum or cell–cell adhesion, stem cells are able to survive and proliferate and we found no significant differences in primary or secondary MS formation of GSK-3 DKO MECs generated by infecting Lin− GSK-3α−/−; GSK-3βFL/FL cells with either Ad-GFP or Ad-Cre-GFP. Interestingly, whereas the CD24:CD49f marker profile of MS dissociated on day 12 post infection was not significantly changed upon loss of GSK-3 across replicate experiments (Figures 3b and c), a prominent CD24hi:CD49flo/− population was observed upon stabilization of β-catenin (Figures 3a and c). To further investigate the functional relevance of these populations, β-cateninActive MECs were sorted into CD24hi:CD49flo/− and CD24lo/−:CD49fhi fractions with only CD24lo/−:CD49fhi cells capable of forming secondary MS, although CD24hi:CD49flo/− MEC population was regenerated (data not shown). On the basis of these in vitro data, we conclude that, unlike direct stabilization of β-catenin, GSK-3 loss does not significantly impact the mammary gland stem cell compartment.

Bottom Line: To uncover possible β-catenin-independent activities of GSK-3, we generated mammary-specific knockouts of GSK-3 and β-catenin.At 10 months, adenocarcinomas that developed in glands lacking GSK-3 and β-catenin displayed elevated levels of γ-catenin/plakoglobin as well as activation of the Hedgehog and Notch pathways.Collectively, these results establish the two isoforms of GSK-3 as essential integrators of multiple developmental signals that act to maintain normal mammary gland function and suppress tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: 1] Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada [2] Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT
Many components of the Wnt/β-catenin signaling pathway have critical functions in mammary gland development and tumor formation, yet the contribution of glycogen synthase kinase-3 (GSK-3α and GSK-3β) to mammopoiesis and oncogenesis is unclear. Here, we report that WAP-Cre-mediated deletion of GSK-3 in the mammary epithelium results in activation of Wnt/β-catenin signaling and induces mammary intraepithelial neoplasia that progresses to squamous transdifferentiation and development of adenosquamous carcinomas at 6 months. To uncover possible β-catenin-independent activities of GSK-3, we generated mammary-specific knockouts of GSK-3 and β-catenin. Squamous transdifferentiation of the mammary epithelium was largely attenuated, however, mammary epithelial cells lost the ability to form mammospheres suggesting perturbation of stem cell properties unrelated to loss of β-catenin alone. At 10 months, adenocarcinomas that developed in glands lacking GSK-3 and β-catenin displayed elevated levels of γ-catenin/plakoglobin as well as activation of the Hedgehog and Notch pathways. Collectively, these results establish the two isoforms of GSK-3 as essential integrators of multiple developmental signals that act to maintain normal mammary gland function and suppress tumorigenesis.

Show MeSH
Related in: MedlinePlus