The Akt pathway is a well-known promoter of tumor malignancy. Akt3 is expressed as two alternatively spliced variants, one of which lacks the key regulatory serine 472 phosphorylation site. Whereas the function of full-length Akt3 isoform (Akt3/+S472) is well-characterized, that of Akt3/−S472 isoform remains unknown. Despite being expressed at a substantially lower level than Akt3/+S472 in triple-negative breast cancer cells, specific ablation of Akt3/−S472 enhanced, whereas overexpression, suppressed mammary tumor growth, consistent with a significant association with patient survival duration relative to Akt3/+S472. These effects were due to striking induction of apoptosis, which was mediated by Bim upregulation, leading to conformational activation of Bax and caspase-3 processing. Bim accumulation was caused by marked endocytosis of EGF receptors with concomitant ERK attenuation, which stabilizes BIM. These findings demonstrate an unexpected function of an endogenously expressed Akt isoform in promoting, as opposed to suppressing, apoptosis, underscoring that Akt isoforms may exert dissonant functions in malignancy.

Significance: These results illuminate an unexpected function for an endogenously expressed Akt isoform in promoting apoptosis, underscoring the likelihood that different Akt isoforms exert distinct functions in human cancer. Cancer Res; 78(1); 103–14. ©2017 AACR.

Akt signaling is frequently deregulated in cancer due to PI3K pathway activation by PIK3CA, PTEN or Akt pleckstrin homology (PH) domain mutations (1, 2). PI3K activation results in Akt phosphorylation leading to transcriptional inhibition of apoptosis genes. Akts phosphorylate a plethora of substrates including GSK3, S6K, mTOR, and FOXOs, resulting in cell proliferation, migration, metabolism, and survival (1, 2, 3). In addition, Akts phosphorylate and inactivate Bad or the Forkhead transcription factors of the FOXO family (1, 4), thus causing FOXO's interaction with 14-3-3 proteins, leading to nuclear exclusion. If Akt signaling is switched off by attenuation of growth factor signaling, FOXOs are retained in the nucleus where they promote transcription of apoptosis genes (4). These include the Fas ligand and the proapoptotic Bcl-2 family member, Bim (5). Bim promotes apoptosis by triggering the conformational activation of BAX, resulting ultimately in pore formation in the mitochondrial outer membrane with the release of cytochrome c to the cytoplasm, caspase activation, and cell death (6, 7).

The Akt kinase family is comprised of three isoforms Akt1, Akt2, and Akt3, which are encoded by distinct genes located on separate chromosomes (8, 9, 10). Akt isoforms contain an N-terminal PH domain, a central kinase domain, which harbors a threonine phosphorylation site in the activation loop, and a C-terminal hydrophobic domain, which contains the serine regulatory phosphorylation site (9, 11). In response to insulin or growth factor signaling, PI3K phosphorylates the membrane associated phosphatidyl (4, 5) -bisphosphate (PIP2) to generate phosphatidyl (3, 4, 5) -trisphosphate (PIP3), which binds the PH domain and recruits Akt to the plasma membrane, where it is phosphorylated on threonine 308 in the kinase activation loop by PDK1, and subsequently on serine 473 by mTORC2 (1, 2). Despite similar effects on cell growth and survival, Akt isoforms exert distinct and even opposite functions on the malignant phenotype. Akt1 and Akt2 may have opposing effects on cell motility, which is inhibited by Akt1 and stimulated by Akt2 (12–17). The conflicting effects of Akt isoforms may explain the failure of clinical trials using pan-Akt inhibitors, which might be otherwise overcome by selective Akt isoform–based therapy (18).

Akt3 was shown to drive the growth of melanomas, ovarian, lung, prostate, and triple-negative breast cancer (TNBC) cell lines (3, 19–22). Mining The Cancer Genome Atlas (TCGA) breast cancer database showed an upregulation of Akt3 mRNA in 28% of triple-negative breast carcinomas (23). Akt3 knockdown in some TNBC cell lines was found to attenuate tumor growth, which was associated with increases in p27KIP1 levels (22). Akt3 was also shown to regulate lung carcinoma cell proliferation and invasion by phosphorylating IWS1, a splicing factor that converts epithelial FGFR2 IIIB into mesenchymal FGFR2 IIIC isoform (3). In contrast, we and others showed that Akt3 inhibits mammary and vascular tumor cell motility, while having no effect on cell growth, thus underscoring the conflicting effects of Akt3 on tumor cell behavior (24, 25).

A major feature distinguishing Akt3 from the otherwise similar Akt1 and Akt2, is that Akt3 is encoded by a gene that gives rise to two almost identical variants via differential splicing of C-terminal exons (9). Whereas the Ser472 phosphorylation site, encoded by exon 13, is present in the full-length Akt3 isoform (Akt3/+S472), it is absent from the second isoform (Akt3/−S472). This isoform excludes exon 13, and instead, encodes at its C-terminus exons 14 and 15 (9). Akt3 is phosphorylated on Thr305, thus creating a conformational change leading to phosphorylation on Ser472, a step necessary for full kinase activation, a process common to all Akts (1, 2).

Here, we show that Akt3/-S472 isoform exerts powerful suppression on mammary tumorigenesis. CRISPR-mediated knockout of Akt3/-S472 in TNBC cells enhanced, whereas overexpression suppressed, mammary tumor growth. By contrast, Akt3/+S472 isoform had no effect on malignant cell growth. Mechanistically, the suppressive effect of Akt3/−S472 was due to dramatic induction of apoptosis that was mediated by increases in Bim abundance, leading to Bax and caspase-3 activation. Increases in Bim expression were, in turn, caused by ERK attenuation due to marked EGFR endocytosis.

Animals

All procedures involving mice were conducted in accordance with NIH regulations concerning the use and care of experimental animals. This study was approved by an Animal Protocol reviewed by the Albert Einstein College of Medicine Animal Use Committee.

Cell Culture

Breast cancer cell lines were purchased from the ATCC and maintained in DMEM (Invitrogen) supplemented with 10% FBS and 1% penicillin/streptomycin (Invitrogen). These include MDA-MB-231, 3475, MDA-MB-436, Hs578T, except for SUM149 that was cultured in DMEM-F12 with 5% FBS 1% penicillin/streptomycin, 5 μg/mL insulin/1 μg/mL hydrocortisone. The 3475 cell line is a lung metastatic MDA-MB-231 subline line that was obtained from Dr. Joan Massagué (Memorial Sloan Kettering Cancer Center, New York, NY). SUM 149 was obtained from Asterand Bioscience.

Antibodies and reagents

Anti Akt3 antibody was from Millipore or ProSci. β-Actin antibody was from Sigma. Antibodies against Akt1, Akt2, Bcl-2, Bim, Bid, Puma, cleaved caspase-3 or PARP, p-ERK, p-Akt S473, p-Akt T308, p-p53, were from Cell Signaling Technology. Bad, Bax, active Bax (6A7) were from Abcam. Anti-ERK, EGFR or EEA1 antibodies were from Santa Cruz Biotechnology. EGF or IGF were from PeproTech. PD 0325901 was from Tocris Bioscience. Drugs were dissolved in DMSO to yield a 1 mmol/L stock solution and stored at −80°.

Subcloning and production of lentiviral particles of hAkt3 isoforms

To subclone the hAkt3 splice variant 1 (Akt3/-S472) into lentiviral expression vector pLVX (Clontech), PCR was performed using 5′-forward: 5′-TCGCTCGAGATGAGCGATGTTACCATTGTG-3′ and 3′ reverse: 5′-CCCTCTAGATTATTTTTT CCAGTTACCCAGC-3′ primers using hAkt3-variant 1 plasmid from Origene as template. Using Xho1/Xba1 restriction sites hAkt3-variant 1 was subcloned into lentiviral vector expression vector pLVX-puro (Clontech). Akt3/+S472 plasmid (HA-hAkt3-WT-pcDNA3) was a kind gift from Dr. Yiling Lu (MD Anderson Cancer Center, Houston, TX). HA-hAkt3-WT clone was transferred from this construct into lentiviral pLVX-puro plasmid, using these primers 5′-GGGCTCGAGATGTACCCATACGATGTTCCAGATT ACGCT-3′ and 3′ region primer: 5′-GGGTCTAGATTATTCTCGTCCACTTGCAGA-3′. Lentiviral particles generated from these constructs were infected into cells. Other constructs include constitutively-active MEK1 retroviral vector, obtained from Dr. Stuart Aaronson at Mount Sinai School of Medicine (New York, NY).

Akt3 isoform CRISPR-Cas9 design and construction

To silence Akt3/+S472 or Akt3/−S472 isoform expression, we performed CRISPR-Cas9 (GeCKO) of human PKB gamma (hAkt3-WT) by targeting exon 13 or 14 respectively using the MIT CRISPR server (http://crispr.mit.edu/). As control, a non-targeting sequence was used (see Supplementary Methods).

Genomic validation of CRISPR-Cas9–mediated Akt3 knockout

To validate the genomic deletion induced by CRISPR-Cas9 to knock out the Akt3 isoforms, we designed primers flanking the gRNA recognition site and used Sanger sequencing as well as Deep Sequencing analysis (see Supplementary Methods).

