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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.
The phosphatidylinositol 3-kinase (PI3K) – protein kinase B (PKB/AKT) – mammalian target of rapamycin complex 1 (mTORC1) kinase cascade transmits signals from ligand stimulated receptor tyrosine kinases to effector molecules that control metabolism, proliferation, size, survival, and motility (Guertin and Sabatini, 2007; Vivanco and Sawyers, 2002). In cancer, this pathway is frequently hyperactivated as a result of: (1) activation of receptor tyrosine kinases by mutation (Epidermal Growth Factor Receptor) or gene amplification (HER2), (2) activating mutations of components of the pathway such as PI3K or AKT, and (3) deletion or decreased function of tumor suppressors such as the PIP3 phosphatase, PTEN (Phosphatase and TENsin homolog) (Hynes and Lane, 2005; Vivanco and Sawyers, 2002). Such lesions are extremely common in cancer.
Tumors with PTEN or PIK3CA mutations or HER2 amplification have been shown to be dependent on PI3K-AKT-mTOR signaling for maintenance of the transformed phenotype and hypersensitive to inhibition of its components. This has led to a major effort to develop inhibitors of PI3K, AKT, mTOR and other components of the pathway (Courtney et al., 2010; Workman et al., 2010). Analogs of the natural product rapamycin, an inhibitor of the mTORC1 complex, were among the first inhibitors of the PI3K kinase pathway to be used for the treatment of cancer. Rapamycin does effectively inhibit mTORC1 signaling; however, it also relieves mTORC1 dependent feedback inhibition of IGF1 receptor signaling. This results in activation of PI3K-AKT signaling and enhanced phosphorylation of non-mTORC1 targets of AKT, such as the FOXO family of transcription factors (Haruta et al., 2000; O’Reilly et al., 2006).
Physiologic activation of signaling is regulated by feedback inhibition of components of the network and is a feature of both normal and oncogene-transformed cells. Relief of this feedback might be a common response to anticancer drugs and could attenuate the therapeutic response (Courtois-Cox et al., 2006; O’Reilly et al., 2006). We reasoned that specific inhibitors of other components of the PI3K-AKT pathway will reactivate different aspects of the network. To examine the role played by AKT in the feedback regulation of activated mitogenic signal transduction, we employed a specific, allosteric AKT inhibitor and examined its effect on the expression and phosphorylation of the receptor tyrosine kinases that commonly stimulate these signaling pathways (DeFeo-Jones et al., 2005; Lindsley et al., 2005; She et al., 2008).
We used selective inhibitors to determine whether AKT mediates feedback inhibition of PI3K signaling in tumors in which it is dysregulated. AKTi-1/2 and AKTi-1/2/3 are PH-domain dependent, non-ATP-competitive inhibitors which potently inhibit AKT1 and AKT2 and not other AGC kinases, with AKTi-1/2/3 having greater potency against AKT3 (Barnett et al., 2005; Lindsley et al., 2005). The drugs share similar potency against activated AKT in the HER2 amplified breast cancer cell line BT-474 as seen in Fig. S1 (She et al., 2008). The drugs prevent binding of AKT to the plasma membrane and thus its phosphorylation by PDK1, so, unlike ATP-competitive inhibitors, they block, rather than activate AKT phosphorylation (Fig. 1A) (Okuzumi et al., 2009).
Breast cancers with HER2-amplification have elevated levels of AKT and ERK phosphorylation and are dependent on the former, but not the latter for maintenance of the transformed phenotype (Basso et al., 2002; Hermanto et al., 2001; She et al., 2008; Solit et al., 2006). In these tumors, PI3K-AKT signaling is HER2-dependent and predominantly driven by HER2-HER3 heterodimers (Holbro et al., 2003; Yakes et al., 2002). HER3 is a kinase-defective member of the HER kinase family that contains six tyrosines in its intracellular domain that, when phosphorylated act as high affinity docking sites for the p85 subunit of PI3K. In breast cancers, overexpressed HER2 dimerizes with and phosphorylates HER3, which binds to and activates PI3K (Holbro et al., 2003).
