. 2011 Dec 15;360(2):329-42.
Epub 2011 Oct 8.
PI3K/mTOR Signaling Regulates Prostatic Branching Morphogenesis
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PI3K/mTOR Signaling Regulates Prostatic Branching Morphogenesis
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Prostatic branching morphogenesis is an intricate event requiring precise temporal and spatial integration of numerous hormonal and growth factor-regulated inputs, yet relatively little is known about the downstream signaling pathways that orchestrate this process. In this study, we use a novel mesenchyme-free embryonic prostate culture system, newly available mTOR inhibitors and a conditional PTEN loss-of-function model to investigate the role of the interconnected PI3K and mTOR signaling pathways in prostatic organogenesis. We demonstrate that PI3K levels and PI3K/mTOR activity are robustly induced by androgen during murine prostatic development and that PI3K/mTOR signaling is necessary for prostatic epithelial bud invasion of surrounding mesenchyme. To elucidate the cellular mechanism by which PI3K/mTOR signaling regulates prostatic branching, we show that PI3K/mTOR inhibition does not significantly alter epithelial proliferation or apoptosis, but rather decreases the efficiency and speed with which the developing prostatic epithelial cells migrate. Using mTOR kinase inhibitors to tease out the independent effects of mTOR signaling downstream of PI3K, we find that simultaneous inhibition of mTORC1 and mTORC2 activity attenuates prostatic branching and is sufficient to phenocopy combined PI3K/mTOR inhibition. Surprisingly, however, mTORC1 inhibition alone has the reverse effect, increasing the number and length of prostatic branches. Finally, simultaneous activation of PI3K and downstream mTORC1/C2 via epithelial PTEN loss-of-function also results in decreased budding reversible by mTORC1 inhibition, suggesting that the effect of mTORC1 on branching is not primarily mediated by negative feedback on PI3K/mTORC2 signaling. Taken together, our data point to an important role for PI3K/mTOR signaling in prostatic epithelial invasion and migration and implicates the balance of PI3K and downstream mTORC1/C2 activity as a critical regulator of prostatic epithelial morphogenesis.
Copyright © 2011 Elsevier Inc. All rights reserved.
Figure 1. PI3K is up-regulated and active during prostatic branching morphogenesis
(A) Immunoblotting of E15.5 female urogenital sinus (UGS) tissues cultured with or without dihydrotestosterone (DHT) for 48 hours reveals mild up-regulation of p-AKT (T308), an indirect measure of PI3K signaling, with a variable increase in levels of the p110α catalytic subunit of PI3K in response to androgen exposure. (B) Quantification of immunoblot in (A); n = 3 independent experiments, 3 UGS/condition/experiment. (C) Immunohistochemistry of E17.5 and E18.5 male UGS tissues demonstrates enrichment of p110α and downstream p-AKT(S473) proteins in the invasive prostatic epithelial buds (top panels, arrows, 200x magnification) compared to surrounding urethral epithelium. Mice transgenic for the PIP 3 biosensor, AKT-PH-GFP, show enrichment of membranous PIP 3 in the emerging prostatic bud epithelium at P4 (bottom panels, arrows, 100x and 400x magnification).
Figure 2. PI3K/mTOR signaling is required for prostatic branching morphogenesis
(A) Treatment of E15.5 male urogenital sinus (UGS) tissues with LY294002, wortmannin and PI-103 (PI3K/mTOR inhibitors) for 7 days in organ culture results in a decrease in prostatic epithelial budding (arrowheads) compared to vehicle control. Scale bar = 1 mm. (B) Immunoblotting of UGS lysates following 24 hours of treatment with vehicle or LY294002 reveals a decrease in PI3K signaling (indirectly measured by p-AKT [T308] levels), mTORC1 signaling (p-p70S6K levels) and mTORC2 signaling (p-AKT [S473] levels). (C) Quantification of immunoblot results from (B); n = 3 independent experiments, 3 UGS/condition/experiment.
Figure 3. PI3K/mTOR inhibition is not toxic and results in fewer and shorter invasive prostatic buds
(A) Treatment of male E15.5 urogenital sinus (UGS) tissue with 25 uM LY294002 for 24 hours followed by drug washout restores prostatic branching compared to UGS tissues treated for 7 days and vehicle controls. Scale bar = 1 mm. (B) Histologic examination of E15.5 UGS tissues treated with LY294002 for 7 days in organ culture reveals a near total absence of the invasive finger-like epithelial buds interspersed with strands of mesenchyme seen in the vehicle control samples, and instead shows broad-based, pushing protrusions into the mesenchyme using hematoxylin and eosin (H&E) staining (buds indicated by black outlines, 100x magnification). Buds are highlighted by pan-keratin (AE1/AE3) immunostaining (200x magnification). Epithelial nuclei appear more crowded in the LY294002-treated samples (400x magnification). ( C and D) Quantification of mean bud number, bud length and nuclear density in LY294002-treated samples reveals a statistically significant decrease in bud number and length and increase in nuclear density (n=10–11 UGS per condition, error bars = standard error of the mean (SEM), p-values using Student’s t-test for unpaired samples with unequal variance are indicated).