TaqMan qRT-PCR

Total RNA was extracted from cell lines and mammary tumors using RNeasy Mini Kit and RNase-free DNase set (Qiagen). Real-time RT-PCR was carried out using TaqMan RNA-to-Ct 1-Step Kit and gene-specific TaqMan probes on a StepOnePlus Real-time PCR system (Applied Biosystems; see Supplementary Methods).

TCGA-based Kaplan–Meier curves

RNASeqv2 level 3 RSEM isoform expression and clinical data were downloaded from TCGA Firehose (Broad Institute TCGA Genome Data Analysis Center (2014): Firehose stddata 2014 06 14 run, Broad Institute of MIT and Harvard. doi:10.7908/C1ZC821V). Invasive ductal carcinoma samples were selected and expression of isoforms uc001hzz.1 and uc001iab.1 used to identify Akt3/-S472 and Akt3/+S472, respectively. SPSS was used to perform Kaplan–Meier Analysis.

RNA extraction from FFPE TNBC specimens

TNBC formalin-fixed and paraffin-embedded (FFPE) tumors were selected by Dr. Susan Fineberg, following approval of the IRB protocol by the Committee of Clinical Investigation of the Albert Einstein College of Medicine (IRB# 2010-366). Ten unstained 10-μm sections from each tumor were microdissected using a pathologically reviewed duplicate hematoxylin and eosin (H&E) section as guide. RNA from microdissected carcinoma tissue, was analyzed and quantified on an Agilent 2100 Bioanalyzer. Akt3 isoform mRNA expression was measured by TaqMan gene expression custom assays and Taqman RNA-to-CT 1-Step Kit, with 50 ng RNA in triplicates. Analysis was performed per the 2(ΔΔCt) (Loudig and colleagues; 2011).

Anoikis assay

Cell monolayers were trypsinized and single cells replated in suspension in DMEM containing 0.5% methylcellulose solution/0.5% FBS on petri dishes coated with 1% agarose, which kept cells in suspension. Cells were incubated for 48 hours in incubator, harvested, spun down, lysed.

TUNEL assay

TUNEL assay was performed using the In Situ Cell Death Detection Kit (Roche). Briefly, after deparaffinization, rehydration, and antigen unmasking, tumor sections were incubated with proteinase-K for 15 minutes at room temperature. Sections were incubated with TUNEL reaction buffer for 1 hour at 37°C, washed three times in Tris buffer for 5 minutes and mounted with media containing DAPI to visualize nuclei. Images were taken by Zeiss Axioskop 2 microscope (Zeiss).

Akt nonradioactive kinase assay

We used the nonradioactive kinase assay kit (Cell Signaling Technology). Cell lysate (0.5-mg protein) was incubated overnight at 4°C with rabbit monoclonal anti phospho-Akt (Ser473) beads conjugate. Beads were washed in lysis buffer and resuspended in kinase buffer including 0.2 mmol/L ATP and 0.5 μg of kinase substrate (GST fusion protein of GSK-3α/β peptide containing the Ser21/9 residue). Beads were incubated for 30 minutes at 30°C; reaction stopped with 25 μL 3× SDS sample buffer and boiling. Bead supernatant was analyzed by SDS-PAGE and immunoblotting using anti phospho-GSK-3α/β (Ser21/9).

EGFR internalization assay using cell surface biotinylation

Cell monolayers were incubated with 100 ng/mL EGF in culture medium at 37°C for various intervals to allow EGFR internalization. Cells were then treated Sulfo-NHS-Biotin (0.5 mg/mL, Thermo Fisher Scientific) for 30 minutes on ice. Excess biotin was quenched with 50 mmol/L NH4Cl in ice-cold PBS-CM (calcium and magnesium). Cells were scrapped, and lysed in RIPA buffer (25 mmol/L Tris-HCL, pH 7.4, with 150 mmol/L NaCL, 0.1% SDS, 1% Triton X-100 supplemented with protease and phosphatases inhibitors. Cell extracts were centrifuged, and the supernatants were incubated with streptavidin beads (Sigma) to collect the biotinylated proteins. After washing the beads, bound proteins were eluted by boiling at 95°C for 10 minutes in 2× sample buffer and analyzed by immunoblotting with anti-EGFR antibody.

All additional procedures, including immunoblotting, immunoprecipitation, immunostaining, tumor formation, and lung metastasis are included as methods in Supplementary Information.

Akt3 isoform expression in TNBC cell lines and tumors

Akt3 was shown to be expressed as two isoforms, which interestingly differ in the carboxyl terminal Ser472 regulatory phosphorylation site, that is present in the full-length Akt3 (Akt3/+S472), whilst absent in the second isoform (Akt3/−S472; ref. 9). In the mammary gland, Akt3/-S472 splice variant comprises approximately 5% of all Akt3 transcripts, implying a low abundance relative to Akt3/+S472 (9). qRT-PCR analysis of six clinical TNBCs using Akt3 isoform specific TaqMan primers revealed lower expression of Akt3/−S472 compared with Akt3/+S472 mRNA in these tumors (Fig. 1A). Differential isoform expression was also observed in TNBC cell lines such as HS578T, MDA-MB-231, 3475 (MDA-MB-231 metastatic subline), MDA-MB-436 and SUM149, where the expression of Akt3/-S472 mRNA was substantially reduced relative to Akt3/+S472, likely due to upregulation Akt3/+S472 in cultured cell lines (Fig. 1B). Furthermore, TCGA mining of invasive duct carcinomas, pointed to a more significant association of Akt3/−S472 mRNA (P = 0.08) relative to Akt3/+S472 (P = 0.99), with patient survival duration (Fig. 1C and D), implying this isoform may negate malignancy.

Figure 1.

Akt3 isoform expression in TNBC specimens and cell lines. A, Quantitative PCR evaluation of Akt3 isoforms in human TNBC. RNA from 6 FFPE TNBCs was evaluated for expression of Akt3/+S472 or Akt3/−S472 mRNA by TaqMan qPCR analysis using Akt3 isoform-specific primers and GAPDH as endogenous control for normalization. Relative isoform expression was obtained by subtracting the mean comparative threshold (Ct; triplicates) obtained for GAPDH from the mean Ct value of the isoform of interest, to generate the ΔCt value. The ΔΔCt value was obtained by subtracting the ΔCt of a sample of interest from the ΔCt value of the Akt3/+S472 from the first sample (value of 0) and the fold change was estimated by the 2(ΔΔCt) formula, shown as black bars for Akt3/+S472 and gray bars for Akt3/−S472. The SD from triplicate experiments was used to generate error bars. B, RNA extracted from Hs578T, MDA-MB-231, 3475, MDA-MB-436, and SUM149 TNBC cell lines was quantified for the expression of Akt3/+S472 and Akt3/−S472 mRNA, as above. The expression is displayed as fold change, following the formula 2(ΔCt Akt3/+S472Hs578T- ΔCtcell line), where the expression of both isoforms in cell lines was compared with the expression of Akt3/+S472 mRNA in Hs578T cells. C–D, TCGA-based Kaplan–Meier survival curves for patients with invasive ductal carcinoma expressing Akt3/−S472 mRNA (P = 0.08; C) or Akt3/+S472 mRNA (P = 0.99; D) comparing the top quartile (solid line; N = 187) versus remaining patients (dashed line; N = 563).

Figure 1.

Akt3 isoform expression in TNBC specimens and cell lines. A, Quantitative PCR evaluation of Akt3 isoforms in human TNBC. RNA from 6 FFPE TNBCs was evaluated for expression of Akt3/+S472 or Akt3/−S472 mRNA by TaqMan qPCR analysis using Akt3 isoform-specific primers and GAPDH as endogenous control for normalization. Relative isoform expression was obtained by subtracting the mean comparative threshold (Ct; triplicates) obtained for GAPDH from the mean Ct value of the isoform of interest, to generate the ΔCt value. The ΔΔCt value was obtained by subtracting the ΔCt of a sample of interest from the ΔCt value of the Akt3/+S472 from the first sample (value of 0) and the fold change was estimated by the 2(ΔΔCt) formula, shown as black bars for Akt3/+S472 and gray bars for Akt3/−S472. The SD from triplicate experiments was used to generate error bars. B, RNA extracted from Hs578T, MDA-MB-231, 3475, MDA-MB-436, and SUM149 TNBC cell lines was quantified for the expression of Akt3/+S472 and Akt3/−S472 mRNA, as above. The expression is displayed as fold change, following the formula 2(ΔCt Akt3/+S472Hs578T- ΔCtcell line), where the expression of both isoforms in cell lines was compared with the expression of Akt3/+S472 mRNA in Hs578T cells. C–D, TCGA-based Kaplan–Meier survival curves for patients with invasive ductal carcinoma expressing Akt3/−S472 mRNA (P = 0.08; C) or Akt3/+S472 mRNA (P = 0.99; D) comparing the top quartile (solid line; N = 187) versus remaining patients (dashed line; N = 563).