AKT inhibitors block AKT signaling in these cells and cause growth arrest and apoptosis (She et al., 2008). We asked whether activation of AKT feedback regulates the HER kinase family members that drive the pathway. In Fig. 1A, we determined the effect of AKT inhibition on HER kinase expression and phosphorylation in BT474, a breast cancer cell line with HER2 amplification. Treatment of BT-474 cells with 1μM AKTi-1/2 inhibits phosphorylation of AKT and its substrate, PRAS40 by 1 hour after drug is added. At 24 hours, AKT phosphorylation begins to rise, but remains significantly inhibited at 72 hours. AKT inhibition causes a marked change in the expression and phosphorylation of HER kinase family members. The expression of EGFR, HER3, and HER4 is induced beginning two to four hours after drug addition and reaches a maximum approximately 24 hours after drug exposure (1.6, 4.1, and 2.5 fold induction). The already high levels of HER2 expression do not change appreciably. There is a concomitant four-fold induction of the tyrosine phosphorylation of HER3 at both the Y1197 and Y1289 sites whereas phosphorylation of EGFR (Y1068), HER2 (Y1221), and HER4 (Y1284) declines over this period (0.4, 0.5, and 0.7 fold). Of the HER family receptors, only HER3 expression and phosphorylation are coordinately upregulated in these cells. Inhibition of the PI3K-AKT pathway using an ATP competitive AKT kinase inhibitor or a PI3K inhibitor also results in upregulation of HER3 and P-HER3 expression (Fig. S1). Moreover, interfering RNA against AKT1, AKT2, and AKT3 result in induction of HER3 expression and phosphorylation 72 hours after transfection (Fig. 1B). HER3 levels in the plasma membrane increase and this is accompanied by increased HER3 heterodimerization with HER2 and association with the p85 subunit of PI3K (Fig. S1). These data suggest that PI3K-AKT activation in response to HER2 amplification and overexpression is associated with an AKT-dependent downregulation of HER family protein expression, which is relieved by the AKT inhibitor. Although AKT and ERK signaling are both driven by HER2 amplification, feedback regulation of receptor expression is mediated by the former but not the latter. Selective inhibition of ERK phosphorylation with the MEK inhibitor, PD325901, has no effect on HER2 or HER3 expression or phosphorylation in tumor cells with HER2 amplification (Fig. 1C).
Induction of HER3 expression in response to AKT inhibition does not require serum factors. Serum starvation of HER2 amplified breast cancer cells is associated with increased HER3 expression, and increased phosphorylation of HER2, HER3 and AKT (Fig. 1D). In these cells, 1μM AKTi-1/2 led to marked induction of HER3 expression, but no further induction of HER3 phosphorylation. The results distinguish induction of phosphorylation of the receptor from induction of expression. Whereas AKT inhibition can further induce HER3 expression in absence of serum factors, it does not affect HER3 phosphorylation.
These results suggest that, in HER2 amplified breast cancer cells, PI3K activation may induce an AKT-dependent feedback inhibition of HER3 expression and activation. To assess the effects of inducing AKT activation, BT-474 cells were treated with DMSO or 100nM AKTi-1/2/3 in serum containing media for 12 hours (lane 2 and lane 7, Fig. 2A) and then the compound was washed out and fresh medium was added (Fig. 2A). Reactivation of AKT phosphorylation occurred four hours after the drug was removed and was sustained. This was associated with decreased HER3 expression and phosphorylation eight hours after drug washout. Levels of HER3 and phosphorylated HER3 declined to near steady state levels. The data suggest that AKT activation negatively regulates HER3 expression and phosphorylation in HER2-amplified breast cancer cells and that inhibition of AKT relieves this feedback.
Multiple receptor tyrosine kinases activate PI3K-AKT signaling in normal and tumor cells. In particular, many of the metabolic effects and other consequences of stimulation of the Insulin and IGF1 receptors (IGF-1R) are mediated via induction of PI3K-AKT signaling. We used an anti-phosphotyrosine receptor antibody array to assess whether receptor tyrosine kinases other than HER3 were induced in response to AKT inhibition (Fig. 2B). These RTK arrays detect phosphorylation of 42 receptor tyrosine kinases (Stommel et al., 2007). In BT-474 cells, HER2 and HER3 were the most prominent phosphorylated RTKs detected (first set of panels, 50μg loaded). Twenty four hours after exposure to the AKT inhibitor, P-HER2 was not significantly changed while P-HER3 was strongly induced. Removing the anti-P-HER2 and anti-P-HER3 dots from the array and increasing the amount of loaded protein (250 μg), increased the sensitivity of the assay. This allowed detection of multiple other receptors in the untreated control. The phosphorylation of a group of RTKs (HER3, FLT-3, HER4, EphA7, MSPR, Insulin receptor (IR), EphA1, IGF-1R, FGFR3, and FGFR1) was induced at least three-fold 24 hours after inhibition of AKT. The induction of the total tyrosine phosphorylation of HER4, in which 19 P-TYR sites have been identified, contrasts with the loss of phosphorylation of the Y1284 site on HER4 in Fig. 1A (Kaushansky et al., 2008). Overall, the data suggest that the phosphorylation of a group of receptors is feedback inhibited in response to PI3K-AKT activation and induced when AKT is inhibited.
