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Regulation of mammary stem/progenitor cells by PTEN/Akt/beta-catenin signaling.

Korkaya H, Paulson A, Charafe-Jauffret E, Ginestier C, Brown M, Dutcher J, Clouthier SG, Wicha MS - PLoS Biol. (2009)

Bottom Line: Akt-driven stem/progenitor cell enrichment is mediated by activation of the Wnt/beta-catenin pathway through the phosphorylation of GSK3-beta.In contrast to chemotherapy, the Akt inhibitor perifosine is able to target the tumorigenic cell population in breast tumor xenografts.These studies demonstrate an important role for the PTEN/PI3-K/Akt/beta-catenin pathway in the regulation of normal and malignant stem/progenitor cell populations and suggest that agents that inhibit this pathway are able to effectively target tumorigenic breast cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Comprehensive Cancer Center, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, USA. hkorkaya@med.umich.edu

ABSTRACT
Recent evidence suggests that many malignancies, including breast cancer, are driven by a cellular subcomponent that displays stem cell-like properties. The protein phosphatase and tensin homolog (PTEN) is inactivated in a wide range of human cancers, an alteration that is associated with a poor prognosis. Because PTEN has been reported to play a role in the maintenance of embryonic and tissue-specific stem cells, we investigated the role of the PTEN/Akt pathway in the regulation of normal and malignant mammary stem/progenitor cell populations. We demonstrate that activation of this pathway, via PTEN knockdown, enriches for normal and malignant human mammary stem/progenitor cells in vitro and in vivo. Knockdown of PTEN in normal human mammary epithelial cells enriches for the stem/progenitor cell compartment, generating atypical hyperplastic lesions in humanized NOD/SCID mice. Akt-driven stem/progenitor cell enrichment is mediated by activation of the Wnt/beta-catenin pathway through the phosphorylation of GSK3-beta. In contrast to chemotherapy, the Akt inhibitor perifosine is able to target the tumorigenic cell population in breast tumor xenografts. These studies demonstrate an important role for the PTEN/PI3-K/Akt/beta-catenin pathway in the regulation of normal and malignant stem/progenitor cell populations and suggest that agents that inhibit this pathway are able to effectively target tumorigenic breast cancer cells.

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Activation of PTEN/PI3-K/Akt/GSK3-β/β-catenin signaling in mammospheres.(A) After 7–10 d of culture, mammospheres and adherent NMECs were analyzed by Western blotting for activation of the PI3-K/Akt pathway and its downstream targets. Mammospheres as compared to adherent NMEC cultures demonstrated increased phosphorylation of PTEN, Akt, GSK3-β, and activated β-catenin (ABC). Mammospheres but not the adherent cells also expressed the marker ALDH1. (B) Knockdown of PTEN expression via shRNA lentivirus infection led to further increases in phospho-Akt, phospho-GSK3-β, and activated β-catenin levels compared with DsRed lentiviral-infected control mammospheres. (C) PTEN knockdown led to an increase in the number of mammosphere forming cells. The efficiency of lentivirus infection was demonstrated by DsRed expression (inserts). (D) DsRed control and PTEN knockdown mammospheres were cultured for three passages, and the number of mammospheres generated per 10,000 cells was determined. (E) Adherent NMECs were infected with control or PTEN lentiviral constructs and maintained in attachment cultures for 7 d. The cells from these attachment cultures were assessed for their mammosphere-forming ability. As indicated, cells with PTEN knockdown generated more mammospheres than control cells Scale bars in (C) = 100 µm. Each data point in (D) and (E) represents the mean±SD of three independent experiments.
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pbio-1000121-g001: Activation of PTEN/PI3-K/Akt/GSK3-β/β-catenin signaling in mammospheres.(A) After 7–10 d of culture, mammospheres and adherent NMECs were analyzed by Western blotting for activation of the PI3-K/Akt pathway and its downstream targets. Mammospheres as compared to adherent NMEC cultures demonstrated increased phosphorylation of PTEN, Akt, GSK3-β, and activated β-catenin (ABC). Mammospheres but not the adherent cells also expressed the marker ALDH1. (B) Knockdown of PTEN expression via shRNA lentivirus infection led to further increases in phospho-Akt, phospho-GSK3-β, and activated β-catenin levels compared with DsRed lentiviral-infected control mammospheres. (C) PTEN knockdown led to an increase in the number of mammosphere forming cells. The efficiency of lentivirus infection was demonstrated by DsRed expression (inserts). (D) DsRed control and PTEN knockdown mammospheres were cultured for three passages, and the number of mammospheres generated per 10,000 cells was determined. (E) Adherent NMECs were infected with control or PTEN lentiviral constructs and maintained in attachment cultures for 7 d. The cells from these attachment cultures were assessed for their mammosphere-forming ability. As indicated, cells with PTEN knockdown generated more mammospheres than control cells Scale bars in (C) = 100 µm. Each data point in (D) and (E) represents the mean±SD of three independent experiments.