Close modal

CRISPR-Cas9–mediated knockout of Akt3/-S472 in TNBC cells enhances mammary tumor growth

To investigate the role of Akt3 isoforms in mammary tumorigenesis, we under or overexpressed each of the isoforms in MDA-MB-231 and 3475 breast cancer cell lines, which are endowed with tumorigenic and metastatic potential (26). Although Akt3/-S472 mRNA was weakly expressed in these cell lines, we tested whether silencing this isoform affects the tumor phenotype. We used the CRISPR-Cas9 technology to knock out endogenous expression of the two Akt3 splice variants (27, 28). The CRISPR-Cas9 genome deletion technique introduces a double-strand break, which leads to DNA repair by introducing small insertions or deletions (indels) at the target region. Small guide RNA (gRNA) sequences targeting Akt3/+S472 on exon 13 or Akt3/−S472 on exon 14, were generated using the CRISPR gRNA design tool at MIT, that excludes off-target genome modification. Twenty nucleotides gRNA sequence for each of the Akt3 isoforms was cloned into Cas9 containing lentiviral constructs that were used for virus production and expression into cancer cells. To validate the targeting efficiency of CRISPR-Cas9, we measured alterations in genomic DNA from bulk cancer cell populations by next-generation sequencing. A 200-bp amplicon spanning the gRNA-binding site on the targeted locus was subjected to multiple sequence alignment to validate CRISPR-Cas9 induced mutations and knockout efficiency in the bulk tumor cell population. Our data confirmed mutations were located close to the targeted sequence in 86% of Akt3/+S472 and 77% of Akt3/−S472 knockout 3475 cell populations (Supplementary Fig. S1A and S1B). In contrast, control 3475 cells showed no modifications in the nucleotide sequence of either isoform, confirming that genomic locus was unaffected by the nontargeting CRISPR sequence (Supplementary Fig. S1C and S1D). Thus, next-generation sequencing of amplicon obtained around the gRNA recognition site clearly shows that Akt3-targeting CRISPR-Cas9 constructs were capable of introducing deletion or insertion (indels) at the genomic locus with high efficiency, thereby rendering defective Akt3 isoform protein.

Limitation in protein detection due to lack of antibody that discriminates the two highly homologous Akt3 splice variants, prompted us to perform TaqMan-based qRT-PCR using isoform-specific primers to confirm the CRISPR-mediated knockout. Akt3/-S472 KO cells exhibited a 4-fold reduction in mRNA relative to control cells, compared with unchanged expression of Akt3/+S472 mRNA (Fig. 2A). Similarly, knockout of Akt3/+S472 led to a 2.2-fold reduction in mRNA, relative to unchanged Akt3/−S472 mRNA (Fig. 2B), implying CRISPR-mediated knockout of Akt3 isoform led to impaired transcript expression.

Figure 2.

CRISPR-mediated knockout of Akt3/−S472 in 3475 TNBC cells increases mammary tumor growth. A and B, RNA extracted from 3475 cells expressing a nontargeting CRISPR sequence (control), CRISPR-Cas9 knockout (KO) lentiviral constructs of Akt3/−S472 or Akt3/+S472 (x-axis), were each tested in triplicates for mRNA expression of Akt3/−S472 (A) or Akt3/+S472 (y-axis; B) by qRT-PCR. C, 3475-Akt3/−S472 KO and 3475-Akt3/+S472 KO cells were compared with control 3475 cells for cell growth rates in vitro. Cells were plated in triplicate wells and subjected for 6 days to cell growth analysis using the WST-1 assay. Results are mean ± SEM from triplicate experiments; P < 0.05. D, Control 3475, 3475-Akt3/−S472 KO and 3475-Akt3/+S472 KO, cells were each injected into the mammary fat pads of 6-female athymic nude mice in duplicate experiments (N = 12). Tumor growth was monitored over 60 days, and final tumor volume was calculated as mean± SEM. Unpaired t test showed significant differences between control and Akt3/−S472 KO tumors (P < 0.05), but not between control and Akt3/+S472 KO tumors (P > 0.05). E and F, RNA from control (E) and Akt3/−S472 KO tumors, or control and Akt3/+S472 KO tumors (N = 3; x-axis; F), was analyzed by qRT-PCR for expression of indicated isoform (y-axis). Results are mean ± SEM; unpaired t test; P < 0.05. **, P < 0.01; ****, P < 0.0001; ns, nonsignificant.

Figure 2.

CRISPR-mediated knockout of Akt3/−S472 in 3475 TNBC cells increases mammary tumor growth. A and B, RNA extracted from 3475 cells expressing a nontargeting CRISPR sequence (control), CRISPR-Cas9 knockout (KO) lentiviral constructs of Akt3/−S472 or Akt3/+S472 (x-axis), were each tested in triplicates for mRNA expression of Akt3/−S472 (A) or Akt3/+S472 (y-axis; B) by qRT-PCR. C, 3475-Akt3/−S472 KO and 3475-Akt3/+S472 KO cells were compared with control 3475 cells for cell growth rates in vitro. Cells were plated in triplicate wells and subjected for 6 days to cell growth analysis using the WST-1 assay. Results are mean ± SEM from triplicate experiments; P < 0.05. D, Control 3475, 3475-Akt3/−S472 KO and 3475-Akt3/+S472 KO, cells were each injected into the mammary fat pads of 6-female athymic nude mice in duplicate experiments (N = 12). Tumor growth was monitored over 60 days, and final tumor volume was calculated as mean± SEM. Unpaired t test showed significant differences between control and Akt3/−S472 KO tumors (P < 0.05), but not between control and Akt3/+S472 KO tumors (P > 0.05). E and F, RNA from control (E) and Akt3/−S472 KO tumors, or control and Akt3/+S472 KO tumors (N = 3; x-axis; F), was analyzed by qRT-PCR for expression of indicated isoform (y-axis). Results are mean ± SEM; unpaired t test; P < 0.05. **, P < 0.01; ****, P < 0.0001; ns, nonsignificant.

Close modal

The effect of Akt3 isoform deletion on tumor cell growth in vitro was evaluated by comparing cell growth kinetics of control and isoform-deficient cells over 5 days in culture; but no differences were noted between the two groups and controls, implying Akt3 isoforms do not regulate cell proliferation in vitro (Fig. 2C). However, evaluation of tumor growth, 60 days postinjection of cancer cells into mammary fat pads of athymic nude mice, revealed a striking difference between the isoforms. Namely, Akt3/−S472 ablation in 3475 cells caused a 2.5-fold increase in mammary tumor volume as compared with control 3475 cells (P < 0.05; Fig. 2D). In contrast, Akt3/+S472 knockout had no effect, as shown by similar sizes of 3475-Akt3/+S472 KO and 3475 control tumors (Fig. 2D). Given the polyclonal nature of the CRISPR-ed cells, we measured whether Akt3 isoform knockout was sustained throughout tumor growth using qRT-PCR analysis. Indeed, Akt3/-S472 KO or Akt3/+S472 KO tumors maintained very low levels of the respective isoform (Fig. 2 E and F). Furthermore, deep sequencing analysis of tumors using genomic DNA, showed multiple indel events occurring near the targeted sequence on several thousands of sequencing reads in 91% of Akt3/+S472 KO and 96% of Akt3/−S472 KO tumors (Supplementary Fig. S2A and S2B). As expected, control tumors exhibited 100% matching of multiple reads with NCBI reference sequence of Akt3/+S472 or Akt3/−S472, with no indels on any of the sequences (Supplementary Fig. S2C and S2D).

Finally, these findings were reproduced in the MDA-MB-231 parental cell line, which is heterogeneous compared with the clonal 3475 subline (Supplementary Fig. S3). Akt3/−S472 or Akt3/+S472 CRISPR-mediated knockout in MDA-MB-231 cells led to a significant reduction in isoform mRNA (Supplementary Fig. S3A–S3B). However, while Akt3/-S472 knockout did not have any effect on cell growth in culture (Supplementary Fig. S3C), it caused a 2-fold increase in tumor burden in vivo (Supplementary Fig. S3D). In contrast, Akt3/+S472 ablation in these cells did not inhibit tumor cell growth in vitro or in vivo (Supplementary Fig. S3C–S3D). Of, note mammary tumors resulting from knockout cells maintained low levels of isoform expression, as indicated by substantially reduced levels of Akt3/−S472 or Akt3/+S472 mRNA (Supplementary Fig. S3E–S3F). These data underscore a robust tumor suppressive effect of Akt3/−S472, especially in light of low basal expression of this spliced variant relative to Akt3/+S472.

Akt3/−S472 overexpression in TNBC cells suppresses the malignant phenotype

To confirm the suppressive effect of Akt3/−S472 on mammary tumorigenesis, this isoform was overexpressed in 3475 cells. Akt3/−S472 overexpression did not affect Akt1 or Akt2 expression, and had interestingly no effect on cell growth in vitro (Fig. 3A and B). However, orthotopic injection of tumor cells into athymic nude mice, revealed a dramatic reduction in mammary tumor volume in mice inoculated with Akt3/−S472–overexpressing 3475 cells relative to 3475 controls (Fig. 3C). This was consistent with the tumor-enhancing effect of Akt3/−S472 knockout (see Fig. 2D). By contrast, Akt3/+S472 overexpression in 3475 cells did not influence tumor growth (Fig. 3D; Supplementary Fig. S4A). As control, overexpression of the prosurvival Akt1 in 3475 cells, enhanced tumor burden (Fig. 3E; Supplementary Fig. S4B).

Figure 3.

