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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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Knockdown of PTEN in NMECs generates disorganized hyperplastic lesions in humanized NOD/SCID mice.(A) Human mammary outgrowths generated from control or PTEN knockdown NMECs in humanized NOD/SCID mice exhibited an altered morphology by hematoxylin and eosin staining. (B) PTEN staining demonstrated reduced PTEN protein expression in outgrowths generated from PTEN knockdown cells as compared to the controls (a and h). Smooth muscle actin (SMA) staining revealed disorganized myoepithelial structures in PTEN knockdown outgrowths compared to an organized layer of myoepithelial cells in controls (b and i). Outgrowths with PTEN knockdown showed increased CK5/6 expression (c and j) and decreased CK18 expression (d and k), as well as a lack of ERα expression compared to control cells (e and l). PTEN knockdown outgrowths displayed increased proliferation characterized by Ki67 staining compared to controls (f and m). Increased ALDH1 expression was demonstrated in PTEN knockdown structures (g and n). Scale bars = 100 µm. Data are representative of experiments with five mice in each group.
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pbio-1000121-g002: Knockdown of PTEN in NMECs generates disorganized hyperplastic lesions in humanized NOD/SCID mice.(A) Human mammary outgrowths generated from control or PTEN knockdown NMECs in humanized NOD/SCID mice exhibited an altered morphology by hematoxylin and eosin staining. (B) PTEN staining demonstrated reduced PTEN protein expression in outgrowths generated from PTEN knockdown cells as compared to the controls (a and h). Smooth muscle actin (SMA) staining revealed disorganized myoepithelial structures in PTEN knockdown outgrowths compared to an organized layer of myoepithelial cells in controls (b and i). Outgrowths with PTEN knockdown showed increased CK5/6 expression (c and j) and decreased CK18 expression (d and k), as well as a lack of ERα expression compared to control cells (e and l). PTEN knockdown outgrowths displayed increased proliferation characterized by Ki67 staining compared to controls (f and m). Increased ALDH1 expression was demonstrated in PTEN knockdown structures (g and n). Scale bars = 100 µm. Data are representative of experiments with five mice in each group.

Mentions: We previously used a mouse model described by Proia and Kuperwasser [25] in which NMECs form outgrowths in NOD/SCID mice whose mammary fat pads have been humanized by the introduction of both irradiated and non-irradiated human mammary fibroblasts. We used this system to examine the effects of PTEN knockdown on mammary development. Serial dilutions of flow cytometry-sorted cells were introduced into the humanized fat pads of NOD/SCID mice. As indicated in Table S1, at all dilutions, NMECs with PTEN knockdown were more efficient in generating outgrowths than DsRed control cells. While at least 10,000 control cells were required for efficient outgrowth formation, as few as 250 PTEN knockdown cells generated outgrowths in 50% of the mice, indicating that PTEN knockdown increased the frequency of multipotent mammary stem/progenitors. In addition, we observed significant morphological alterations in structures generated by PTEN knockdown in NMECs compared to DsRed controls (Figure 2A). PTEN knockdown cells produced much larger structures that displayed significant morphologic alterations. Knockdown of PTEN was confirmed by the lack of PTEN expression in PTEN knockdown outgrowths compared to controls (Figure 2B, a and h). We used immunohistochemical staining for markers of myoepithelial, basal, and luminal epithelial cells to ascertain the effects of PTEN knockdown on cellular differentiation. Outgrowths generated by control-infected NMECs consisted of ductal structures that were characterized by a single layer of myoepithelial cells, which expressed smooth muscle actin recapitulating the architecture of normal mammary ducts in humans. In contrast, structures generated by PTEN shRNA-infected cells were characterized by gross disorganization with increased numbers of smooth muscle actin expressing myoepithelial cells distributed throughout the gland (Figure 2B, b and i). Glands produced by control cells contained only a small number of cells expressing the primitive cytokeratins 5/6, whereas the frequency of these cells was greatly increased in PTEN knockdown structures (Figure 2B, c and j). Examination of epithelial markers also revealed significant differences between structures derived from PTEN knockdown and control cells. In control structures, the majority of the luminal epithelial cells expressed the luminal marker CK18, whereas expression of this marker occurred only in a subfraction of PTEN knockdown cells (Figure 2B, d and k). Estrogen receptor (ER) was expressed in luminal epithelial cells in structures generated from DsRed control cells, but not in structures derived from PTEN knockdown cells (Figure 2B, e and l). Furthermore, structures with PTEN knockdown displayed significant increases in proliferating cells as determined by Ki67 expression (Figure 2B, f and m). Consistent with the in vitro experiments, PTEN knockdown also increased the proportion of cells expressing ALDH1 (Figure 2B, g and n). These experiments confirm and extend the in vitro findings and suggest that in addition to resulting in enrichment of the stem/progenitor cell pool, activation of the PTEN/PI3-K/Akt pathway affects cellular growth and differentiation. This results in the generation of cells displaying increased proliferation, with aberrant differentiation resulting in increased expression of primitive and basal markers and decreased expression of luminal epithelial markers. All of these histopathologic features are characteristic of atypical ductal hyperplasia, a premalignant lesion that may progress to invasive breast cancer.