Figure 4. PI3K/mTOR activity is not required for epithelial proliferation, apoptosis or specification
(A) E15.5 male and female UGS tissues were cultured for 4 days in vehicle or 25 μM LY294002 and treated with BrdU for 2 hours prior to fixation. BrdU-positive epithelial nuclei are localized to epithelial buds in both LY294002- and vehicle-treated specimens (left panels, 200x magnification). Immunohistochemistry for cleaved caspase-3 using the same specimens reveals a total absence of apoptotic epithelial cells in both conditions, although rare apoptotic mesenchymal cells are present in vehicle- and LY294002-treated controls and serve as an internal positive control (middle panels, arrows, 400x magnification). Finally, immunohistochemistry for NKX3.1, an androgen-induced homeobox domain-containing transcription factor expressed during early prostatic development, shows positive nuclei confined to the emerging prostatic buds in both vehicle- and LY294002-treated specimens, suggesting that appropriate prostatic epithelial cell specification occurs in the presence of PI3K/mTOR inhibition (right panels, 200x magnification). (B) Quantification of proportion of BrdU-positive cells/epithelial bud in vehicle- and LY294002-treated specimens reveals no statistical difference between the two conditions (n=7–9 UGS/condition, error bars = SEM, p-value using Student’s t-test for unpaired samples with unequal variance).
Figure 5. PI3K/mTOR activity is required specifically in epithelial cells
(A) A combination of enzymatic and manual dissection allows dissociation of intact urogenital sinus epithelium (UGE) from mesenchymal tissue and epithelial branching following 48 hours of culture in Matrigel with androgen (DHT, 1 × 10 −8 M) and FGF10 (500 ng/mL) and FGF7 (200 ng/mL). (B) Hematoxylin and eosin (H&E) staining of UGE cultures shows individual epithelial buds surrounded by a prominent basal cell layer, with most cells expressing NKX3.1, a prostatic differentiation marker (200x; inset at 400x magnification). (C) Immunofluorescence for K8 demonstrates that most interior cells are luminal cells, while exterior cells predominantly express basal marker p63 with minimal overlap between these populations in merged images (600x magnification). (D) Time-lapse DIC microscopy of mesenchyme-free UGE cultures demonstrates the emergence of large prostatic buds in the distribution of the anterior and ventral prostate lobes (arrows) in the vehicle control (arrows, top panels), while only small and abortive buds are visible in the 25 μM LY294002-treated specimens (arrowheads, bottom panels). Scale bars=100 μm. Images shown are representative of one experiment (n=5).
Figure 6. PI3K/mTOR activity is required for efficient epithelial cell migration in mesenchyme-free epithelial cultures
(A) Urogenital sinus epithelial (UGE) tissues from R26ERCre;mT/mG transgenic mice show mosaic expression of membranous EGFP allowing three-dimensional tracking of epithelial cell migration over time using time-lapse epifluorescence imaging of mesenchyme-free cultures. While individual cells in vehicle-treated controls demonstrate efficient motility with cytoplasmic protrusions in the direction of migration (top panels, arrows) as well as readily apparent cell divisions (top panels, arrowheads), LY294002-treated epithelial cells assume an elongated shape with extension and retraction of protrusions, but are relatively immobile (bottom panels, arrows). (B) Positions of individual epithelial cells can be tracked over time in three dimensions (arrows indicate the net displacement over a 30 hour time period). Individual cell tracks in LY294002-treated UGEs are visibly shorter than those in vehicle controls. Scale bars = 100 um. (C) Net track displacement and mean velocity are significantly decreased with LY294002 treatment. Data shown are from one representative experiment out of 5 (n= 16–35 cells/condition, error bars = SEM, p-value using Student’s t-test for unpaired samples with unequal variance).
Specific inhibition of mTOR kinase without PI3K inhibition abrogates prostatic branching and phenocopies combined PI3K/mTOR inhibition.