Akt3/−S472 overexpression in 3475 TNBC cells suppresses mammary tumor growth and metastasis. A, Akt3/−S472 or control lentiviral construct was expressed in 3475 cells; cell lysates were immunoblotted with anti Akt3, Akt1, Akt2, or β-actin antibody. B, Control and Akt3/−S472 overexpressing 3475 cells (OE) were plated at 5 × 104 in 6-well plates and counted over 6 days. Results are shown as mean ± SEM from triplicate experiments. C, Control 3475 cells or 3475-Akt3/−S472 OE cells were each implanted into mammary glands of athymic nude mice at 1 × 106 cells per site in two sites in 6 mice, each in duplicate experiments (N = 12). Comparisons of tumor growth (volume) over 30 days (mean ± SEM) showed significant pair-wised differences between control and 3475-Akt3/−S472 OE cells; unpaired t test (****, P < 0.0001). D and E, Control 3475 and 3475-Akt3/+S472 OE cells (D) or control 3475 and 3475-Akt1 OE cells (E) were implanted into mammary fat pads of athymic nude mice at 1 × 106 cells in 5 to 10 mice. Tumor volumes (mean ± SEM), over 30 days, showed no difference between control and Akt3/+S472 OE tumors (*, P > 0.05), but a significant increase was observed between control and Akt1 OE tumors. ***, P < 0.001; unpaired t test. F, To evaluate metastasis from the orthotopic site, mammary tumors were surgically removed when the tumor reached 1 cm. Mice were then incubated for 14 weeks; tumor foci in 6 to 12 mouse lungs were counted. G and H, Lung colonization (G), 5 weeks post tail vein injection of 1 × 106 control 3475 or 3475-Ak3/−S472 OE cells into 6-12 athymic nude mice, was measured in H&E-stained lung sections (H). The number and distribution of foci produced by orthotopic or intravenous injection showed significant differences between control and 3475-Akt3/−S472 OE cells (Wilcoxon Rank Sum test; P < 0.05).

Figure 3.

Akt3/−S472 overexpression in 3475 TNBC cells suppresses mammary tumor growth and metastasis. A, Akt3/−S472 or control lentiviral construct was expressed in 3475 cells; cell lysates were immunoblotted with anti Akt3, Akt1, Akt2, or β-actin antibody. B, Control and Akt3/−S472 overexpressing 3475 cells (OE) were plated at 5 × 104 in 6-well plates and counted over 6 days. Results are shown as mean ± SEM from triplicate experiments. C, Control 3475 cells or 3475-Akt3/−S472 OE cells were each implanted into mammary glands of athymic nude mice at 1 × 106 cells per site in two sites in 6 mice, each in duplicate experiments (N = 12). Comparisons of tumor growth (volume) over 30 days (mean ± SEM) showed significant pair-wised differences between control and 3475-Akt3/−S472 OE cells; unpaired t test (****, P < 0.0001). D and E, Control 3475 and 3475-Akt3/+S472 OE cells (D) or control 3475 and 3475-Akt1 OE cells (E) were implanted into mammary fat pads of athymic nude mice at 1 × 106 cells in 5 to 10 mice. Tumor volumes (mean ± SEM), over 30 days, showed no difference between control and Akt3/+S472 OE tumors (*, P > 0.05), but a significant increase was observed between control and Akt1 OE tumors. ***, P < 0.001; unpaired t test. F, To evaluate metastasis from the orthotopic site, mammary tumors were surgically removed when the tumor reached 1 cm. Mice were then incubated for 14 weeks; tumor foci in 6 to 12 mouse lungs were counted. G and H, Lung colonization (G), 5 weeks post tail vein injection of 1 × 106 control 3475 or 3475-Ak3/−S472 OE cells into 6-12 athymic nude mice, was measured in H&E-stained lung sections (H). The number and distribution of foci produced by orthotopic or intravenous injection showed significant differences between control and 3475-Akt3/−S472 OE cells (Wilcoxon Rank Sum test; P < 0.05).

Close modal

Limitations in primary tumor growth do not necessarily impair metastasis, which is dependent on invasiveness, that may not be inhibited by Akt3/−S472. We therefore measured the effect of Akt3/−S472 on distant metastasis, 4 months following surgical removal of the primary tumor, to mimic the clinical setting. In addition, we measured lung colonization following intravenous injection of tumor cells, which is independent of primary tumor growth. In both instances, metastasis burden, as gauged by the number and distribution of pulmonary foci, was dramatically reduced in mice inoculated with 3475-Akt3/-S472 OE cells relative to controls (rank-sum analysis; P < 0.001; Fig. 3F–H). These findings demonstrate that Akt3/−S472 suppresses the tumor phenotype by inhibiting primary and metastatic tumor growth.

Akt3/−S472 suppresses mammary tumorigenesis by stimulating apoptosis

The mechanism whereby Akt3/−S472 inhibits tumor growth remained to be addressed. Although Akt3/−S472 had no effect on cell growth in vitro, it suppressed tumor burden in vivo (see Fig. 3B and C). To rule out effects on tumor cell proliferation in vivo, we measured Ki67 staining in mammary carcinomas, which showed no difference between 3475-Akt3/-S472 OE and control tumors (Fig. 4A). Because the balance between cell proliferation and cell death regulates tumor size, we measured apoptosis in non-necrotic carcinoma areas. This revealed a substantial increase in TUNEL staining in 3475-Akt3/-S472 OE tumors relative to controls (Fig. 4B). Consistent with this effect, CRISPR-mediated knockout of Akt3/−S472 in 3475 cells, resulted in mammary tumors expressing lower TUNEL activity, yet similar levels of Ki67 staining relative to control carcinomas (Supplementary Fig. S4C–S4D). By contrast, Akt3/+S472 or Akt1-overexpressing tumors did not exhibit any change in TUNEL staining relative to controls (Supplementary Fig. S4E–S4F).

Figure 4.

Akt3/−S472 overexpression in 3475 TNBC cells promotes apoptosis via Bim upregulation and Bax activation in vivo. A, Control and 3475-Akt3/−S472 OE tumors were immunostained with anti-Ki67 antibody using TRITC secondary detection and DAPI. The number of Ki67-positive cells was determined in each tumor by counting 6 (×40) microscopic fields in nonduplicating sections from 5 individual tumors, expressed as the percentage of positive cells as mean ± SEM; P > 0.05. B, Control 3475 and 3475-Akt3/−S472 OE mammary tumors (N = 5) were stained for TUNEL. The percentage of TUNEL-positive tumor cells is shown as mean ± SEM; two-tailed t test; ****, P < 0.0001. C, Lysates from control and 3475-Akt3/−S472 OE tumors (N = 4) were immunoblotted for Akt3, cleaved caspase-3 or PARP, Bim, Bax, Akt1, Akt2, β-Actin. D, Bim mRNA from control 3475 and 3475-Akt3/−S472 OE tumors was measured by qRT-PCR. Data are shown as mean ± SEM (N = 4); unpaired t test, *, P < 0.05. E, Control and 3475-Akt3/−S472 OE tumor lysates were immunoblotted for Bid, Bad, Puma, p-p53, or BCL-xL expression, followed by densitometry of immunoblots. Bar graphs represent values of expression in 3475-Akt3/−S472 OE relative to control 3475 tumors for the indicated proteins in four independent tumors; results are expressed as mean ± SEM. Unpaired t test shows significant differences for Bim (**, P < 0.01) or Bid (*, P < 0.05) expression, but not for other indicated proteins. F, Lysates from control 3475 or 3475-Akt3/−S472 OE tumors were either immunoblotted with anti Bax (Input; lanes 1, 2), immunoprecipitated with control IgG (lanes 3, 4), or with antiactive-Bax (6A7) antibody (lanes 5, 6), followed by anti-Bax immunoblotting. G, Lysates from control and 3475-Akt3/−S472 OE cells grown in suspension cultures, in low serum media for 48 hours to mimic anoikis, were immunoblotted for Akt3, cleaved-PARP, Bim, or β-Actin expression.

Figure 4.

Akt3/−S472 overexpression in 3475 TNBC cells promotes apoptosis via Bim upregulation and Bax activation in vivo. A, Control and 3475-Akt3/−S472 OE tumors were immunostained with anti-Ki67 antibody using TRITC secondary detection and DAPI. The number of Ki67-positive cells was determined in each tumor by counting 6 (×40) microscopic fields in nonduplicating sections from 5 individual tumors, expressed as the percentage of positive cells as mean ± SEM; P > 0.05. B, Control 3475 and 3475-Akt3/−S472 OE mammary tumors (N = 5) were stained for TUNEL. The percentage of TUNEL-positive tumor cells is shown as mean ± SEM; two-tailed t test; ****, P < 0.0001. C, Lysates from control and 3475-Akt3/−S472 OE tumors (N = 4) were immunoblotted for Akt3, cleaved caspase-3 or PARP, Bim, Bax, Akt1, Akt2, β-Actin. D, Bim mRNA from control 3475 and 3475-Akt3/−S472 OE tumors was measured by qRT-PCR. Data are shown as mean ± SEM (N = 4); unpaired t test, *, P < 0.05. E, Control and 3475-Akt3/−S472 OE tumor lysates were immunoblotted for Bid, Bad, Puma, p-p53, or BCL-xL expression, followed by densitometry of immunoblots. Bar graphs represent values of expression in 3475-Akt3/−S472 OE relative to control 3475 tumors for the indicated proteins in four independent tumors; results are expressed as mean ± SEM. Unpaired t test shows significant differences for Bim (**, P < 0.01) or Bid (*, P < 0.05) expression, but not for other indicated proteins. F, Lysates from control 3475 or 3475-Akt3/−S472 OE tumors were either immunoblotted with anti Bax (Input; lanes 1, 2), immunoprecipitated with control IgG (lanes 3, 4), or with antiactive-Bax (6A7) antibody (lanes 5, 6), followed by anti-Bax immunoblotting. G, Lysates from control and 3475-Akt3/−S472 OE cells grown in suspension cultures, in low serum media for 48 hours to mimic anoikis, were immunoblotted for Akt3, cleaved-PARP, Bim, or β-Actin expression.