Mentions: To determine whether this pathway was activated in primitive mammary cells, we compared the levels of PTEN and Akt phosphorylation and its downstream targets in normal mammary stem and progenitor cells in mammospheres compared with those in cells induced to undergo differentiation in monolayer cultures. Activation of the PTEN/PI3-K/Akt pathway was assessed by Western blotting using phospho-specific antibodies. As compared to adherent cultures, normal mammary epithelial cells (NMECs) in mammosphere cultures expressed increased Ser380 phosphorylation of PTEN (Figure 1A), which results in its conformational changes masking the PDZ binding domain [22]. PTEN, through its lipid phosphatase activity, antagonizes PI3-K/Akt signaling. We detected increased Akt Ser473 phosphorylation in mammospheres as compared with monolayer cultures, suggesting that inactivation of PTEN results in increased Akt phosphorylation in more primitive cells (Figure 1A). Akt has a number of known downstream targets including GSK3-β, which regulates the Wnt/β-catenin pathway. As compared to differentiated cells, cells in mammospheres displayed increased levels of GSK3-β phosphorylation and β-catenin activation (Figure 1A). β-catenin has been demonstrated to play an important role in the development of mammary stem cells in mouse models [23], suggesting that this pathway may also be active in human mammary stem/progenitor cells in mammospheres.


Regulation of mammary stem/progenitor cells by PTEN/Akt/beta-catenin signaling.

Korkaya H, Paulson A, Charafe-Jauffret E, Ginestier C, Brown M, Dutcher J, Clouthier SG, Wicha MS - PLoS Biol. (2009)

Activation of PTEN/PI3-K/Akt/GSK3-β/β-catenin signaling in mammospheres.(A) After 7–10 d of culture, mammospheres and adherent NMECs were analyzed by Western blotting for activation of the PI3-K/Akt pathway and its downstream targets. Mammospheres as compared to adherent NMEC cultures demonstrated increased phosphorylation of PTEN, Akt, GSK3-β, and activated β-catenin (ABC). Mammospheres but not the adherent cells also expressed the marker ALDH1. (B) Knockdown of PTEN expression via shRNA lentivirus infection led to further increases in phospho-Akt, phospho-GSK3-β, and activated β-catenin levels compared with DsRed lentiviral-infected control mammospheres. (C) PTEN knockdown led to an increase in the number of mammosphere forming cells. The efficiency of lentivirus infection was demonstrated by DsRed expression (inserts). (D) DsRed control and PTEN knockdown mammospheres were cultured for three passages, and the number of mammospheres generated per 10,000 cells was determined. (E) Adherent NMECs were infected with control or PTEN lentiviral constructs and maintained in attachment cultures for 7 d. The cells from these attachment cultures were assessed for their mammosphere-forming ability. As indicated, cells with PTEN knockdown generated more mammospheres than control cells Scale bars in (C) = 100 µm. Each data point in (D) and (E) represents the mean±SD of three independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2683567&req=5

