Akt activation disrupts mammary acinar architecture and enhances proliferation in an mTOR-dependent manner

Abstract

Activation of the serine/threonine kinase Akt/PKB positively impacts on three cellular processes relevant to tumor progression: proliferation, survival, and cell size/growth. Using a three-dimensional culture model of MCF-10A mammary cells, we have examined how Akt influences the morphogenesis of polarized epithelial structures. Activation of a conditionally active variant of Akt elicits large, misshapen structures, which primarily arise from the combined effects of Akt on proliferation and cell size. Importantly, Akt activation amplifies proliferation during the early stages of morphogenesis, but cannot overcome signals suppressing proliferation in late-stage cultures. Akt also cooperates with oncoproteins such as cyclin D1 or HPV E7 to promote proliferation and morphogenesis in the absence of growth factors. Pharmacological inhibition of the Akt effector, mammalian target of rapamycin (mTOR), with rapamycin prevents the morphological disruption elicited by Akt activation, including its effect on cell size and number, and the cooperative effect of Akt on oncogene-driven proliferation, indicating that mTOR function is required for the multiple biological effects of Akt activation during morphogenesis.

Figure 1.

Effects of Akt activation on mammary acinar morphogenesis and luminal apoptosis. (A) MCF10A cells expressing ER-Akt were cultured on Matrigel for 15–16 d in the presence of ethanol as vehicle control (top) or 1 μM OHT (bottom). Shown are representative phase images (left), confocal cross sections through the middle of DAPI-stained structures (center), and equatorial DAPI-stained confocal cross sections of structures immunostained with α-phospho Akt Ser⁴⁷³ (green, right). Bars, 25 μm. (B) ER-Akt structures grown in 3D with or without 1 μM OHT for the indicated times were immunostained with α-activated caspase 3 (green) and α-laminin 5 (red) and counterstained with DAPI (blue). Shown are equatorial confocal cross sections. Bars, 25 μm. (C) Structures of the indicated cell types were fixed and immunostained as illustrated in B. For both graphs, cells occupying the lumen were defined as those lacking direct contact with basement membrane as delineated by α-laminin 5 staining. (C, left) The percent of cells positive for activated caspase-3 observed in the lumen at various times was quantified; each time point represents the mean ± SD of 90 structures obtained from three independent experiments. (C, right) The mean ± SEM number of viable cells, defined as cells negative for activated caspase-3 present in the lumens of individual structures was enumerated; each time point represents 90 structures obtained from three independent experiments. (D) ER-Akt cells were cultured on EHS for 6 d in the presence of ethanol control (top) or 1 μM OHT and immunostained with α-P-Akt Ser⁴⁷³ (green, top) or α-P-FKHR/FKHR-L1 (green, bottom). Shown are equatorial cross sections counterstained with DAPI (blue). Bars, 25 μm.

Figure 2.

Cell size and proliferation in activated Akt structures. (A) After 18 d of 3D culture, structures grown with or without OHT were immunostained with αP-ERM (left), to mark cell membranes to delineate cell size/shape, and DAPI (right). Confocal cross sections through the equators of acini are shown. Bars, 25 μm. (B) Day 6 and 20 acini were immunostained with α-Ki-67. Representative fields show control and OHT-activated Akt structures with occasional Ki-67–positive cells at day 20, while HPV E7–expressing structures continue to proliferate. Corresponding phase contrast images are shown to the right of each Ki-67 stain. Bars, 50 μm. (C) Single cell suspensions of the indicated cell types were prepared from day 6 and 20 cultures, labeled with propidium iodide, and analyzed by flow cytometry to quantify the percent of cells with DNA content corresponding to the S and G2/M (S + G2/M) phases of the cell cycle. The mean ± SEM of three experiments is shown; statistical significance was determined by t test, with P > 0.05 defined as not significant (NS).

Figure 3.

Akt activation amplifies proliferation in mammary structures coexpressing cyclin D1 and HPV E7. MCF-10A cells coexpressing ER-Akt with LXSN (vector, left), cyclin D1 (middle), or HPV E7 (right) were cultured on EHS for 20 d with ethanol control (top) or 1 μM OHT (bottom). Representative images of α-Ki-67–immunostained structures along with corresponding phase images are shown. Similar results were obtained with pBABE as the vector control (not depicted). Bars, 50 μm.

Figure 4.

Akt and HPV E7 cooperate to promote proliferation and morphogenesis in the absence of exogenously provided growth factors. (A) The indicated cell types were cultured in 3D without EGF along with ethanol control (left) or 1 μM OHT (right) for the indicated number of days; representative phase-contrast images illustrate the development of acinar structures in MCF-10A cells coexpressing cyclin D1 or HPV E7 where ER-Akt has been activated with OHT. Bars, 50 μm. (B) The indicated cell types were cultured in 3D without EGF (or without both EGF and insulin) for 25 d, and cell numbers were quantified. Results represent the mean ± SD from three to five independent experiments.

Figure 5.

Rapamycin prevents Akt-mediated disruption of glandular morphology. (A) ER-Akt cells were cultured on Matrigel for 6 d in the presence of ethanol control (top) or 1 μM OHT; immunostained with α-P-Akt Ser⁴⁷³ (green, left) or α-P-mTOR (green, center and right). Shown are DAPI-stained equatorial cross sections. Bars, 25 μm. (B) Phase morphology of day 20 ER-Akt–expressing structures cultured with ethanol control (top) or 1 μM OHT (bottom) along with the indicated doses of rapamycin. Bars, 50 μm. (C) Day 12 ER-Akt structures treated with ethanol control or 1 μM OHT, as well as the indicated doses of rapamycin, were immunostained with α-activated caspase 3 (green) and α-laminin 5 (red). Representative DAPI-stained equatorial confocal cross sections are shown. Bars, 25 μm. (D) Day 20 ER-Akt structures were grown as indicated in the presence of ethanol (control), 1μM OHT, 20 nM rapamycin, or both and immunostained with α-P-ERM to outline cell membranes and illustrate the size and shape of individual cells within these structures. Bar, 25 μm. (E) ER-Akt cells grown as monolayers were treated with OHT (1 μM), rapamycin (20 nM), or both for 6 h, lysed, immunoblotted with α-P-Akt Ser⁴⁷³ and α-P-p70S6K Thr³⁸⁹, stripped, and reprobed with α-Akt and α-S6K. (F) ER-Akt structures grown on EHS for the indicated days in vehicle control, 1 μM OHT, or 1 μM OHT + 20 nM rapamycin (Rap) were immunostained with α-P-Akt Ser⁴⁷³; shown are DAPI-stained equatorial confocal cross sections. Bars, 25 μm.

Figure 6.

Rapamycin precludes the cooperative effects of Akt on oncogene-driven, EGF- independent proliferation. (A) Cells coexpressing ER-Akt along with LXSN (top) or HPV E7 (bottom) were 3D cultured for 20 d in the absence of EGF but with the indicated drug treatments; representative phase-contrast images are shown. Bar, 50 μm. (B) Cells coexpressing ER-Akt with LXSN or HPV E7 were cultured in 3D for 20 d without EGF but with the indicated drug treatments; thereafter, cell numbers were quantified. Results represent the mean ± SD from three independent experiments.