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)

Knockdown of PTEN in NMECs generates disorganized hyperplastic lesions in humanized NOD/SCID mice.(A) Human mammary outgrowths generated from control or PTEN knockdown NMECs in humanized NOD/SCID mice exhibited an altered morphology by hematoxylin and eosin staining. (B) PTEN staining demonstrated reduced PTEN protein expression in outgrowths generated from PTEN knockdown cells as compared to the controls (a and h). Smooth muscle actin (SMA) staining revealed disorganized myoepithelial structures in PTEN knockdown outgrowths compared to an organized layer of myoepithelial cells in controls (b and i). Outgrowths with PTEN knockdown showed increased CK5/6 expression (c and j) and decreased CK18 expression (d and k), as well as a lack of ERα expression compared to control cells (e and l). PTEN knockdown outgrowths displayed increased proliferation characterized by Ki67 staining compared to controls (f and m). Increased ALDH1 expression was demonstrated in PTEN knockdown structures (g and n). Scale bars = 100 µm. Data are representative of experiments with five mice in each group.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2683567&req=5

pbio-1000121-g002: Knockdown of PTEN in NMECs generates disorganized hyperplastic lesions in humanized NOD/SCID mice.(A) Human mammary outgrowths generated from control or PTEN knockdown NMECs in humanized NOD/SCID mice exhibited an altered morphology by hematoxylin and eosin staining. (B) PTEN staining demonstrated reduced PTEN protein expression in outgrowths generated from PTEN knockdown cells as compared to the controls (a and h). Smooth muscle actin (SMA) staining revealed disorganized myoepithelial structures in PTEN knockdown outgrowths compared to an organized layer of myoepithelial cells in controls (b and i). Outgrowths with PTEN knockdown showed increased CK5/6 expression (c and j) and decreased CK18 expression (d and k), as well as a lack of ERα expression compared to control cells (e and l). PTEN knockdown outgrowths displayed increased proliferation characterized by Ki67 staining compared to controls (f and m). Increased ALDH1 expression was demonstrated in PTEN knockdown structures (g and n). Scale bars = 100 µm. Data are representative of experiments with five mice in each group.
Mentions: We previously used a mouse model described by Proia and Kuperwasser [25] in which NMECs form outgrowths in NOD/SCID mice whose mammary fat pads have been humanized by the introduction of both irradiated and non-irradiated human mammary fibroblasts. We used this system to examine the effects of PTEN knockdown on mammary development. Serial dilutions of flow cytometry-sorted cells were introduced into the humanized fat pads of NOD/SCID mice. As indicated in Table S1, at all dilutions, NMECs with PTEN knockdown were more efficient in generating outgrowths than DsRed control cells. While at least 10,000 control cells were required for efficient outgrowth formation, as few as 250 PTEN knockdown cells generated outgrowths in 50% of the mice, indicating that PTEN knockdown increased the frequency of multipotent mammary stem/progenitors. In addition, we observed significant morphological alterations in structures generated by PTEN knockdown in NMECs compared to DsRed controls (Figure 2A). PTEN knockdown cells produced much larger structures that displayed significant morphologic alterations. Knockdown of PTEN was confirmed by the lack of PTEN expression in PTEN knockdown outgrowths compared to controls (Figure 2B, a and h). We used immunohistochemical staining for markers of myoepithelial, basal, and luminal epithelial cells to ascertain the effects of PTEN knockdown on cellular differentiation. Outgrowths generated by control-infected NMECs consisted of ductal structures that were characterized by a single layer of myoepithelial cells, which expressed smooth muscle actin recapitulating the architecture of normal mammary ducts in humans. In contrast, structures generated by PTEN shRNA-infected cells were characterized by gross disorganization with increased numbers of smooth muscle actin expressing myoepithelial cells distributed throughout the gland (Figure 2B, b and i). Glands produced by control cells contained only a small number of cells expressing the primitive cytokeratins 5/6, whereas the frequency of these cells was greatly increased in PTEN knockdown structures (Figure 2B, c and j). Examination of epithelial markers also revealed significant differences between structures derived from PTEN knockdown and control cells. In control structures, the majority of the luminal epithelial cells expressed the luminal marker CK18, whereas expression of this marker occurred only in a subfraction of PTEN knockdown cells (Figure 2B, d and k). Estrogen receptor (ER) was expressed in luminal epithelial cells in structures generated from DsRed control cells, but not in structures derived from PTEN knockdown cells (Figure 2B, e and l). Furthermore, structures with PTEN knockdown displayed significant increases in proliferating cells as determined by Ki67 expression (Figure 2B, f and m). Consistent with the in vitro experiments, PTEN knockdown also increased the proportion of cells expressing ALDH1 (Figure 2B, g and n). These experiments confirm and extend the in vitro findings and suggest that in addition to resulting in enrichment of the stem/progenitor cell pool, activation of the PTEN/PI3-K/Akt pathway affects cellular growth and differentiation. This results in the generation of cells displaying increased proliferation, with aberrant differentiation resulting in increased expression of primitive and basal markers and decreased expression of luminal epithelial markers. All of these histopathologic features are characteristic of atypical ductal hyperplasia, a premalignant lesion that may progress to invasive breast cancer.

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