pbio-1000121-g001: Activation of PTEN/PI3-K/Akt/GSK3-β/β-catenin signaling in mammospheres.(A) After 7–10 d of culture, mammospheres and adherent NMECs were analyzed by Western blotting for activation of the PI3-K/Akt pathway and its downstream targets. Mammospheres as compared to adherent NMEC cultures demonstrated increased phosphorylation of PTEN, Akt, GSK3-β, and activated β-catenin (ABC). Mammospheres but not the adherent cells also expressed the marker ALDH1. (B) Knockdown of PTEN expression via shRNA lentivirus infection led to further increases in phospho-Akt, phospho-GSK3-β, and activated β-catenin levels compared with DsRed lentiviral-infected control mammospheres. (C) PTEN knockdown led to an increase in the number of mammosphere forming cells. The efficiency of lentivirus infection was demonstrated by DsRed expression (inserts). (D) DsRed control and PTEN knockdown mammospheres were cultured for three passages, and the number of mammospheres generated per 10,000 cells was determined. (E) Adherent NMECs were infected with control or PTEN lentiviral constructs and maintained in attachment cultures for 7 d. The cells from these attachment cultures were assessed for their mammosphere-forming ability. As indicated, cells with PTEN knockdown generated more mammospheres than control cells Scale bars in (C) = 100 µm. Each data point in (D) and (E) represents the mean±SD of three independent experiments.
Mentions: To determine whether this pathway was activated in primitive mammary cells, we compared the levels of PTEN and Akt phosphorylation and its downstream targets in normal mammary stem and progenitor cells in mammospheres compared with those in cells induced to undergo differentiation in monolayer cultures. Activation of the PTEN/PI3-K/Akt pathway was assessed by Western blotting using phospho-specific antibodies. As compared to adherent cultures, normal mammary epithelial cells (NMECs) in mammosphere cultures expressed increased Ser380 phosphorylation of PTEN (Figure 1A), which results in its conformational changes masking the PDZ binding domain [22]. PTEN, through its lipid phosphatase activity, antagonizes PI3-K/Akt signaling. We detected increased Akt Ser473 phosphorylation in mammospheres as compared with monolayer cultures, suggesting that inactivation of PTEN results in increased Akt phosphorylation in more primitive cells (Figure 1A). Akt has a number of known downstream targets including GSK3-β, which regulates the Wnt/β-catenin pathway. As compared to differentiated cells, cells in mammospheres displayed increased levels of GSK3-β phosphorylation and β-catenin activation (Figure 1A). β-catenin has been demonstrated to play an important role in the development of mammary stem cells in mouse models [23], suggesting that this pathway may also be active in human mammary stem/progenitor cells in mammospheres.

Bottom Line: Akt-driven stem/progenitor cell enrichment is mediated by activation of the Wnt/beta-catenin pathway through the phosphorylation of GSK3-beta.In contrast to chemotherapy, the Akt inhibitor perifosine is able to target the tumorigenic cell population in breast tumor xenografts.These studies demonstrate an important role for the PTEN/PI3-K/Akt/beta-catenin pathway in the regulation of normal and malignant stem/progenitor cell populations and suggest that agents that inhibit this pathway are able to effectively target tumorigenic breast cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Comprehensive Cancer Center, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, USA. hkorkaya@med.umich.edu

ABSTRACT
Recent evidence suggests that many malignancies, including breast cancer, are driven by a cellular subcomponent that displays stem cell-like properties. The protein phosphatase and tensin homolog (PTEN) is inactivated in a wide range of human cancers, an alteration that is associated with a poor prognosis. Because PTEN has been reported to play a role in the maintenance of embryonic and tissue-specific stem cells, we investigated the role of the PTEN/Akt pathway in the regulation of normal and malignant mammary stem/progenitor cell populations. We demonstrate that activation of this pathway, via PTEN knockdown, enriches for normal and malignant human mammary stem/progenitor cells in vitro and in vivo. Knockdown of PTEN in normal human mammary epithelial cells enriches for the stem/progenitor cell compartment, generating atypical hyperplastic lesions in humanized NOD/SCID mice. Akt-driven stem/progenitor cell enrichment is mediated by activation of the Wnt/beta-catenin pathway through the phosphorylation of GSK3-beta. In contrast to chemotherapy, the Akt inhibitor perifosine is able to target the tumorigenic cell population in breast tumor xenografts. These studies demonstrate an important role for the PTEN/PI3-K/Akt/beta-catenin pathway in the regulation of normal and malignant stem/progenitor cell populations and suggest that agents that inhibit this pathway are able to effectively target tumorigenic breast cancer cells.

Show MeSH
Related in: MedlinePlus