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

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.

Conflict of interest statement

MSW holds equity in and is a scientific consultant for OncoMed Pharmaceuticals.

Figures

Figure 1. 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.

Figure 2. 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.

Figure 3. Effect of PI3-K/Akt inhibitors on mammosphere formation, ALDH1 expression, and mammary development in NOD/SCID mice.

(A) Treatment of mammospheres with the PI3-K inhibitor LY294002 (1 µM), Akt inhibitor IV (2 µM), or perifosine (5 µM) inhibited mammosphere formation in control cells, whereas PTEN knockdown NMECs were sensitive to lower doses of these inhibitors: LY294002 (0.5 µM), Akt inhibitor IV (1 µM), or perifosine (2 µM). In contrast, the mTOR inhibitor rapamycin (0.5 µM) had little effect on these cells. (B) Perifosine dose response was tested in both control and PTEN knockdown cells. As indicated, PTEN knockdown cells with higher Akt activity are more sensitive to perifosine than control cells with an IC50 of 5 µM. (C) The effect of perifosine on the Aldefluor-positive population of NMECs was measured by the Aldefluor assay. Treatment of NMECs with perifosine over 5 d reduced the Aldefluor-positive population in primary mammospheres by more than 50%. (D) Perifosine treatment of mice implanted with control or PTEN knockdown NMECs completely blocked the formation of outgrowths in NOD/SCID humanized fat pads compared to saline-treated control mice. High magnification (insets) shows persistence of the inoculated cells. Scale bars = 100 µm. Data represent the mean±SD of three independent experiments.

Figure 4. 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.

Figure 5. 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.

Figure 6. Perifosine targets the Aldefluor-positive cell population in breast cancer xenografts.

(A–C). 50,000 cells from SUM159 cell line or two breast cancer xenografts, MCI, or UM2 were injected into NOD/SCID mice and the tumor size was monitored. When the tumors were approximately 4 mm, perifosine (30 mg/kg) or docetaxel (10 mg/kg), or a combination of the two agents were administered intraperitoneally once per week. Tumor size before and during the course of each indicated treatment is shown. (D) ALDH activity was assessed by the Aldefluor assay. Control or docetaxel-treated SUM159, MCI, or UM2 tumor xenografts contained a fraction of cells ranging from 4–8%, which displayed Aldefluor activity. In contrast, perifosine treatment alone or in combination with docetaxel produced a 75–90% decrease in the proportion of cancer stem/progenitor cells as assessed by the Aldefluor assay. (E) Effect of treatment on β-catenin expression. Control and docetaxel-treated tumors displayed abundant nuclear β-catenin expression. This was significantly reduced by perifosine treatment. Scale bars = 100 µm. Data represent the mean±SD of experiments with five mice in each group.

Figure 7. Perifosine treatment reduces the tumorigenic cell population as assessed by reimplantation in secondary NOD/SCID mice.

(A and B). Serial dilutions of cells obtained from primary Sum159 (A) or MCI (B) tumors treated with saline control, perifosine, docetaxel, or both were implanted into secondary NOD/SCID mice. Control and docetaxel-treated primary tumors formed secondary tumors at all dilutions. In contrast, primary tumors treated with perifosine alone or in combination with docetaxel demonstrated growth delay with secondary tumors that were three times smaller in size than the control or docetaxel-treated tumors. Furthermore, 1,000 cells from perifosine or perifosine- plus docetaxel-treated primary tumors failed to form any secondary tumors, whereas tumors grew in mice inoculated with an equivalent number of cells from control or docetaxel-treated tumors. (C) Primary treated tumors were sorted by the Aldefluor assay and 5×103 cells from each of the Aldelfuor-positive and Aldelfuor-negative populations were reimplanted in secondary NOD/SCID mice. Only Aldefluor-positive cells generated secondary tumors that remained sensitive to perifosine treatment. Data represent the mean±SD of experiments with five mice in each group.