Critical role for PI3-kinase in regulating the use of proteins as an amino acid source

Roles of Growth-Factor Signaling Pathways in Supporting Cell Proliferation During Amino Acid Limitation.

It remains unclear how growth-factor signaling regulates cellular adaptation to limiting availability of extracellular amino acids. Two arms of signaling downstream of growth-factor–activated receptor tyrosine kinases have been demonstrated to promote cellular nutrient uptake: Akt, which promotes expression and surface presentation of nutrient transporters (5), and Ras, which induces macropinocytosis of extracellular macromolecules as an alternative amino acid source (12). To study consequences of activating either signaling pathway, we generated immortalized mouse embryonic fibroblasts (MEFs) that harbor an oncogenic K-Ras^G12D allele or express myristoylated Akt1 (myr-Akt1), which is constitutively active. As Akt and Ras participate in signaling pathways by which PI3-kinase transduces growth-factor signals, we also examined MEFs in which the negative PI3-kinase pathway regulator, PTEN, was deleted by CRISPR/Cas9. When placed in medium containing 10% serum and high amino acid levels (complete medium), control cells proliferated well (Fig. 1A). Expression of myr-Akt1 or deletion of PTEN did not affect serum-stimulated cell proliferation, and K-Ras^G12D expression caused a slight enhancement of growth in complete medium. When serum-stimulated in medium containing low levels of all amino acids (10% of complete medium), wild-type and K-Ras^G12D MEFs were not able to proliferate (Fig. 1B). In contrast, myr-Akt1 expression or PTEN KO enabled cells to sustain robust proliferation in low amino acid medium for several consecutive days.

Besides importing amino acids in their monomeric form, cells can also acquire amino acids through macropinocytosis and lysosomal catabolism of extracellular proteins. Of note, standard tissue culture media formulations disfavor the nutritional utilization of extracellular proteins, because compared with mammalian plasma and interstitial fluids they contain supraphysiological amino acid levels, but strongly reduced protein levels. To shift the balance of amino acid availability in favor of extracellular proteins, low amino acid medium was thus supplemented with 3% albumin to mimic its physiological concentration (2–5% in human interstitial fluids and plasma). K-Ras^G12D MEFs could not proliferate in low amino acid medium; albumin supplementation caused a striking rescue of proliferation (Fig. 1C). These results confirm the ability of activating Ras mutations to support cell proliferation that uses extracellular proteins as an amino acid source (14). Myr-Akt1 cells proliferated in low amino acid medium, but their growth was not improved by albumin addition. Growth rates of PTEN KO cells were modestly increased when albumin was supplemented at physiological levels (Fig. 1C). Albumin supplementation also allowed wild-type controls to proliferate in low amino acid medium, but only to a minor extent (Fig. S1A).

Akt Activation or PTEN Deletion Results in Increased Levels of Amino Acid Transporters.

As amino acid-deprived wild-type MEFs progressively lose viability, Akt might sustain cell proliferation during amino acid limitation through its ability to suppress apoptosis (21). To test this, we blocked apoptosis in MEFs by overexpression of antiapoptotic Bcl-xl or genetic ablation of the apoptosis mediators Bax and Bak. Over a range of amino acid concentrations, Bcl-xl overexpressing cells and controls proliferated at the same rate (Fig. S1B). Moreover, Bax/Bak deletion did not improve cell proliferation under amino acid limitation, whereas myr-Akt1 expression restored proliferation of wild-type controls (Fig. S1C). Thus, myr-Akt1 does not sustain cell proliferation in low amino acid medium by blocking cell death. Another way by which growth-factor signaling could promote cellular amino acid acquisition is the induction of plasma membrane amino acid transporters such as CD98. Indeed, myr-Akt1 expression or PTEN KO, which increased phosphorylation of the Akt target tuberous sclerosis complex 2 (TSC2), caused a significant elevation of CD98 protein levels (Fig. 2A). In contrast, K-Ras^G12D MEFs had slightly decreased CD98 levels. Previous work had shown that myr-Akt sustains cell-surface levels of CD98 in growth-factor–deprived lymphocytes in an mTORC1-dependent manner (5). To ask whether mTORC1 was required for CD98 expression in MEFs, we inhibited mTORC1 by genetic ablation of raptor or treatment with the mTOR kinase inhibitor Torin 1. Raptor knockout or Torin 1 treatment caused a strong reduction in CD98 (Fig. 2 B and C). The PI3-kinase class IA inhibitor GDC0941 and the pan-Akt inhibitor MK-2206, which moderately decreased mTORC1 activity, had less of an effect on CD98 levels. These data indicate that PTEN KO and myr-Akt1 increase CD98 levels through activation of mTORC1.

