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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 breast cancer cell lines results in enrichment of breast cancer stem/progenitor cells via the Akt/GSK3-β/β-catenin pathway.(A) PTEN knockdown in MCF7 or SUM159 cells resulted in increased Akt phosphorylation as assessed by Western blotting. (B) PTEN knockdown resulted in increased secondary tumorsphere formation in MCF7 and SUM159 cells. (C) PTEN knockdown secondary tumorspheres contained an increased proportion of Aldefluor-positive cells as compared with the tumorspheres from parental lines. (D) Flow cytometry analyses of TOP-GFP-infected SUM159 tumorspheres treated with indicated inhibitors. Perifosine treatment decreased the proportion of GFP-positive cells by more than 50%, whereas Bio treatment increased the proportion of GFP-positive cells more than 2-fold and reversed the inhibitory effect of perifosine. Data represent the mean±SD of three independent experiments.
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pbio-1000121-g005: Knockdown of PTEN in breast cancer cell lines results in enrichment of breast cancer stem/progenitor cells via the Akt/GSK3-β/β-catenin pathway.(A) PTEN knockdown in MCF7 or SUM159 cells resulted in increased Akt phosphorylation as assessed by Western blotting. (B) PTEN knockdown resulted in increased secondary tumorsphere formation in MCF7 and SUM159 cells. (C) PTEN knockdown secondary tumorspheres contained an increased proportion of Aldefluor-positive cells as compared with the tumorspheres from parental lines. (D) Flow cytometry analyses of TOP-GFP-infected SUM159 tumorspheres treated with indicated inhibitors. Perifosine treatment decreased the proportion of GFP-positive cells by more than 50%, whereas Bio treatment increased the proportion of GFP-positive cells more than 2-fold and reversed the inhibitory effect of perifosine. Data represent the mean±SD of three independent experiments.

Mentions: By using primary mammary carcinoma xenografts, we previously demonstrated that cancer cells with stem cell-like properties were contained within the Aldefluor-positive population [19]. Recent studies have suggested that established breast cancer cell lines also contain subpopulations with stem cell-like properties. In MCF7 and SUM159 cell lines, we previously demonstrated that only the Aldefluor-positive populations were able to form tumors capable of serial passaging in NOD/SCID mice [31]. To determine whether the PTEN/PI3-K/Akt pathway played a similar role in the regulation of malignant mammary stem/progenitor cells to that of normal mammary stem/progenitor cells, we determined the effect of knocking down PTEN expression on the cancer stem/progenitor cell populations in these cell lines. As shown by Western blotting (Figure 5A), control MCF7 cells expressed PTEN, but not phospho-Akt. Knockdown of PTEN with an shRNA lentivirus resulted in Akt activation (Figure 5A). SUM159 cells display a baseline level of phospho-Akt expression, which was further enhanced by PTEN knockdown (Figure 5A). To determine whether reduction of PTEN expression affected the mammary cancer stem/progenitor cell components of these cell lines, we used tumorsphere and Aldefluor assays. As shown in Figure 5B, knockdown of PTEN increased tumorsphere formation in both MCF7 and SUM159 breast carcinoma cells. Furthermore, this knockdown resulted in more than a 2-fold increase in the Aldefluor-positive population (p<0.01) in these cell lines (Figure 5C). We confirmed that PTEN knockdown in MCF7 xenographs resulted in increased Akt phosphorylation (Figure S4B). Subsequently we determined whether the increase in the cancer stem/progenitor cell population resulting from PTEN knockdown resulted in increased tumorigenicity. As shown in Figure S4A and S4C, PTEN knockdown in MCF7 or SUM159 cells increased their tumorigenicity in NOD/SCID mice. Since Zhou et al. previously reported that the side population (SP) in MCF7 contained tumor-initiating cells, we examined the overlap between the SP and Aldefluor-positive populations. As demonstrated in Figure S4, we found a 2-fold enrichment of Aldefluor-positive population in the MCF7 SP population as compared to non-SP cells. This suggests that the Aldefluor and SP assays detect distinct, but partially overlapping, cell populations. We previously reported that tumorigenicity in these cell lines is mediated by the Aldefluor-positive population, which is enriched for tumorigenic cancer stem/progenitor cells [31]. Thus, these results suggest that Akt activation increases tumorigenicity through effects on the cancer stem/progenitor cell population. We next used the TOP-GFP reporter system to determine whether Akt signals through the Wnt/β-catenin pathway in breast carcinoma cells. Perifosine treatment of TOP-GFP-infected SUM159 breast cancer cells resulted in a significant decrease in GFP-positive cells (p<0.01), whereas treatment of these cells with Bio increased the proportion of GFP-positive cells 3-fold, p<0.01 (Figure 5D). As was the case for normal mammary cells, Bio treatment was able to reverse the effect of perifosine in these mammary carcinoma cells (Figure 5D).