(A) Treatment of male urogenital sinus (UGS) tissues with either of two different mTOR kinase inhibitors (1000 nM torin1 or 80 μM DMK-1) for 5–7 days results in decreased prostatic branching grossly identical to that seen after 25 μM LY294002 treatment. Arrowheads indicate prostatic branches in vehicle control, while seminal vesicle morphogenesis is grossly unaffected by any inhibitors (arrows). Images are representative of 3 independent experiments. (B) Histologic sectioning of urogenital sinuses treated with DMK-1 for 5 days reveals absence of invasive and elongated epithelial buds (arrows), as compared to vehicle-treated controls. (C) Immunoblotting of UGS tissues treated for 24 hours with mTOR kinase inhibitors demonstrates decreased mTORC1 signaling (represented by decreased p-p70S6K levels) and mTORC2 signaling (represented by decreased p-AKT[S473] levels), while PI3K signaling (as measured by p-AKT[T308] levels) remains unchanged. (D) Quantitation of immunoblot from (B); n = 3 independent experiments, 3 UGS/condition/experiment.
Figure 8. Specific inhibition of mTORC1 increases the number and length of prostatic branches
(A) Culture of E15.5 urogenital sinus (UGS) tissues in 200 nM rapamycin results in increased numbers of prostatic branches (top panels, arrowheads) by day 11 of culture. Excess branches in rapamycin-treated specimens are highlighted on cytokeratin 14 (CK14) immunostained histologic sections (middle panels, arrowheads, 200x magnification). Immunohistochemistry for pAKT(S473) after 11 days of culture in rapamycin reveals increased mTORC2 activity in prostatic buds relative to vehicle-treated control (bottom panels, 200x magnification). (B) Quantification of bud number and length in day 11 samples (n= 4 UGS/condition, error bars = SEM, p-value using Student’s t-test for unpaired samples with unequal variance.) ( C and D) Immunoblots with quanitification of vehicle- and rapamycin-treated UGS tissues after 24 hours of culture reveals decreased mTORC1 activity (p-p70S6K levels), and slightly increased mTORC2 activity (p-AKT[S473]) levels, consistent with rapamycin-mediated relief of negative feedback between p70S6K and mTORC2 signaling; n = 3 independent experiments, 3 UGS/condition/experiment.
Figure 9. PTEN loss-of-function decreases prostatic epithelial bud number and length in an mTORC1-dependent fashion
(A) Cre is efficiently induced prior to extensive prostatic branching in E15.5 R26ERCre; mT/mG reporter mouse urogenital sinus (UGS) tissues cultured for 4 days in 6 μM 4-OHT. Vehicle-treated control UGS tissues express a membrane-bound tomato red fluorophore using the mT/mG reporter allele, consistent with the absence of inducible Cre expression (left panel, merged image), while 4-OHT-treated UGS epithelial cells express membrane-bound EGFP, consistent with Cre expression in the majority of cells (middle panel, merged image). Only small epithelial buds are visible in in vitro cultures at this early timepoint (right panel, merged image). The mesenchymal cells in the 4-OHT-treated specimens demonstrate a lower level of inducible Cre than the epithelial cells (middle and right panels, merged images). (B) Immunoblotting for PTEN and PI3K/mTOR signaling components in PTEN fl/fl ( PTEN+/+) and R26ERCre;PTEN fl/fl ( PTEN−/−) UGS tissues treated with 4-OHT for 7 days during in vitro organ culture. PTEN protein is dramatically decreased while PI3K and mTOR signaling is increased in UGS specimens with inducible PTEN loss ( PTEN−/−). (C) Prostatic bud number and length are decreased by inducible PTEN loss-of-function and this effect is reversible by mTORC1 inhibition with rapamycin (n= 5–16 UGS/condition, error bars = SEM, * p<0.01; ** p < 0.0001; *** p < 0.01 using Student’s t-test for unpaired samples with unequal variance).
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Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Epithelial Cells / metabolism
Fluorescent Antibody Technique
Mechanistic Target of Rapamycin Complex 1
PTEN Phosphohydrolase / genetics
PTEN Phosphohydrolase / metabolism
Phosphatidylinositol 3-Kinases / genetics
Phosphatidylinositol 3-Kinases / metabolism*
Phosphoinositide-3 Kinase Inhibitors
Prostate / growth & development*
Proteins / antagonists & inhibitors
TOR Serine-Threonine Kinases / antagonists & inhibitors
TOR Serine-Threonine Kinases / genetics
TOR Serine-Threonine Kinases / metabolism*
Trans-Activators / antagonists & inhibitors
Trans-Activators / metabolism
Phosphoinositide-3 Kinase Inhibitors
TOR Serine-Threonine Kinases
Mechanistic Target of Rapamycin Complex 1
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