Rac1 and PLC Are Required for Extracellular Protein-Dependent Cell Proliferation.

The small GTPase Rac1 and phospholipase C (PLC) are intracellular effectors downstream of growth-factor signaling that regulate actin-driven membrane ruffling and macropinosome closure (11, 22, 23). We therefore asked whether Rac1 and PLC were required for proliferation of growth-factor–stimulated cells that depended on extracellular proteins as nutrients. We first determined requirements for Rac1 and PLC in cellular albumin uptake in response to growth-factor stimulation. Serum-deprived MEFs were incubated for 30 min with fluorescently labeled albumin and concomitantly stimulated with platelet-derived growth factor (PDGF) in the presence of the Rac1 inhibitor NSC23766 or the PLCγ inhibitor U-73122. Inhibiting either Rac1 or PLCγ caused a significant decrease in intracellular albumin (Fig. 3 A and B). Treating cells concomitantly with both inhibitors decreased albumin uptake even further. To ask if Rac1 or PLCγ inhibition affected lysosomal catabolism of internalized albumin, we used a self-quenched albumin that fluoresces upon degradation (DQ-BSA). NSC23766 and U-73122 significantly decreased DQ-BSA fluorescence, consistent with the effect of these inhibitors to suppress cellular albumin uptake (Fig. S2 A and B). While we cannot exclude the possibility that NSC23766 and U-73122 could have additional targets, these data indicate that inhibition of Rac1 and PLCγ limits ingestion of extracellular proteins, consistent with the role of these proteins in macropinosome formation.

We next asked whether Rac1- and PLC-mediated albumin uptake contributed to extracellular protein-dependent cell proliferation. To address this question, we turned to a nutritional setting where albumin is an obligatory amino acid source. When wild-type MEFs are placed in medium lacking the essential amino acid leucine, the most abundant amino acid in the mammalian proteome, they cease to proliferate and progressively lose viability, despite constant stimulation with serum-provided growth factors. Cell survival in leucine-free medium can be rescued by supplementing 3% albumin as an alternative source for leucine (14) (Fig. S2C). To examine the relevance of Rac1 and PLCγ for extracellular protein-dependent cell survival, increasing concentrations of NSC23766 or U-73122 were added to leucine-free medium + 3% albumin and cell numbers determined after 4 d. Cell viability was decreased by either inhibitor in a concentration-dependent manner and almost completely lost when both inhibitors were added together (Fig. 3C). In contrast, inhibiting Rac1 or PLCγ did not impair cell proliferation when free leucine was added to the medium (Fig. 3D). Thus, Rac1 and PLC are specifically required when cell survival depends on extracellular proteins as a source for leucine.

PI3-Kinase but Not Ras Is Required for Growth-Factor–Induced Macropinocytosis.

