AKT inhibition relieves feedback suppression of receptor tyrosine kinase expression and activity

Abstract

Activation of the PI3K-AKT pathway in tumors is modulated by negative feedback, including mTORC1-mediated inhibition of upstream signaling. We now show that AKT inhibition induces the expression and phosphorylation of multiple receptor tyrosine kinases (RTKs). In a wide spectrum of tumor types, inhibition of AKT induces a conserved set of RTKs, including HER3, IGF-1R, and insulin receptor. This is in part due to mTORC1 inhibition and in part secondary to a FOXO-dependent activation of receptor expression. PI3K-AKT inhibitors relieve this feedback and activate RTK signaling; this may attenuate their antitumor activity. Consistent with this model, we find that, in tumors in which AKT suppresses HER3 expression, combined inhibition of AKT and HER kinase activity is more effective than either alone.

Fig. 1. AKT inhibition promotes HER3 expression and phosphorylation

(A) BT474 cells were treated with AKTi-1/2 (1μM) and collected at indicated times. AKT inhibition as measured by loss of S473 phosphorylation, along with AKT targets S6K and PRAS40, is associated with increases in HER3 and P-HER3. (B) BT-474 cells treated with siRNAs against AKT1, AKT2 and AKT3 (AKT3 was not detectable) for 72 hours were collected and lysates immunoblotted demonstrating loss of AKT expression is associated with increased expression of RTKs. (C) The HER2 amplified cancer cell lines BT-474 and SK-BR3 were treated with the MEK inhibitor PD325901 (50nM). Inhibition of ERK1/2 phosphorylation was not associated with an induction of HER3 or P-HER3. (D) BT-474 and SK-BR3 cell lines were serum starved for 12 hours followed by treatment with 1μM AKTi. The induction of HER3 expression occurred upon AKT inhibition despite the lack of serum. See also Fig S1.

Fig. 2. Activation of AKT represses HER3 while inhibition induces several phosphorylated RTKs

(A) BT-474 cells (at time = -12) were treated with AKTi-1/2/3 (100nM) or dmso for 12 hours (until t=0). Cells were then washed 4x with PBS, placed in fresh media, and collected at indicated times after (t=4, 8, 24, 48hr). Immunoblots demonstrate loss of HER3 and P-HER3 correlated with activation of AKT. (B) BT474 cells were left untreated (0hr) or treated with AKTi-1/2/3 (1μM) for 24 hours and lysates applied to Phospho-RTK arrays. Spots are in duplicate and each pair corresponds to a specific P-RTK. Top set of blots correspond to 50μg lysates while bottom two correspond to 250μg lysates with HER2 and HER3 dot blots excised from the membrane. Phospho-HER3 corresponds to the doublet at B5 and shows increased expression with prolonged treatment with the AKT inhibitor.

Fig. 3. RTKs expression and phosphorylation are induced by AKT inhibition in multiple tumor types

(A) Shown are immunoblots from representative Phospho-RTK arrays from cancer cell lines treated with 1μM AKTi-1/2/3 (24hr) compared to untreated (0hr) demonstrating that phosphorylation of several receptors is induced with inhibitor treatment. The HER2 and HER3 dot blots were excised from the membrane in the case of SKBR-3 cell line shown here. See Supp. Methods for list of receptors corresponding to labels (e.g. B5 = HER3) and Fig. S2 for other cell lines tested. (B) Listed are the receptors in which average tyrosine phosphorylation (10 cell lines) induced >2.9 fold after 1μM AKTi-1/2/3 (24hr) as measured by densitometry on P-RTK arrays. Mean induction and the number of cell lines with an induction (>1.3) are listed (Cell lines – BT-474, H292, MCF10A, SKBR3, MDA231, MDA453, LNCAP, H3255, H1975, IGROV1). (C) A panel of 7 non-HER2 amplified cell lines was treated with 1μM AKTi-1/2/3 and immunoblots demonstrate induction of the expression of several RTKs. (D) Listed is the fold induction of protein expression of HER3, Insulin Receptor, and IGF-1R after AKT inhibition calculated by densitometry from immunoblots. See also Table S1.

Fig. 4. HER kinase inhibition does not block AKTi stimulated RTK expression but can alleviate AKTi induction of P-RTKs

(A) BT-474 cells were treated with dmso, 1μM AKTi-1/2/3, 1μM Lapatinib, or the combination. Immunoblots of P-RTK arrays were quantified by densitometry. The effect of the HER1/2 kinase inhibitor upon the induction caused by AKT inhibition is shown with the induced expression of most P-RTKs blocked in the combination treatment. (B) NCI-H292 cells were treated with dmso, 1μM AKTi-1/2/3, 5μM Gefitinib, or the combination. The effect of the HER kinase inhibitor upon the induction caused by the AKTi is shown with the induced expression of some P-RTKs blocked in the combination treatment. (C) BT-474 cells were treated with dmso, 1μM AKTi-1/2/3, 1μM Lapatinib, or the combination and collected at 8 and 24 hours post treatment. Immunoblotting of lysates demonstrates AKTi or Lapatinib or the combination induces HER3, IGF-1R, and Insulin receptor expression but phosphorylation of these receptors is blocked by Lapatinib treatment. (D) NCI-H292 cells were treated with 1μM Lapatinib, 1μM AKTi-1/2/3, or the combination and collected at 4, 8, or 24 hours post-treatment and lysates immunoblotted showing induction HER3 and P-HER3 in response to AKT inhibition and blockade of HER3 phosphorylation by Lapatinib. See also Fig. S3.