Close modal

Akt3/−S472 promotes apoptosis via Bim upregulation, leading to Bax and PARP/caspase-3 activation

The proapoptotic effect of Akt3/−S472 overexpression was associated with robust caspase-3 activation and PARP cleavage in several tumors, an effect that was independent of Akt1 or Akt2 expression, which was unchanged (Fig. 4C). Moreover, Akt3/−S472 caused a dramatic upregulation in the levels of proapoptotic Bim protein in several independent tumors (Fig. 4C), but had no effect on the level of Bax apoptotic effector protein, which is normally activated by Bim (Fig. 4C). Interestingly, Bim protein upregulation was matched by higher expression of Bim mRNA in tumors overexpressing Akt3/−S472 relative to controls (Fig. 4D). By contrast, immunoblotting analysis of mammary tumors showed that Akt3/−S472 overexpression led to Bid downregulation, but had no effect on the expression of other Bcl-2 family genes including the proapoptotic Bad and Puma, or the antiapoptotic Bcl-xL (Fig. 4E). In addition, Akt3/−S472 did not affect p53 phosphorylation, which results from DNA damage induced apoptosis (Fig. 4E; ref. 29).

Bim is known to bind the N-terminus of Bax, inducing conformational changes that culminate in the exposure of Bax α-helix 9 containing a transmembrane domain (30). These events trigger Bax translocation to mitochondria and insertion of this protein in the outer mitochondrial membrane, where it oligomerizes and forms pores that allow the release of cytochrome c to the cytoplasm, resulting in caspase activation and apoptosis (31). To test whether Akt3/−S472 stimulates Bax activation in vivo, we measured the levels of active-Bax using immunoprecipitation of tumor extract under native conditions with an antibody that recognizes the active conformation (6A7) followed by anti-Bax immunoblotting. As control, Bax expression in tumors was unchanged by Akt3/−S472 overexpression (Fig. 4F, lanes 1–2). However, the level of active-Bax protein was dramatically increased in 3475-Akt3/-S472 OE tumors relative to controls (Fig. 4F, lanes 5–6). These findings indicate that Akt3/−S472 triggers tumor apoptosis via Bim/Bax/caspase-3 activation.

Although apoptosis was prominent in mammary tumors in vivo, it was not detected in vitro in cell monolayers (ECM attached), even in response to apoptotic stimuli. We therefore tested the effect of ECM detachment on apoptotic stimulation by stress involving growing cells in suspension in low serum containing media (0.5%) for 48 hours, thus mimicking anoikis-like conditions. This interestingly resulted in a marked increase in Bim protein and cleavage of the caspase-3 substrate PARP in Akt3/−S472–overexpressing 3475 cells relative to control cells (Fig. 4G).

Importantly, these findings were reproduced in the MDA-MB-436 cell line, which as 3475, is a triple negative and highly tumorigenic breast cancer cell line. Akt3/−S472 overexpression in MDA-MB-436 attenuates mammary tumor growth, an effect that was accompanied by increases in TUNEL, Bim, and active–caspase-3 levels, as compared with unchanged Ki67 levels in tumors (Supplementary Fig. S5), thus underscoring the role of Akt3/−S472 in tumorigenic suppression via induction of apoptosis.

The proapoptotic effect of Akt3/−S472 is not due to a dominant negative effect on pan-Akt kinase activation

To determine whether Akt3/−S472 causes apoptosis by inhibiting the prosurvival effect of other Akts, involving Akt3/+S472, Akt1 or Akt2, we measured overall Akt phosphorylation in response to IGF treatment for 10 and 20 minutes (Fig. 5A). pAkt-T308 immunoblotting of cell lysates showed phosphorylation of a single Akt protein band in control cells, and a doublet in Akt3/−S472 overexpressing 3475 cells, corresponding to endogenous Akts, and overexpressed Akt3/−S472, which is only phosphorylated on threonine 305 (Fig. 5A). By comparison, pAkt-S473 immunoblotting showed phosphorylation of a single Akt protein band, which might include all Akts, except Akt3/−S472, which is lacking the S472 phosphorylation site. These data demonstrated that Akt3/−S472 overexpression does not inhibit overall Akt phosphorylation. Importantly, pan-Akt kinase activity, as measured by exogenous phosphorylation of GSK3α/β peptide in vitro by pAkt-S473 IPs, showed similar levels of phosphorylated GSK3α/β substrate in 3475-Akt3/−S472 OE and control cells, consistent with comparable levels of pAkt-S473 (Fig. 5B). These data imply that Akt3/−S472 stimulates apoptosis via a mechanism that does not involve a dominant negative effect on pan-Akt kinase activity.

Figure 5.

Akt3/−S472 overexpression does not affect pan-Akt kinase activation but attenuates ERK phosphorylation, leading to Bim upregulation. A, Serum-starved control 3475 and 3475-Akt3/−S472 OE cells were untreated or treated with 50 ng/mL IGF for 10 and 20 minutes; cell lysates were immunoblotted with anti-pAkt-T308, anti-pAkt-S473, or anti-β-actin antibody. Arrowheads indicate pan-Akt and Akt3/−S472 phosphorylated bands on Thr308 and T305, respectively. B, Control 3475 or 3475-Akt3/−S472 OE cells were serum-starved and treated with 50 ng/mL IGF for 10 minutes; cell lysates were immunoprecipitated with anti-pAkt-S473 antibody; immunoprecipitations were either assayed for Akt phosphorylation by anti-pAkt-S473 immunoblotting (top) or for Akt kinase activity using an in vitro nonradioactive assay that tests exogenous GSK3 peptide phosphorylation using p-GSK3α/β immunoblotting (middle). β-Actin input of each cell lysate is shown (bottom). C, Lysate from control or 3475-Akt3/−S472 OE adherent cells cultured in growth media was immunoblotted with anti–p-MEK1, MEK1, p-ERK, or ERK antibody. D, 3475-Akt3/−S472 OE cells were infected with control or active-MEK1 retroviral vector; cell lysates were Western blotted with anti MEK1, p-ERK, Bim, or β-actin antibody. E, 3475 control cells were treated with DMSO or the MEK1 inhibitor PD0325901 (ERKi) at 0.5 or 1.0 μmol/L in DMSO for 18 hours; levels of p-ERK, ERK, and Bim were determined by immunoblotting. F, Lysate from control and 3475-Akt3/−S472 OE cells, grown for 48 hours under anoikis-like conditions, were immunoblotted for Akt3, p-ERK, ERK, and Bim expression.

Figure 5.

Akt3/−S472 overexpression does not affect pan-Akt kinase activation but attenuates ERK phosphorylation, leading to Bim upregulation. A, Serum-starved control 3475 and 3475-Akt3/−S472 OE cells were untreated or treated with 50 ng/mL IGF for 10 and 20 minutes; cell lysates were immunoblotted with anti-pAkt-T308, anti-pAkt-S473, or anti-β-actin antibody. Arrowheads indicate pan-Akt and Akt3/−S472 phosphorylated bands on Thr308 and T305, respectively. B, Control 3475 or 3475-Akt3/−S472 OE cells were serum-starved and treated with 50 ng/mL IGF for 10 minutes; cell lysates were immunoprecipitated with anti-pAkt-S473 antibody; immunoprecipitations were either assayed for Akt phosphorylation by anti-pAkt-S473 immunoblotting (top) or for Akt kinase activity using an in vitro nonradioactive assay that tests exogenous GSK3 peptide phosphorylation using p-GSK3α/β immunoblotting (middle). β-Actin input of each cell lysate is shown (bottom). C, Lysate from control or 3475-Akt3/−S472 OE adherent cells cultured in growth media was immunoblotted with anti–p-MEK1, MEK1, p-ERK, or ERK antibody. D, 3475-Akt3/−S472 OE cells were infected with control or active-MEK1 retroviral vector; cell lysates were Western blotted with anti MEK1, p-ERK, Bim, or β-actin antibody. E, 3475 control cells were treated with DMSO or the MEK1 inhibitor PD0325901 (ERKi) at 0.5 or 1.0 μmol/L in DMSO for 18 hours; levels of p-ERK, ERK, and Bim were determined by immunoblotting. F, Lysate from control and 3475-Akt3/−S472 OE cells, grown for 48 hours under anoikis-like conditions, were immunoblotted for Akt3, p-ERK, ERK, and Bim expression.