Oncogenic Ras variants induce macropinocytosis in mammalian cells independently of growth-factor stimulation (10, 11). However, lack of inhibitors and genetic redundancy between different Ras homologs have made it difficult to establish whether Ras was required for growth-factor–induced macropinocytosis. The recent generation of Rasless MEFs, which are devoid of all Ras proteins, allowed us to address this question (24). Inducing Cre recombinase in H-Ras, N-Ras double-knockout MEFs harboring conditional K-Ras alleles reduced Ras protein levels below detection limit within a week (Fig. 4A). Rasless MEFs had strongly diminished MAP kinase signaling, as shown by loss of Erk phosphorylation, but had normal levels of Akt and mTORC1 activity (Fig. 4A and Fig. S3A). We also introduced inducible H-Ras^G12V to compare deletion and oncogenic activation of Ras. Macromolecular uptake was assessed by internalization of fluorescently labeled high molecular weight dextran, which marks macropinosomes. To determine cell-intrinsic macropinocytic activity, inputs from growth-factor signaling were reduced by placing cells in medium containing 0.1% serum. Under these conditions, dextran uptake was almost undetectable in Rasless MEFs and controls (Fig. 4 B and C). In contrast, H-Ras^G12V MEFs displayed robust dextran uptake, consistent with the ability of oncogenic Ras mutants to trigger macropinocytosis in the absence of serum-provided growth factors. To examine growth-factor–induced macropinocytosis, dextran uptake was examined in the presence of PDGF. Stimulation with PDGF only slightly increased dextran uptake in H-Ras^G12V MEFs, presumably because it was already high in the absence of growth factors (Fig. 4 B and C). Unexpectedly, Rasless MEFs responded to PDGF stimulation with avid dextran uptake. PDGF-induced dextran uptake was consistently higher in Rasless MEFs than in controls.

We next asked whether loss of Ras signaling affected lysosomal catabolism of internalized albumin. Dequenching of DQ-BSA fluorescence was similar in Rasless MEFs and controls, while being moderately elevated in H-Ras^G12V MEFs (Fig. S3B). Moreover, Rasless MEFs and controls displayed a comparable increase in DQ-BSA degradation in response to mTOR inhibition and decrease upon treatment with lysosomal protease inhibitors (Fig. S3C). Rasless MEFs also had normal levels of autophagy induction upon amino acid starvation, as assessed by lipidation and turnover of LC3 (Fig. S3D). Levels of processed cathepsins were even slightly elevated in Rasless MEFs (Fig. S3A). Thus, complete loss of all Ras proteins does not cause any overt defects in lysosomal proteolysis. Together, these data establish that Ras GTPases are dispensable for macropinocytosis and lysosomal catabolism of albumin in response to growth-factor stimulation. Whether Ras proteins were required for the cell proliferation that uses proteins as nutrients could not be determined, because Rasless MEFs do not proliferate (24).

Besides Ras, PI3-kinase is an important regulator of macropinosome formation, and pharmacological inhibition of PI3-kinase suppresses macropinocytosis (11, 17). Moreover, PI3-kinase can activate Rac1 and PLC (25, 26). We therefore wondered whether in the absence of Ras, PDGF induced macropinocytosis through activating PI3-kinase. Indeed, pharmacological inhibition of PI3-kinase strongly decreased PDGF-induced dextran uptake in Rasless MEFs (Fig. 4 D and E) and MEFs harboring wild-type Ras proteins (Fig. 4F and Fig. S4A). Macropinocytosis induced by K-Ras^G12V expression in the absence of growth factors was also significantly decreased by PI3-kinase inhibition (Fig. 4 G and H and Fig. S4B). Thus, PI3-kinase is required for macropinocytosis that is either induced by growth factors or triggered by oncogenic Ras independently of growth-factor stimulation.

Akt Suppresses Lysosomal Catabolism of Extracellular Proteins.

The above data demonstrate that Rac1 and PLC are required when cell proliferation relies on macropinocytosis as an amino acid acquisition strategy. However, Akt is the PI3-kinase effector pathway with the best-established role in promoting nutrient acquisition and cell growth (1, 21). We therefore asked whether Akt regulated the utilization of extracellular proteins as nutrients. To address this, wild-type MEFs were placed in leucine-free medium supplemented with 3% albumin and treated with the Akt inhibitor MK-2206. Control cells maintained viability in leucine-free medium + 3% albumin, but could not proliferate. However, inhibiting Akt with MK-2206 enabled cells to proliferate over several consecutive days (Fig. 5A). Moreover, increasing MK-2206 concentrations in leucine-free medium + albumin caused cells to accumulate at progressively higher numbers (Fig. S5A). Conversely, in leucine-containing medium MK-2206 caused a concentration-dependent decrease of cell numbers (Fig. S5B). Therefore, Akt inhibition promotes serum-induced cell proliferation that relies on albumin as a source for leucine, but suppresses growth in complete medium. Concomitant addition of MK-2206 with the Rac1 inhibitor NSC23766 or the PLCγ inhibitor U-73122 blunted cell growth in leucine-free medium + 3% albumin (Fig. S5A). Thus, the ability of Akt inhibition to enhance growth that uses albumin as a source of leucine remains dependent on Rac1 and PLCγ.

