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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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PTEN regulates β-catenin activity in mammary stem/progenitor cells.(A) Effects of the GSK3-β inhibitor (Bio) and the Akt inhibitor (perifosine) on activation of Akt/GSK3-β/β-catenin signaling as assessed by Western blotting using phospho-specific antibodies. Perifosine inhibits pAkt, pGSK3-β, and activated β-catenin expression. The GSK3-β inhibitor Bio restores β-catenin activation even in the presence of perifosine. (B) After 5 d treatment of primary mammospheres with either 10 µM perifosine or 0.5 µM Bio alone or in combination, cells were dissociated and passaged to form secondary mammospheres. Bio treatment increased the number of secondary mammospheres more than 2-fold, whereas perifosine treatment or down-regulation of β-catenin via infection with an shRNA lentivirus decreased the number of secondary mammospheres by more than 50%. Bio reversed the inhibitory effect of perifosine. (C) To monitor β-catenin activity, TOP-GFP reporter lentivirus-infected mammospheres were treated with Bio or co-transfected with PTEN shRNA. Control mammospheres cultured for 7 d contained one–four GFP-positive cells. The proportion of GFP-positive cells was increased more than 2-fold by Bio treatment. Knockdown of PTEN also resulted in more than a 2-fold increase in the proportion of GFP-positive cells. (D) TOP-GFP infected mammospheres were treated for 5 d with indicated compounds either alone or in combination and analyzed by flow cytometry. Perifosine treatment decreased the proportion of GFP-positive cells by more than 50%, whereas Bio treatment increased them more than 2-fold. The inhibition produced by perifosine was abrogated when the mammospheres were also treated with Bio. (E) Outgrowths generated in NOD/SCID mice from PTEN shRNA lentivirus-infected cells displayed increased phospho-Akt expression as well as increased nuclear β-catenin localization as compared to control outgrowths. Scale bars = 100 µm. Data represent the mean±SD of three independent experiments.
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pbio-1000121-g004: PTEN regulates β-catenin activity in mammary stem/progenitor cells.(A) Effects of the GSK3-β inhibitor (Bio) and the Akt inhibitor (perifosine) on activation of Akt/GSK3-β/β-catenin signaling as assessed by Western blotting using phospho-specific antibodies. Perifosine inhibits pAkt, pGSK3-β, and activated β-catenin expression. The GSK3-β inhibitor Bio restores β-catenin activation even in the presence of perifosine. (B) After 5 d treatment of primary mammospheres with either 10 µM perifosine or 0.5 µM Bio alone or in combination, cells were dissociated and passaged to form secondary mammospheres. Bio treatment increased the number of secondary mammospheres more than 2-fold, whereas perifosine treatment or down-regulation of β-catenin via infection with an shRNA lentivirus decreased the number of secondary mammospheres by more than 50%. Bio reversed the inhibitory effect of perifosine. (C) To monitor β-catenin activity, TOP-GFP reporter lentivirus-infected mammospheres were treated with Bio or co-transfected with PTEN shRNA. Control mammospheres cultured for 7 d contained one–four GFP-positive cells. The proportion of GFP-positive cells was increased more than 2-fold by Bio treatment. Knockdown of PTEN also resulted in more than a 2-fold increase in the proportion of GFP-positive cells. (D) TOP-GFP infected mammospheres were treated for 5 d with indicated compounds either alone or in combination and analyzed by flow cytometry. Perifosine treatment decreased the proportion of GFP-positive cells by more than 50%, whereas Bio treatment increased them more than 2-fold. The inhibition produced by perifosine was abrogated when the mammospheres were also treated with Bio. (E) Outgrowths generated in NOD/SCID mice from PTEN shRNA lentivirus-infected cells displayed increased phospho-Akt expression as well as increased nuclear β-catenin localization as compared to control outgrowths. Scale bars = 100 µm. Data represent the mean±SD of three independent experiments.

Mentions: Activated Akt has been demonstrated to be capable of phosphorylating and inactivating GSK3-β, leading to nuclear translocation and activation of β-catenin [28],[29]. In addition, Akt can directly phosphorylate β-catenin, an event which further facilitates its nuclear translocation [21]. Since Wnt signaling through β-catenin has been shown to play a role in mammary development and stem cell self-renewal, we examined whether Akt effects on mammary stem/progenitor cells were mediated by this pathway. To examine the role of Wnt/β-catenin signaling, we used the GSK3-β inhibitor 6-bromoindirubin-3′-oxime (Bio), which has been shown to be able to maintain pluripotency of human and mouse embryonic stem cells through activation of β-catenin signaling [30]. We first confirmed that addition of Bio to NMECs resulted in phosphorylation of GSK3-β with subsequent activation of β-catenin (Figure 4A). To determine the biological relevance of this pathway, we examined the effects of Bio on mammosphere formation. As shown in Figure 4B, the addition of Bio increased mammosphere formation 2-fold. In addition, the reduction of mammosphere formation induced by perifosine was reversed by the addition of Bio (Figure 4B). Reversal of inhibition occurred despite the persistent inhibition of Akt activity in the presence of both compounds (Figure 4A). To confirm the importance of β-catenin signaling in stem/progenitor cell self-renewal, we knocked down β-catenin expression by using an shRNA lentivirus (Figure S3). Lentiviral shRNA-mediated down-regulation of β-catenin expression in NMECs reduced the number of mammospheres by approximately 70% (Figure 4B). These experiments suggest that the effects of Akt on mammary stem/progenitor cell self-renewal are mediated through GSK3-β/β-catenin signaling.