Close modal

Akt3/−S472 enhances Bim expression via ERK inhibition

Our data point to Bim as the predominant proapoptotic gene upregulated by Akt3/−S472, at the mRNA and protein level (see Fig. 4C–E). Bim gene transcription is known to be often driven by FOXO activation in response to Akt inhibition, causing nuclear localization and transcriptional activation (32). However, no meaningful change in FOXO3a phophorylation or nuclear localization was observed in response to Akt3/−S472 overexpression (data not shown), consistent with our finding that Akt3/−S472 overexpression does not inhibit pan-Akt kinase activation.

MAPK/ERK pathway activation is known to destabilize Bim protein via phosphorylation, resulting in Bim ubiquitylation and degradation (33). We therefore tested whether Akt3/−S472 regulates Bim via ERK inhibition. Indeed, we found that ERK and MEK1 phosphorylation were both reduced in 3475-Akt3/−S472 OE cells (Fig. 5C). In support of this notion, expression of constitutively active MEK1 in 3475-Akt3/−S472 OE cells, which rescued ERK phosphorylation, caused a marked reduction in Bim protein (Fig. 5D). By contrast, treatment of control 3475 cells with the MEK1 inhibitor, PD0325901, which suppressed ERK phosphorylation at 0.5 and 1.0 μmol/L, markedly increased Bim expression (Fig. 5E). Interestingly, growing cells under anoikis conditions, led to further inhibition of ERK phosphorylation, which was concomitant with Bim upregulation in 3475-Akt3/−S472 OE relative to control 3475 cells (Fig. 5F). In contrast with ERK, Akt phosphorylation was not observed in anoikis/nutrient–deprived cultures of 3475-Akt3/−S472 OE or control cells (data not shown), supporting the notion that ERK, and not Akt, inhibition by Akt3/−S472 mediates Bim upregulation. In aggregate, these results clearly demonstrate a role for Akt3/−S472 in ERK attenuation leading to Bim stabilization.

Akt3/−S472 enhances EGF receptor endocytosis

Bim upregulation following ERK inhibition was found to drive anoikis of MCF10A nontumorigenic breast cells as a result of EGFR downregulation (34). Hence, it is plausible that Akt3/−S472 inhibits ERK by suppressing EGFR, which is overexpressed in TNBC cells. EGFR protein expression was, however, unchanged in 3475-Akt3/−S472 OE cells growing either as adherent or suspension cultures (Fig. 6A). We, therefore, tested whether Akt3/−S472 regulates EGFR signaling by affecting EGFR endocytosis. We measured EGFR internalization in response to EGF stimulation using cell surface biotinylation. Cells were treated with 100 ng/mL EGF in growth media for 0 to 40 minutes, and cell surface biotinylated EGFR was captured by avidin-coupled beads, and levels assessed by EGFR immunoblotting and densitometry. Interestingly, EGF stimulated a striking attenuation of surface EGFR relative to overall EGFR expression in 3475-Akt3/−S472 OE cells as compared to control 3475 cells (Fig. 6B). Densitometric analysis revealed that EGFR was internalized by 46% and 88% in 3475-Akt3/−S472 OE cells relative to 1.4% and 35% in control 3475 cells by 5 and 40 minutes post EGF treatment (Fig. 6C). Consistent with these findings, staining of cells, showed EGFR membrane localization in ligand-untreated control 3475 and 3475-Akt3/−S472 OE cells (Fig. 6D). However, stimulation with EGF for 10 minutes caused dramatic re-localization of EGFR from the cell surface to early endosomes, as shown by costaining of EGFR with EEA1, an effect that was intensified in 3475-Akt3/−S472 OE relative to control 3475 cells (Fig. 6D). Indeed, quantification of endocytosed EGFR at 10 min post EGF stimulation showed a 3-fold increase in endocytic vesicles containing EGFR and EEA1 in 3475-Akt3/−S472 OE relative to control cells (Fig. 6E). To evaluate whether increased EGFR endocytosis affects ERK phosphorylation, we measured p-ERK levels at various time points following EGF treatment. ERK was phosphorylated to a similar extent in control and 3475-Akt3/−S472 OE cells at 10 to 40 minutes post EGF stimulation; however, at 80 minutes, there was a sharp reduction in p-ERK levels in 3475-Akt3/−S472 OE cells relative to control cells (Fig. 6F).

Figure 6.

Akt3/−S472 enhances EGF-stimulated EGFR endocytosis. A, Control and 3475-Akt3/−S472 OE cells from adherent or suspension (anoikis) cultures were immunoblotted with anti-EGFR or β-actin antibody. B, Control and 3475-Akt3/−S472 OE cells were stimulated in growth media with 100 ng/mL EGF for the indicated time points. Cells were then biotinylated and lysates incubated with avidin-coupled beads, followed by immunoblotting with anti-EGFR. Inputs of EGFR or β-actin from total cell lysates are shown. C, Densitometric plots of EGFR immunoblots from triplicate experiments are shown as mean ± SEM; **, P < 0.001. D, Control and 3475-Akt3/−S472 OE cells were untreated or treated with 100 ng/mL EGF for 10 minutes, fixed, and coimmunostained with anti-EGFR (TRITC) and anti EEA1 (FITC) antibody, followed by DAPI counterstaining. E, Costaining of EGFR with EEA1 was quantified in EGF-treated control and 3475-Akt3/−S472 OE cells in 25 to 50 cells per group; the number of EGFR/EEA1-positive endosomes per cell are shown as mean ± SEM; ****, P < 0.0001. F, Control and 3475-Akt3/−S472 OE cells were incubated in growth media with 100 ng/mL EGF for 0, 10, 20, 40, and 80 minutes; cell lysates were immunoblotted with anti–p-ERK or ERK.

Figure 6.

Akt3/−S472 enhances EGF-stimulated EGFR endocytosis. A, Control and 3475-Akt3/−S472 OE cells from adherent or suspension (anoikis) cultures were immunoblotted with anti-EGFR or β-actin antibody. B, Control and 3475-Akt3/−S472 OE cells were stimulated in growth media with 100 ng/mL EGF for the indicated time points. Cells were then biotinylated and lysates incubated with avidin-coupled beads, followed by immunoblotting with anti-EGFR. Inputs of EGFR or β-actin from total cell lysates are shown. C, Densitometric plots of EGFR immunoblots from triplicate experiments are shown as mean ± SEM; **, P < 0.001. D, Control and 3475-Akt3/−S472 OE cells were untreated or treated with 100 ng/mL EGF for 10 minutes, fixed, and coimmunostained with anti-EGFR (TRITC) and anti EEA1 (FITC) antibody, followed by DAPI counterstaining. E, Costaining of EGFR with EEA1 was quantified in EGF-treated control and 3475-Akt3/−S472 OE cells in 25 to 50 cells per group; the number of EGFR/EEA1-positive endosomes per cell are shown as mean ± SEM; ****, P < 0.0001. F, Control and 3475-Akt3/−S472 OE cells were incubated in growth media with 100 ng/mL EGF for 0, 10, 20, 40, and 80 minutes; cell lysates were immunoblotted with anti–p-ERK or ERK.

Close modal

In support of these findings, CRISPR-mediated knockout of Akt3/−S472 caused a delay in EGFR internalization, as shown by the higher levels of surface-biotinylated EGFR in Akt3/−S472 KO cells relative to control cells, expressing a non-targeting CRISPR sequence (Fig. 7A). As control, the total levels of EGFR post EGF stimulation were similar to those in control cells (Fig. 7A). Changes in EGFR internalization were especially striking at 20 minutes post EGF treatment, but less obvious at later time points (Fig. 7A and B). Immunostaining for EGFR and EEA1 confirmed these data; EGF clearly induced a less efficient intracellular localization of EGFR in 3475-Akt3/−S472 KO cells than in control 3475 cells (Fig. 7C). Indeed, Akt3/−S472 KO cells displayed fewer EGFR/EEA1 containing endosomes relative to control cells (Fig. 7D). These findings underscore that Akt3/−S472 attenuates ERK signaling by enhancing EGFR endocytosis.

Figure 7.

Akt3/−S472 CRISPR–mediated knockout delays EGFR endocytosis. A, Control and 3475-Akt3/−S472 KO cells were untreated (time zero) or treated in growth media with 100 ng/mL EGF for indicated time points. Cells were then biotinylated and lysates incubated with avidin-coupled beads, followed by immunoblotting with anti-EGFR. Inputs of EGFR in total cell lysates are shown. B, Densitometric plots of EGFR immunoblots from triplicate experiments are shown as mean ± SEM. C, Control and 3475-Akt3/−S472 KO cells were untreated or treated with 100 ng/mL EGF for 10 minutes, fixed, and coimmunostained with anti-EGFR (TRITC) and anti-EEA1 (FITC) antibody, and DAPI counterstaining. D, Costaining of EGFR with EEA1 was measured in EGF-treated 3475 controls and 3475-Akt3/−S472 KO cells in 25 to 50 cells per group and the EGFR/EEA1-positive endosomes per cell are shown as mean ± SEM; *, P < 0.05.

Figure 7.