We next asked whether Akt signaling suppressed albumin-dependent growth by regulating macropinocytosis or lysosomal proteolysis. Modulating Akt activity in MEFs via myr-Akt1 expression or MK-2206 treatment did not affect dextran uptake (Fig. S5 C and D). However, inhibiting Akt with MK-2206 caused a strong increase in dequenching of DQ-BSA fluorescence (Fig. 5 B and C). Thus, although Akt does not regulate macropinocytosis, it suppresses lysosomal proteolysis of internalized albumin. These phenotypes are reminiscent of previous findings that mTORC1 inhibition promotes cell proliferation that uses extracellular proteins as an amino acid source by enhancing their lysosomal catabolism. Consistently, MK-2206 and Torin 1 promoted DQ-BSA degradation to a similar extent (Fig. S5E). Besides MEFs, various cancer cell lines respond to Akt and mTOR inhibitors with increased DQ-BSA proteolysis and albumin-dependent cell proliferation (14) (Fig. S5F).

To study the effects of increased Akt signaling, we expressed wild-type Akt1 or myristoylated Akt1, Akt2, or Akt3 in K-Ras^G12D MEFs (Fig. S6A). While K-Ras^G12D MEFs are normally able to grow in leucine-free medium + 3% albumin, they ceased to proliferate and rapidly lost viability when overexpressing an activated Akt variant (Fig. 5D). In contrast, expression of Akt1 or the different myr-Akt variants did not have adverse effects on cell proliferation in leucine-containing medium (Fig. S6B). As mTORC1 is a central effector pathway of Akt signaling, we asked whether suppression of albumin-dependent cell proliferation by myr-Akt required mTORC1. Rapamycin completely rescued proliferation of K-Ras^G12D MEFs expressing myr-Akt variants in leucine-free medium + 3% albumin (Fig. 5E). Moreover, addition of MK-2206 or rapamycin alone or in combination caused a similar increase in numbers of wild-type MEFs in leucine-free medium + 3% albumin (Fig. 5F). Thus, the Akt/mTORC1 signaling axis opposes the action of other branches of PI3-kinase signaling, which promote albumin-dependent cell proliferation.

Loss of PTEN and Activating Mutations in PIK3CA Support Extracellular Protein-Dependent Cell Proliferation.

Activation of the PI3-kinase pathway by loss of the negative regulator PTEN or activating mutations in the catalytic subunit PIK3CA are highly prevalent in cancer (27). Because extracellular proteins could function as important alternative nutrients in nutrient-deprived tumor microenvironments, we determined the consequences of increased PI3-kinase signaling on extracellular protein-dependent cell proliferation. First, PI3-kinase pathway activity was increased by CRISPR/Cas9-mediated deletion of PTEN. Lentiviral delivery of three different gRNAs in wild-type MEFs resulted in robust depletion of PTEN (Fig. S7A). PTEN KO increased PDGF-induced macropinocytosis, as assessed by dextran uptake, which was sensitive to the PI3-kinase inhibitor GDC0941 (Fig. 6 A and B). Despite this, when placed in leucine-free medium + 3% albumin containing 10% serum, PTEN KO MEFs did not proliferative faster than controls (Fig. S7B). However, PTEN KO enhanced Akt signaling (Fig. S7A), which might antagonize the nutritional utilization of proteins. Thus, we asked whether PTEN KO could improve extracellular protein-dependent cell proliferation if Akt/mTORC1 signaling was suppressed. When placed in leucine-free medium + 3% albumin, serum-induced proliferation of control cells was slightly improved by rapamycin or Torin 1 (Fig. S7B). However, PTEN KO MEFs responded to mTOR inhibition with dramatically improved proliferation (Fig. S7B) and PTEN KO synergized with mTORC1 inhibition to promote albumin-dependent cell proliferation (Fig. 6 C and D). To compare how Akt/mTORC1 signaling regulated proliferation of PTEN KO MEFs when using either free amino acids or proteins, cells were placed in leucine-containing or leucine-free medium + 3% albumin and treated with MK-2206, rapamycin, or Torin 1. When free leucine was provided extracellularly, all these inhibitors suppressed serum-induced cell proliferation (Fig. 6E). In contrast, they increased cell proliferation in medium where albumin was the leucine source (Fig. 6F). Together, these data demonstrate that PTEN KO confers a growth advantage when leucine must be derived from extracellular amino acids, if the Akt/mTORC1 signaling axis is suppressed (Fig. 7A).