In HER2 amplified tumor cells, overexpression of HER2 dysregulates PI3K-AKT signaling by promoting HER2-HER3 heterodimer formation. Our data suggests that AKT activation feedback downregulates HER3 and a set of other receptor tyrosine kinases in these cells. We asked whether this is a general phenomenon or peculiar to tumor cells in which HER2 drives PI3K-AKT signaling. We used the phospho-RTK arrays to determine if the effects of AKT inhibition on receptor phosphorylation were shared among different cancer models (Fig. 3A, Fig. S2). Two additional HER2 amplified cell lines (SK-BR3 and MDA-MB-453) were evaluated. When the results were quantified and the mean of the three cell lines taken, twelve receptors (HER3, IGF-1R, Insulin receptor, HER2, HER4, MSPR, SCFR, EphA1, EphA7, FLT-3, ROR1, and RET) had an induction greater than 3-fold after 24 hours of AKT inhibition. P-HER3 was highly induced in all three of these models. These results were compared with six tumor cell lines lacking HER2 amplification and representing four different cell lineages (LNCAP - prostate, IGROV-1 - ovarian, MDA-MB-231 - breast, and NCI-H292, NCI-H3255, NCI-H1975 - lung). In these six cell lines, nine receptors had average inductions above 3 fold (HER3, IGF-1R, Insulin receptor, MER, MET, RET, ROR1, TIE-2, and EphA7). Comparing the HER2 and non-HER2 lists, we noted overlap of IGF-1R, Insulin receptor, HER3, EphA7, and RET, which were significantly induced (>3 fold mean induction) in the majority of these cell lines. We evaluated the effect of AKT inhibition in a non-transformed, breast epithelial cell line, MCF10A, and found that only, IGF-1R, Insulin receptor and PDGFRa were induced after AKT inhibition. Fig. 3B lists the receptors in the ten cell lines for which average phosphorylation was induced at least 3 fold. The number of cell lines in which each receptor was induced and the average degree of induction are shown. IGF-1R, Insulin receptor, HER3, EphA7, and RET were induced in the majority of models, whereas HER4, EphA1, ROR1, and MET phosphorylations were induced in a subset. We conclude that inhibition of AKT induces the phosphorylation of a group of receptor tyrosine kinases and induction is not confined to a particular type of tumor or to tumors in which signaling is driven by HER2.
In BT-474 cells, AKT inhibition caused a parallel and equivalent induction of HER3 phosphorylation and expression, consistent with the possibility that the former is due to the latter. We evaluated whether induction of HER3, IR, and IGF-1R phosphorylation could be explained by induction in the expression of these proteins. In six out of seven cell lines without HER2 amplification, AKT inhibition caused increased levels of HER3 protein (Fig. 3C). Increases in Insulin receptor were detected in 5 out of 7 of these cell lines and IGF-1R was detected and induced in 6 out of 7 cell lines. In contrast, RET protein was detected and induced in only 2 out of the 7 cell lines and HER4 protein was not induced in any of the 7 (Fig. 3C). The induction in HER3, IGF-1R and Insulin receptor protein occurred in the majority of tested models and was not restricted to a specific tumor lineage. Moreover, in the non-transformed model, MCF10A, upregulation in the protein levels of HER3, Insulin receptor, and IGF-1R was also observed. It was notable that induction of phosphorylated IGF-1R and Insulin receptor, but not P-HER3 was seen in this model, which may be related to the comparably low levels of activated HER kinases in this cell. The average induction in protein level of these three receptors across these cell lines and additional HER2 amplified or EGFR mutant cancer cells was quantified by densitometry. All three receptors are commonly induced (HER3 – 13/14, IGF-1R – 12/14, Insulin Receptor – 14/14) with average inductions of HER3-2.8 fold, IGF-1R-2.2 fold, and Insulin receptor-2.5 fold.
Induction of expression of HER3, IGF1R or insulin receptor was almost always associated with increased phosphorylation (in 25 out of 28 instances). However, in most cases, induction of phosphorylation was out of proportion to induction of expression (Table S1). Therefore, the induction of phosphorylation of these proteins can only be partially explained by increased protein expression. Nevertheless, the data suggest that the expression of a common set of receptors is upregulated among cell lines and this correlates with the induction of their phosphorylation in response to AKT inhibition.