Akt3/−S472 CRISPR–mediated knockout delays EGFR endocytosis. A, Control and 3475-Akt3/−S472 KO cells were untreated (time zero) or treated in growth media with 100 ng/mL EGF for indicated time points. Cells were then biotinylated and lysates incubated with avidin-coupled beads, followed by immunoblotting with anti-EGFR. Inputs of EGFR in total cell lysates are shown. B, Densitometric plots of EGFR immunoblots from triplicate experiments are shown as mean ± SEM. C, Control and 3475-Akt3/−S472 KO cells were untreated or treated with 100 ng/mL EGF for 10 minutes, fixed, and coimmunostained with anti-EGFR (TRITC) and anti-EEA1 (FITC) antibody, and DAPI counterstaining. D, Costaining of EGFR with EEA1 was measured in EGF-treated 3475 controls and 3475-Akt3/−S472 KO cells in 25 to 50 cells per group and the EGFR/EEA1-positive endosomes per cell are shown as mean ± SEM; *, P < 0.05.

Close modal

Akt isoforms regulate various cellular processes including cell proliferation, migration, and survival (12, 22, 35, 36). Akt3 has been largely associated with progression of several tumor types especially melanomas, due to widespread PTEN mutations (37, 38). In TNBC, Akt3 was found to be expressed in a third of cancers, implying a potential benefit for Akt3-based therapy (39). However, Akt3 was found to have paradoxical effects on breast and other malignancies (22, 3, 24, 25). Akt3 was shown to promote breast cancer cell proliferation as well as Akt drug resistance (22, 40). By contrast, we and others showed that like Akt1, Akt3 ablation in cancer cells enhanced cell motility, but not metastasis (16, 36, 24), likely due to events, beyond cell migration, that are not regulated by Akt3/−S472.

Most studies on Akt3 inputs into malignant progression focused on the full-length Akt3 (Akt3/+S472), but overlooked the Akt3/−S472 isoform, likely because of low abundance or potential redundancy with full-length Akt3. qRT-PCR analysis of clinical TNBCs, revealed a low expression of Akt3/−S472 relative to Akt3/+S472, consistent with the scarcity of this isoform (5%) in the mammary gland (9), which could be due to inherent proapoptotic activity that may cause cell toxicity. In support of a physiologic role for Akt3/−S472, we speculate that this isoform regulates mammary morphogenesis by causing apoptotsis or clearance of epithelial cells from the luminal cavity, thus driving lumen formation and glandular differentiation (41). Consistent with this idea, TCGA breast cancer datasets revealed a borderline association of Akt3/−S472 expression with improved patient survival, implying a potential role in maintenance of a benign phenotype.

In contrast with our findings, Akt3 was shown to regulate TNBC cell growth in vitro and in vivo, using MDA-MB-468 and BT-549 cell lines, which are however PTEN mutated, and are thus likely to express a constitutively active Akt3 that renders an oncogenic function (22). By contrast, MDA-MB-231 and 3475 cells are PTEN wild-type, and unlikely to express a dominant active Akt3 kinase, hence supporting our findings that ablation of Akt3/+S472 in these cell lines is dispensable for tumor growth (42).

These findings convey an unconventional role for Akt in apoptosis, which is inconsistent with its classical prosurvival function. What clearly differentiates the two Akt3 splice variants is the carboxyl terminal serine 472, which is missing in Akt3/−S472, a site that is necessary for Akt kinase activation. Namely, although T308 phosphorylation by PDK1 activates the Akt kinase, subsequent phosphorylation on S472 by mTORC1 leads to full kinase activation (43, 44). Indeed, it was shown that T308 phosphorylation causes a 10-fold increase in kinase activation, whereas subsequent phosphorylation on S472 results in a 1,000-fold activation (9). This implies that Akt3/−S472, may not efficiently compete substrates shared with other Akts including Akt3/+S472, making it unlikely to act as a dominant negative Akt. In support of this notion, exogenous GSK3 peptide phosphorylation by IGF-activated Akts in vitro, was not affected by Akt3/−S472 overexpression. Moreover, under or over expression of Akt3/+S472 isoform did not influence tumor growth, implying this isoform may not act as a driver oncogene in MDA-MB-231 or 3475 cells, and is therefore unlikely to be inhibited by Akt3/−S472. Finally, Akt3/−S472 may phosphorylate substrates that cause apoptosis, that are not shared with other Akts, and that interact with Akt3/−S472 on sequences encoded by exons 14/15.

We propose that Akt3/−S472 triggers apoptosis via Bim upregulation due to EGFR–ERK axis inhibition. These data underscore findings from the Brugge laboratory, showing that apoptosis underlying mammary gland lumen formation is mediated by Bim stabilization due to ERK inhibition caused by EGFR attenuation (41). Interestingly, Akt3/−S472–mediated apoptosis was observed in vivo, but not in vitro in adherent cultures, a limitation that was overcome by growing cells under anoikis conditions. Interestingly, ERK, but not Akt, was phosphorylated in control 3475 cells growing under anoikis- or nutrient-deprived conditions, thus supporting the contention that Akt3/−S472 targets ERK, and not Akt, to provoke apoptosis.

How Akt3/−S472 regulates EGFR endocytosis remains unknown. Others have shown that Akt facilitates EGFR trafficking and degradation by phosphorylating and activating PIKfyve (45). Therefore, one might speculate that Akt3/−S472 phosphorylates PIKfyve or other substrates involved in EGFR endocytosis, a question that may be resolved by expressing a kinase-dead Akt3/−S472. Alternatively, Akt3/−S472 may activate clathrin-mediated endocytosis, which was shown to be affected by Akt phosphorylation, resulting in enhanced EGFR endocytosis (46). In sum, this study demonstrates a highly unusual function for an Akt molecule, in promoting, and not inhibiting, apoptosis through negative regulation of the EGFR–ERK survival pathway.

No potential conflicts of interest were disclosed.

Conception and design: K. Suyama, R.N. Kitsis, R.B. Hazan

Development of methodology: K. Suyama, J. Yao, H. Liang, O. Benard, S. Fineberg, R.N. Kitsis

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): K. Suyama, J. Yao, H. Liang, O. Benard, O. Loudig, D. Amgalan, W.M. McKimpson, G.R. Phillips, Y. Wang, S. Fineberg, R.N. Kitsis

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): K. Suyama, J. Yao, H. Liang, O. Benard, O. Loudig, D. Amgalan, W.M. McKimpson, G.R. Phillips, J. Segall, Y. Wang, S. Fineberg, R.N. Kitsis, R.B. Hazan

Writing, review, and/or revision of the manuscript: O. Loudig, D. Amgalan, J. Segall, L. Norton, R.N. Kitsis, R.B. Hazan

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): L. Norton

We are very grateful to Dr. Yiling Lu (MD Anderson Cancer Center) for fruitful discussions and generous Akt plasmids. We thank Christina Liu for technical expertise. We thank Dr. Jonathan Backer for data discussion. We thank the Cancer Center Support Grant (P30 CA013330) for the use of shared resources.