Lastly, we investigated the role of PI3-kinase directly. Inhibition of PI3-kinase prevented proliferation of K-Ras^G12D MEFs in leucine-free medium + 3% albumin (Fig. 7B). Thus, PI3-kinase signaling is required for extracellular protein-dependent growth, even in cells that harbor an activating Ras mutation. To determine effects of increased PI3-kinase activity, we generated MEFs expressing activated variants of PIK3CA, which were discovered in cancer genome sequencing efforts (PIK3CA^E545K and PIK3CA^H1047R) (28) as well as genetically engineered myr-PIK3CA. When placed in complete medium, cells expressing activated PIK3CA accumulated to higher numbers (Fig. 7C). Similarly, all activated PIK3CA variants increased cell numbers in leucine-free medium + 3% albumin and in response to Torin 1 treatment (Fig. 7 D and E). Thus, activating PIK3CA mutations confer a proliferative advantage to cells when leucine is provided either in its free form or when it must be recovered from extracellular proteins.

Reagents.

Antibodies were from the following suppliers: Cell Signaling (2280 Raptor, 2708 S6K1, 2920 Akt, 2983 mTOR, 3617 phospho-T1462 TCS2, 3965 pan-Ras, 4060 phospho-S473 Akt, 4308 TSC2, 4377 phospho-T389 S6K1, 9101 phospho-T202/Y204 Erk1/2, 9107 Erk1/2, 9552 PTEN, 13038 phospho-T308 Akt), R&D Systems (AF965 cathepsin B, AF1515 cathepsin L), Santa Cruz Biotechnology (CD98 M-20), and Sigma (A2228 β-actin). Secondary antibodies were from GE Healthcare (HRP-linked anti-rabbit, HRP-linked anti-mouse) and Thermo Fisher Scientific (HRP-linked anti-goat). Alexa Fluor 647 BSA, Oregon Green dextran (70 kDA), DQ Green BSA, Hoechst 33342, Lysotracker Red, and mouse PDGF-BB were from Thermo Fisher Scientific. Inhibitors were from Cayman Chemical (GDC0941, MK-2206), Cell Signaling (LY294002), EMD Chemicals (rapamycin), Selleck Chemicals (BKM-120), Sigma (bafilomycin A1, leupeptin, E-64, pepstatin A, U-73122), and Tocris Bioscience (NSC 23766, Torin 1). Amino acid starvation media were from Athena (DMEM/F12 0423) or prepared by the Memorial Sloan Kettering Cancer Center media core facility. Dialyzed FBS was from Gemini Biosystems and essential amino acids (M5550) and BSA (A1470) were from Sigma.

Cell Lines and cDNA and gRNA Constructs.

K-Ras^G12D MEFs were described in ref. 14 and Bax ^−/−; Bak ^−/− MEFs in ref. 40. K-Ras^lox; H-Ras^−/−; N-Ras^−/− MEFs were kindly provided by Barbacid and colleagues (24) and Raptor^lox MEFs by Hall and colleagues (41). Expression of CreERT2 was induced by adding 1 µM 4′-hydroxytamoxifen to culture medium, and experiments were performed after 4 d (Raptor KO) or 7–9 d (Ras KO) after induction of Cre.