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)

PTEN regulates β-catenin activity in mammary stem/progenitor cells.(A) Effects of the GSK3-β inhibitor (Bio) and the Akt inhibitor (perifosine) on activation of Akt/GSK3-β/β-catenin signaling as assessed by Western blotting using phospho-specific antibodies. Perifosine inhibits pAkt, pGSK3-β, and activated β-catenin expression. The GSK3-β inhibitor Bio restores β-catenin activation even in the presence of perifosine. (B) After 5 d treatment of primary mammospheres with either 10 µM perifosine or 0.5 µM Bio alone or in combination, cells were dissociated and passaged to form secondary mammospheres. Bio treatment increased the number of secondary mammospheres more than 2-fold, whereas perifosine treatment or down-regulation of β-catenin via infection with an shRNA lentivirus decreased the number of secondary mammospheres by more than 50%. Bio reversed the inhibitory effect of perifosine. (C) To monitor β-catenin activity, TOP-GFP reporter lentivirus-infected mammospheres were treated with Bio or co-transfected with PTEN shRNA. Control mammospheres cultured for 7 d contained one–four GFP-positive cells. The proportion of GFP-positive cells was increased more than 2-fold by Bio treatment. Knockdown of PTEN also resulted in more than a 2-fold increase in the proportion of GFP-positive cells. (D) TOP-GFP infected mammospheres were treated for 5 d with indicated compounds either alone or in combination and analyzed by flow cytometry. Perifosine treatment decreased the proportion of GFP-positive cells by more than 50%, whereas Bio treatment increased them more than 2-fold. The inhibition produced by perifosine was abrogated when the mammospheres were also treated with Bio. (E) Outgrowths generated in NOD/SCID mice from PTEN shRNA lentivirus-infected cells displayed increased phospho-Akt expression as well as increased nuclear β-catenin localization as compared to control outgrowths. Scale bars = 100 µm. Data represent the mean±SD of three independent experiments.
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Related In: Results  -  Collection

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pbio-1000121-g004: PTEN regulates β-catenin activity in mammary stem/progenitor cells.(A) Effects of the GSK3-β inhibitor (Bio) and the Akt inhibitor (perifosine) on activation of Akt/GSK3-β/β-catenin signaling as assessed by Western blotting using phospho-specific antibodies. Perifosine inhibits pAkt, pGSK3-β, and activated β-catenin expression. The GSK3-β inhibitor Bio restores β-catenin activation even in the presence of perifosine. (B) After 5 d treatment of primary mammospheres with either 10 µM perifosine or 0.5 µM Bio alone or in combination, cells were dissociated and passaged to form secondary mammospheres. Bio treatment increased the number of secondary mammospheres more than 2-fold, whereas perifosine treatment or down-regulation of β-catenin via infection with an shRNA lentivirus decreased the number of secondary mammospheres by more than 50%. Bio reversed the inhibitory effect of perifosine. (C) To monitor β-catenin activity, TOP-GFP reporter lentivirus-infected mammospheres were treated with Bio or co-transfected with PTEN shRNA. Control mammospheres cultured for 7 d contained one–four GFP-positive cells. The proportion of GFP-positive cells was increased more than 2-fold by Bio treatment. Knockdown of PTEN also resulted in more than a 2-fold increase in the proportion of GFP-positive cells. (D) TOP-GFP infected mammospheres were treated for 5 d with indicated compounds either alone or in combination and analyzed by flow cytometry. Perifosine treatment decreased the proportion of GFP-positive cells by more than 50%, whereas Bio treatment increased them more than 2-fold. The inhibition produced by perifosine was abrogated when the mammospheres were also treated with Bio. (E) Outgrowths generated in NOD/SCID mice from PTEN shRNA lentivirus-infected cells displayed increased phospho-Akt expression as well as increased nuclear β-catenin localization as compared to control outgrowths. Scale bars = 100 µm. Data represent the mean±SD of three independent experiments.
Mentions: Activated Akt has been demonstrated to be capable of phosphorylating and inactivating GSK3-β, leading to nuclear translocation and activation of β-catenin [28],[29]. In addition, Akt can directly phosphorylate β-catenin, an event which further facilitates its nuclear translocation [21]. Since Wnt signaling through β-catenin has been shown to play a role in mammary development and stem cell self-renewal, we examined whether Akt effects on mammary stem/progenitor cells were mediated by this pathway. To examine the role of Wnt/β-catenin signaling, we used the GSK3-β inhibitor 6-bromoindirubin-3′-oxime (Bio), which has been shown to be able to maintain pluripotency of human and mouse embryonic stem cells through activation of β-catenin signaling [30]. We first confirmed that addition of Bio to NMECs resulted in phosphorylation of GSK3-β with subsequent activation of β-catenin (Figure 4A). To determine the biological relevance of this pathway, we examined the effects of Bio on mammosphere formation. As shown in Figure 4B, the addition of Bio increased mammosphere formation 2-fold. In addition, the reduction of mammosphere formation induced by perifosine was reversed by the addition of Bio (Figure 4B). Reversal of inhibition occurred despite the persistent inhibition of Akt activity in the presence of both compounds (Figure 4A). To confirm the importance of β-catenin signaling in stem/progenitor cell self-renewal, we knocked down β-catenin expression by using an shRNA lentivirus (Figure S3). Lentiviral shRNA-mediated down-regulation of β-catenin expression in NMECs reduced the number of mammospheres by approximately 70% (Figure 4B). These experiments suggest that the effects of Akt on mammary stem/progenitor cell self-renewal are mediated through GSK3-β/β-catenin signaling.

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