This work was supported by grants from the National Cancer Institute R01 CA135061-01A1 (to R.B. Hazan), R01CA136854-01A1 (to R.B. Hazan), and the Breast Cancer Research Foundation (to R.B. Hazan and L. Norton).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Manning
BD
,
Cantley
LC
. 
AKT/PKB signaling: navigating downstream
.
Cell
2007
;
129
:
1261
74
.
2.
Vivanco
I
,
Sawyers
CL
. 
The phosphatidylinositol 3-Kinase AKT pathway in human cancer
.
Nat Rev Cancer
2002
;
2
:
489
501
.
3.
Sanidas
I
,
Polytarchou
C
,
Hatziapostolou
M
,
Ezell
SA
,
Kottakis
F
,
Hu
L
, et al
Phosphoproteomics screen reveals akt isoform-specific signals linking RNA processing to lung cancer
.
Mol Cell
2014
;
53
:
577
90
.
4.
Eijkelenboom
A
,
Burgering
BM
. 
FOXOs: signalling integrators for homeostasis maintenance
.
Nat Rev Mol Cell Biol
2013
;
14
:
83
97
.
5.
Harada
H
,
Grant
S
. 
Apoptosis regulators
.
Rev Clin Exp Hematol
2003
;
7
:
117
38
.
6.
Scorrano
L
,
Korsmeyer
SJ
. 
Mechanisms of cytochrome c release by proapoptotic BCL-2 family members
.
Biochem Biophys Res Commun
2003
;
304
:
437
44
.
7.
Harada
H
,
Grant
S
. 
Targeting the regulatory machinery of BIM for cancer therapy
.
Crit Rev Eukaryot Gene Expr
2012
;
22
:
117
29
.
8.
Dummler
B
,
Hemmings
BA
. 
Physiological roles of PKB/Akt isoforms in development and disease
.
Biochem Soc Trans
2007
;
35
:
231
5
.
9.
Brodbeck
D
,
Hill
MM
,
Hemmings
BA
. 
Two splice variants of protein kinase B gamma have different regulatory capacity depending on the presence or absence of the regulatory phosphorylation site serine 472 in the carboxyl-terminal hydrophobic domain
.
J Biol Chem
2001
;
276
:
29550
8
.
10.
Du
K
,
Tsichlis
PN
. 
Regulation of the Akt kinase by interacting proteins
.
Oncogene
2005
;
24
:
7401
9
.
11.
Yang
ZZ
,
Tschopp
O
,
Hemmings-Mieszczak
M
,
Feng
J
,
Brodbeck
D
,
Perentes
E
, et al
Protein kinase B alpha/Akt1 regulates placental development and fetal growth
.
J Biol Chem
2003
;
278
:
32124
31
.
12.
Chin
YR
,
Toker
A
. 
Function of Akt/PKB signaling to cell motility, invasion and the tumor stroma in cancer
.
Cell Signal
2009
;
21
:
470
6
.
13.
Dillon
RL
,
Muller
WJ
. 
Distinct biological roles for the akt family in mammary tumor progression
.
Cancer Res
2010
;
70
:
4260
4
.
14.
Maroulakou
IG
,
Oemler
W
,
Naber
SP
,
Tsichlis
PN
. 
Akt1 ablation inhibits, whereas Akt2 ablation accelerates, the development of mammary adenocarcinomas in mouse mammary tumor virus (MMTV)-ErbB2/neu and MMTV-polyoma middle T transgenic mice
.
Cancer Res
2007
;
67
:
167
77
.
15.
Chin
YR
,
Toker
A
. 
Akt2 regulates expression of the actin-bundling protein palladin
.
FEBS Lett
2010
;
584
:
4769
74
.
16.
Chin
YR
,
Toker
A
. 
The actin-bundling protein palladin is an Akt1-specific substrate that regulates breast cancer cell migration
.
Mol Cell
2010
;
38
:
333
44
.
17.
Lin
HP
,
Lin
CY
,
Huo
C
,
Jan
YJ
,
Tseng
JC
,
Jiang
SS
, et al
AKT3 promotes prostate cancer proliferation cells through regulation of Akt, B-Raf, and TSC1/TSC2
.
Oncotarget
2015
;
6
:
27097
112
.
18.
Hennessy
BT
,
Smith
DL
,
Ram
PT
,
Lu
Y
,
Mills
GB
. 
Exploiting the PI3K/AKT pathway for cancer drug discovery
.
Nat Rev Drug Discov
2005
;
4
:
988
1004
.
19.
Stahl
JM
,
Sharma
A
,
Cheung
M
,
Zimmerman
M
,
Cheng
JQ
,
Bosenberg
MW
, et al
Deregulated Akt3 activity promotes development of malignant melanoma
.
Cancer Res
2004
;
64
:
7002
10
.
20.
Cristiano
BE
,
Chan
JC
,
Hannan
KM
,
Lundie
NA
,
Marmy-Conus
NJ
,
Campbell
IG
, et al
A specific role for AKT3 in the genesis of ovarian cancer through modulation of G(2)-M phase transition
.
Cancer Res
2006
;
66
:
11718
25
.
21.
Madhunapantula
SV
,
Robertson
GP
. 
The PTEN-AKT3 signaling cascade as a therapeutic target in melanoma
.
Pigment Cell Melanoma Res
2009
;
22
:
400
19
.
22.
Chin
YR
,
Yoshida
T
,
Marusyk
A
,
Beck
AH
,
Polyak
K
,
Toker
A
. 
Targeting Akt3 signaling in triple-negative breast cancer
.
Cancer Res
2014
;
74
:
964
73
.
23.
Perou
CM
,
Sorlie
T
,
Eisen
MB
,
van de Rijn
M
,
Jeffrey
SS
,
Rees
CA
, et al
Molecular portraits of human breast tumours
.
Nature
2000
;
406
:
747
52
.
24.
Chung
S
,
Yao
J
,
Suyama
K
,
Bajaj
S
,
Qian
X
,
Loudig
OD
, et al
N-cadherin regulates mammary tumor cell migration through Akt3 suppression
.
Oncogene
2013
;
32
:
422
30
.
25.
Phung
TL
,
Du
W
,
Xue
Q
,
Ayyaswamy
S
,
Gerald
D
,
Antonello
Z
, et al
Akt1 and akt3 exert opposing roles in the regulation of vascular tumor growth
.
Cancer Res
2015
;
75
:
40
50
.
26.
Minn
AJ
,
Gupta
GP
,
Siegel
PM
,
Bos
PD
,
Shu
W
,
Giri
DD
, et al
Genes that mediate breast cancer metastasis to lung
.
Nature
2005
;
436
:
518
24
.
27.
Horvath
P
,
Barrangou
R
. 
CRISPR/Cas, the immune system of bacteria and archaea
.
Science
2010
;
327
:
167
70
.
28.
Sander
JD
,
Joung
JK
. 
CRISPR-Cas systems for editing, regulating and targeting genomes
.
Nat Biotechnol
2014
;
32
:
347
55
.
29.
Youle
RJ
,
Strasser
A
. 
The BCL-2 protein family: opposing activities that mediate cell death
.
Nat Rev Mol Cell Biol
2008
;
9
:
47
59
.
30.
Gavathiotis
E
,
Suzuki
M
,
Davis
ML
,
Pitter
K
,
Bird
GH
,
Katz
SG
, et al
BAX activation is initiated at a novel interaction site
.
Nature
2008
;
455
:
1076
81
.
31.
Korsmeyer
SJ
,
Wei
MC
,
Saito
M
,
Weiler
S
,
Oh
KJ
,
Schlesinger
PH
. 
Pro-apoptotic cascade activates BID, which oligomerizes BAK or BAX into pores that result in the release of cytochrome c
.
Cell Death Differ
2000
;
7
:
1166
73
.
32.
Tzivion
G
,
Dobson
M
,
Ramakrishnan
G
. 
FoxO transcription factors; regulation by AKT and 14–3-3 proteins
.
Biochim Biophys Acta
2011
;
1813
:
1938
45
.
33.
Ley
R
,
Balmanno
K
,
Hadfield
K
,
Weston
C
,
Cook
SJ
. 
Activation of the ERK1/2 signaling pathway promotes phosphorylation and proteasome-dependent degradation of the BH3-only protein, Bim
.
J Biol Chem
2003
;
278
:
18811
6
.
34.
Schmelzle
T
,
Mailleux
AA
,
Overholtzer
M
,
Carroll
JS
,
Solimini
NL
,
Lightcap
ES
, et al
Functional role and oncogene-regulated expression of the BH3-only factor Bmf in mammary epithelial anoikis and morphogenesis
.
Proc Natl Acad Sci U S A
2007
;
104
:
3787
92
.
35.
Chin
YM
,
Yuan
X
,
Balk
SP
,
Toker
A
. 
Pten-deficient tumors depend on akt2 for maintenance and survival
.
Cancer Discov
;
4
:
942
55
.
36.
Maroulakou
IG
,
Oemler
W
,
Naber
SP
,
Klebba
I
,
Kuperwasser
C
,
Tsichlis
PN
. 
Distinct roles of the three Akt isoforms in lactogenic differentiation and involution
.
J Cell Physiol
2008
;
217
:
468
77
.
37.
Robertson
GP
. 
Functional and therapeutic significance of Akt deregulation in malignant melanoma
.
Cancer Metastasis Rev
2005
;
24
:
273
85
.
38.
Madhunapantula
SV
,
Robertson
GP
. 
Targeting protein kinase-b3 (akt3) signaling in melanoma
.
Expert Opin Ther Targets
2017
;
21
:
273
90
.
39.
Banerji
S
,
Cibulskis
K
,
Rangel-Escareno
C
,
Brown
KK
,
Carter
SL
,
Frederick
AM
, et al
Sequence analysis of mutations and translocations across breast cancer subtypes
.
Nature
2012
;
486
:
405
9
.
40.
Stottrup
C
,
Tsang
T
,
Chin
YR
. 
Upregulation of AKT3 Confers Resistance to the AKT Inhibitor MK2206 in Breast Cancer
.
Mol Cancer Ther
2016
;
15
:
1964
74
.
41.
Mailleux
AA
,
Overholtzer
M
,
Schmelzle
T
,
Bouillet
P
,
Strasser
A
,
Brugge
JS
. 
BIM regulates apoptosis during mammary ductal morphogenesis, and its absence reveals alternative cell death mechanisms
.
Dev Cell
2007
;
12
:
221
34
.
42.
Saal
LH
,
Gruvberger-Saal
SK
,
Persson
C
,
Lovgren
K
,
Jumppanen
M
,
Staaf
J
, et al
Recurrent gross mutations of the PTEN tumor suppressor gene in breast cancers with deficient DSB repair
.
Nat Genet
2008
;
40
:
102
7
.
43.
Scheid
MP
,
Woodgett
JR
. 
PKB/AKT: functional insights from genetic models
.
Nat Rev Mol Cell Biol
2001
;
2
:
760
8
.
44.
Alessi
DR
,
Andjelkovic
M
,
Caudwell
B
,
Cron
P
,
Morrice
N
,
Cohen
P
, et al
Mechanism of activation of protein kinase B by insulin and IGF-1
.
EMBO J
1996
;
15
:
6541
51
.
45.
Er
EE
,
Mendoza
MC
,
Mackey
AM
,
Rameh
LE
,
Blenis
J
. 
AKT facilitates EGFR trafficking and degradation by phosphorylating and activating PIKfyve
.
Sci Signal
2013
;
6
:
ra45
.
46.
Tomas
A
,
Futter
CE
,
Eden
ER
. 
EGF receptor trafficking: consequences for signaling and cancer
.
Trends Cell Biol
2014
;
24
:
26
34
.