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 breast cancer cell lines results in enrichment of breast cancer stem/progenitor cells via the Akt/GSK3-β/β-catenin pathway.(A) PTEN knockdown in MCF7 or SUM159 cells resulted in increased Akt phosphorylation as assessed by Western blotting. (B) PTEN knockdown resulted in increased secondary tumorsphere formation in MCF7 and SUM159 cells. (C) PTEN knockdown secondary tumorspheres contained an increased proportion of Aldefluor-positive cells as compared with the tumorspheres from parental lines. (D) Flow cytometry analyses of TOP-GFP-infected SUM159 tumorspheres treated with indicated inhibitors. Perifosine treatment decreased the proportion of GFP-positive cells by more than 50%, whereas Bio treatment increased the proportion of GFP-positive cells more than 2-fold and reversed the inhibitory effect of perifosine. Data represent 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-g005: Knockdown of PTEN in breast cancer cell lines results in enrichment of breast cancer stem/progenitor cells via the Akt/GSK3-β/β-catenin pathway.(A) PTEN knockdown in MCF7 or SUM159 cells resulted in increased Akt phosphorylation as assessed by Western blotting. (B) PTEN knockdown resulted in increased secondary tumorsphere formation in MCF7 and SUM159 cells. (C) PTEN knockdown secondary tumorspheres contained an increased proportion of Aldefluor-positive cells as compared with the tumorspheres from parental lines. (D) Flow cytometry analyses of TOP-GFP-infected SUM159 tumorspheres treated with indicated inhibitors. Perifosine treatment decreased the proportion of GFP-positive cells by more than 50%, whereas Bio treatment increased the proportion of GFP-positive cells more than 2-fold and reversed the inhibitory effect of perifosine. Data represent the mean±SD of three independent experiments.
Mentions: By using primary mammary carcinoma xenografts, we previously demonstrated that cancer cells with stem cell-like properties were contained within the Aldefluor-positive population [19]. Recent studies have suggested that established breast cancer cell lines also contain subpopulations with stem cell-like properties. In MCF7 and SUM159 cell lines, we previously demonstrated that only the Aldefluor-positive populations were able to form tumors capable of serial passaging in NOD/SCID mice [31]. To determine whether the PTEN/PI3-K/Akt pathway played a similar role in the regulation of malignant mammary stem/progenitor cells to that of normal mammary stem/progenitor cells, we determined the effect of knocking down PTEN expression on the cancer stem/progenitor cell populations in these cell lines. As shown by Western blotting (Figure 5A), control MCF7 cells expressed PTEN, but not phospho-Akt. Knockdown of PTEN with an shRNA lentivirus resulted in Akt activation (Figure 5A). SUM159 cells display a baseline level of phospho-Akt expression, which was further enhanced by PTEN knockdown (Figure 5A). To determine whether reduction of PTEN expression affected the mammary cancer stem/progenitor cell components of these cell lines, we used tumorsphere and Aldefluor assays. As shown in Figure 5B, knockdown of PTEN increased tumorsphere formation in both MCF7 and SUM159 breast carcinoma cells. Furthermore, this knockdown resulted in more than a 2-fold increase in the Aldefluor-positive population (p<0.01) in these cell lines (Figure 5C). We confirmed that PTEN knockdown in MCF7 xenographs resulted in increased Akt phosphorylation (Figure S4B). Subsequently we determined whether the increase in the cancer stem/progenitor cell population resulting from PTEN knockdown resulted in increased tumorigenicity. As shown in Figure S4A and S4C, PTEN knockdown in MCF7 or SUM159 cells increased their tumorigenicity in NOD/SCID mice. Since Zhou et al. previously reported that the side population (SP) in MCF7 contained tumor-initiating cells, we examined the overlap between the SP and Aldefluor-positive populations. As demonstrated in Figure S4, we found a 2-fold enrichment of Aldefluor-positive population in the MCF7 SP population as compared to non-SP cells. This suggests that the Aldefluor and SP assays detect distinct, but partially overlapping, cell populations. We previously reported that tumorigenicity in these cell lines is mediated by the Aldefluor-positive population, which is enriched for tumorigenic cancer stem/progenitor cells [31]. Thus, these results suggest that Akt activation increases tumorigenicity through effects on the cancer stem/progenitor cell population. We next used the TOP-GFP reporter system to determine whether Akt signals through the Wnt/β-catenin pathway in breast carcinoma cells. Perifosine treatment of TOP-GFP-infected SUM159 breast cancer cells resulted in a significant decrease in GFP-positive cells (p<0.01), whereas treatment of these cells with Bio increased the proportion of GFP-positive cells 3-fold, p<0.01 (Figure 5D). As was the case for normal mammary cells, Bio treatment was able to reverse the effect of perifosine in these mammary carcinoma cells (Figure 5D).

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