Fig. 5. Inhibition of AKT but not mTORC1 promotes the expression and phosphorylation of several RTKs

(A) BT-474 cells were treated with 1μM AKTi-1/2, 50nM Rapamycin, or 500nM Lapatinib and collected at indicated times. Loss of AKT S473 phosphorylation is associated with an induction in HER3 protein levels while treatment with Rapamycin is not associated with significantly increased levels of HER3. (B) BT474 cells were treated with 1μM AKTi-1/2 or 50nM Rapamycin and lysates applied to Phospho-RTK arrays. The HER2 and HER3 dot blots were excised from the membranes shown here. Densitometry was performed to calculate fold induction over untreated cells. All receptors induced >1.9 fold are listed. See also Fig. S4.

Fig. 6. AKT but not mTORC1 inhibition induces RTK RNA expression in a FOXO dependent manner

(A) BT-474 cells treated with 1μM AKTi-1/2, 50nM Rapamycin, or 500nM Lapatinib and RT-PCR was performed using reverse transcribed cellular RNA. Fluorescence was normalized to housekeeping gene expression (β-Actin and GAPDH) and is displayed as the percentage of the value for untreated lysate. Expression of HER3, IGF-IR, and Insulin Receptor was induced by effective suppression of activated AKT but minimally with Rapamycin treatment. (B) BT-474 cells were treated with interfering RNAs against FOXO1 (siF1), FOXO3 (siF3), FOXO4 (siF4), FOXO1 + 3 + 4 (siFall) or mock (M) with or without AKTi-1/2/3 (1μM). Cells were collected 72 hours post-siRNA transfection (24 hours post AKTi) and RT-PCR performed with the indicated probes. Inhibition of AKT is associated with enhanced expression of the HER3, IR, and IGF-1R RNAs. RNA induction is suppressed by FOXO1/3/4 knockdown. (C) BT-474 cells were treated with FOXO1/3/4 siRNAs or mock for 48 hours followed by treatment with AKTi-1/2/3 (1μM) or DMSO for 24 hours followed by formalin-fixation and isolation of chromatin. Immunoprecipitation was performed using antibodies against FOXO 1, 3, and 4 and RT-PCR done with oligonucleotides complementary to a sequence 1kb 5’ to the HER3, INSR, and IGF-1R start sites. Expression was normalized to the non-immunoprecipitated (input) fraction and reported as fold enrichment (± SEM) and demonstrate increased binding of FOXO proteins to the RTK 5’ UTRs upon AKT inhibition. (D) BT-474 cells were treated as in (C) and collected for immunoblotting demonstrating knockdown of FOXO proteins associated with diminished induction of the RTKs. See also Fig S5.

Fig. 7. HER1/2 kinase inhibition improves antitumor efficacy when given in combination with the AKTi

(A) Mice bearing BT-474 tumors were randomized to (1) no treatment, (2) Lapatinib 150mpk 3 times/week, (3) AKTi-1/2 100mpk 3 times/week, or (4) combination of (2) and (3) and tumor size measured 2x/week with the combination treatment demonstrating superior antitumor efficacy compared to the single agents. (B) Mice bearing NCI-H292 tumors were randomized to (1) no treatment, (2) Gefitinib (Iressa) 150mpk 3 times/week, (3) AKTi-1/2/3 100mpk 3 times/week, or (4) combination of (2) and (3) and tumor size measured by vernier calipers 2x/week with the combination treatment demonstrating superior antitumor effects to the single agents. The results in (A) and (B) are presented as the mean tumor volume ± SEM (n = 5 mice/group). (C) Mice (n=12) bearing BT-474 tumors were randomized to no treatment, treatment with a single dose of Lapatinib 150mpk, AKTi-1/2/3 100mpk, or the combination and collected at 6 and 12 hours post treatment. Immunoblotting of tumor lysates demonstrates AKT inhibition induces P-HER3 and P-IGF-1R/INSR in vivo and this is attenuated by Lapatinib coadministration. Note, two mice treated for the 12 hour time points lacked adequate tumor tissue at retrieval and were omitted. (D) Mice bearing NCI-H292 tumors were treated with a single dose of Gefitinib 150mpk, AKTi-1/2/3 100mpk, or the combination and collected at 6 and 12 hours post treatment. Immunoblotting of lysates demonstrates AKT inhibition induces P-HER3 and P-IGF-1R/INSR and this is attenuated by Gefitinib coadministration. See also Fig. S6.

Fig. 8. AKT inhibition and mTORC1 inhibition relieve feedback inhibition at unique nodes of oncogenic growth factor signaling pathways

Depicted in middle panel is the steady state feedback program elicited by activation of AKT and/or mTORC1. Receptor activation stimulates PI3K-AKT activity which inhibits FOXO mediated activation of RTK transcription. Receptor stimulation also activates mTORC1 which directs inhibitory feedback upon the expression of IRS-1, thereby attenuating PI3K-AKT activity. In the left panel, Rapamycin blocks mTORC1 action, relieving the feedback inhibition upon IRS-1 and causing hyperactivation of PI3K-AKT signaling. In the right panel, AKT inhibition blocks both AKT and the downstream mTORC1. The consequence is to relieve mTORC1 feedback but also to activate FOXO driven transcription of RTKs and stimulate PI3K-AKT activity through higher RTK expression and activity.