Retroviral plasmids for constitutive expression of Akt variants (pBabe-Akt, myr-Akt1, myr-Akt2, and myr-Akt3) were from Addgene (42, 43). cDNAs of activated PIK3CA^E545K, PIK3CA^H1047R, and myr-PIK3CA were from Addgene (44); for inducible expression, PIK3CA variants were subcloned into a modified version of the retroviral vector pTRE-Tight (Clontech) (45). pTRE-Tight–based plasmids for inducible expression of K-Ras^G12V and H-Ras^G12V are described in ref. 14. pBabe Bcl-xl is described in ref. 46. Ras expression was induced by addition of 100 ng/mL doxycycline (Sigma) to culture medium and PIK3CA expression was induced by addition of 1 µg/mL doxycyline. gRNAs targeting PTEN were cloned into LentiCRISPR v2 (47).

Plasmids were cotransfected with lentivirus or retrovirus packaging plasmids into HEK293T cells using Lipofectamine 2000 Transfection Reagent (Life Technologies), fresh media were added after 16 h, and viral supernatants were collected 2 d after infection. Target cells were infected by addition of viral supernatant and 8 µg/mL polybrene. Twenty-four hours after infection, cells were selected with appropriate antibiotics.

Cell Culture and Nutrient Starvation Experiments.

Cell culture experiments were performed at 37 °C and 5% CO₂ in DMEM/F12 with 10% dialyzed FBS (molecular weight cutoff 10,000; Gemini Biosystems), 100 units/mL penicillin, 100 μg/mL streptomycin, 17.5 mM glucose, and 4 mM glutamine.

For proliferation assays, MEFs were plated in complete medium, 5 h later briefly rinsed, and then cultured in starvation medium as indicated. For amino acid (AA) titration experiments, AAs were supplemented at indicated percentages of 1× minimum essential medium (MEM) amino acid solution (M5550, Sigma) and 4 mM glutamine. Numbers of adherent cells were determined using a Multisizer 3 Coulter Counter (Beckman).

Western Blotting.

Cells were rinsed with ice-cold PBS and detached from culture plates by incubation with ice-cold trypsin (0.05%). Trypsin was inactivated with ice-cold serum and cells were pelleted and rinsed with PBS. Cells were lysed in ice-cold lysis buffer (50 mM Hepes, pH 7.4, 40 mM NaCl₂, 2 mM EDTA, 1 mM Na orthovandanate, 50 mM NaF, 10 mM Na pyrophosphate, 10 mM Na glycerophosphate, 1% Triton X-100, 1× Halt protease and phosphatase inhibitor mixtures; Thermo Scientific) for 15 min, and soluble lysate fractions were isolated by centrifugation at 16,000 × g for 10 min. Protein concentrations were determined with the Pierce BCA Protein Assay (Thermo Scientific) and equal amounts of proteins were analyzed by SDS gel electrophoresis and Western blotting following standard protocols.

Fluorescence Microscopy.

Fluid phase uptake of fluorescently labeled macromolecules was determined by incubating cells with 0.1 mg/mL 70 kDa dextran or 0.1 mg/mL BSA for 30 min. Subsequently, cells were washed three times with ice-cold PBS and fixed with 4% formaldehyde in PBS for 15 min. After fixation, cells were labeled for 5 min with PBS + 10 µg/mL Hoechst, then washed in PBS and imaged immediately.

For live imaging of DQ-BSA fluorescence dequenching, medium was supplemented with 0.1 mg/mL DQ Green BSA. One hour before imaging, 10 nM LysoTracker Red and 0.2 µg/mL Hoechst were added. Cells were imaged either immediately after addition of DQ-BSA or after a 6-h incubation with DQ-BSA.

Images were acquired with a Zeiss LSM 5 LIVE or a Zeiss LSM 880 confocal microscope. Cellular uptake/degradation of fluorescently labeled dextran or albumin was quantified using the particle analyzer function of Fiji (48). Mean fluorescence intensity was determined by calculating the integrated signal density per cell in randomly chosen fields of view. Due to large size differences between MEFs deficient for Ras or expressing activated Ras, integrated signal density was normalized by cell area in experiments concerning Rasless MEFs.

Data Analysis.

Graphs were created using GraphPad Prism software, and statistical analysis was performed with GraphPad Prism. No statistical method was used to predetermine sample size.