In most of the tumor cells we examined, the induction of HER3 phosphorylation was the most prominent effect of AKT inhibition. We used selective HER1/2 kinase inhibitors to determine whether induction of phosphorylation of receptors was HER kinase dependent. Lapatinib, a HER2/EGFR inhibitor that potently blocks HER2 kinase was used to address this question in breast cancer cells with HER2 amplification (Karaman et al., 2008). Gefitinib, which preferentially inhibits EGFR kinase activity, was used in H292, a non-small cell lung cancer cell line with overexpression of wild type EGFR. We compared the effects of the AKTi-1/2/3, Lapatinib, or the combination on the induction of receptor phosphorylation in the HER2-amplified breast cancer cell (BT-474) (Fig. 4A). After twenty four hours exposure to AKTi-1/2/3 (1μM, 2nd panel) phosphorylation of HER3, IGF-1R, IR and several other receptors was induced. When the HER1/2 kinase inhibitor (1μM) was combined with the AKT inhibitor, levels of phosphorylated HER3, IGF-1R, and Insulin receptor were repressed below basal levels. Treatment with Lapatinib alone blocked the phosphorylation of all the assessed RTKs below steady state levels. The effect of the HER kinase inhibitors is unlikely to be due to direct inhibition of IGF1R or insulin receptor kinases, to which they do not bind avidly (Karaman et al., 2008). Moreover, these inhibitors do not suppress IGF1R or insulin receptor phosphorylation in H1975 in which the T790M mutant EGFR is resistant to Gefitinib (Fig. S3). These data suggest that induction of IGF1R and IR phosphorylation is HER2-dependent in breast cancers in which this receptor is amplified.
We evaluated the HER kinase dependence of induction of receptor phosphorylation in a non-HER2 dependent model as well, NCI-H292. AKT inhibition resulted in induction of phosphorylated HER3, IGF-1R, Insulin receptor and several other receptors including FGFR1 and EphA7 (Fig. 4B, Fig. S3). In this case, the AKTi-induction of phosphorylation of some but not all of the RTKs could be suppressed by the HER1/2 inhibitor Gefitinib (5μM). For instance, phosphorylation of HER3 and Insulin receptor are suppressed below basal levels by the combination, and induction of P-EphA7 is significantly weaker. In contrast, induction of P-FGFR1 is unaffected by Gefitinib and IGF-1R phosphorylation is supra-induced by the combination. The data suggests that in cells in which HER2 is not the dominant receptor tyrosine kinase, the HER kinase-dependence of induction of receptor phosphorylation is variable.
AKT inhibition leads to a parallel induction of the expression and phosphorylation of IGF-1R, Insulin receptor, and HER3. We asked whether the induction of expression of these RTKs is dependent upon HER kinase activity as well. BT-474 cells were treated for 8 or 24 hours with 1μM AKTi-1/2/3, 1μM Lapatinib, or the combination (Fig. 4C). As expected, both inhibitors block AKT phosphorylation (P-S473). The induction of HER3 and IGF-1R/insulin receptor phosphorylation (antibody detects both receptors) by the AKTi was completely blocked by Lapatinib. In contrast, the induction of IGF-1R, Insulin receptor, and HER3 protein expression was unaffected by the addition of Lapatinib. Similarly, the AKTi-mediated induction of HER3 phosphorylation but not protein expression was blocked by Lapatinib in the H292 lung cancer cell line (Fig. 4D). These data show that the upregulation of receptor expression caused by AKT-inhibition is not dependent on HER kinase activity while the induction of phosphorylation is.
The mTORC1 complex is one of the primary downstream targets of AKT signaling. Direct inhibition of mTORC1 with rapamycin blocks S6Kinase-dependent feedback inhibition of IRS-1 expression and activates IGF-1R signaling (O’Reilly et al., 2006). We asked whether induction of receptor expression in response to AKT inhibition was mediated by mTORC1 inhibition. The effects of HER2, AKT, and mTORC1 inhibition on HER kinase expression and signaling were examined in BT474 cells (Fig. 5A). Cells were exposed to AKTi-1/2, Lapatinib, or Rapamycin for up to 24 hours. Each inhibitor causes potent inhibition of its respective target (P-HER2 for Lapatinib, P-S6K for Rapamycin, and P-AKT for AKTi-1/2) at 8 and 24 hours. Rapamycin and AKTi-1/2 each induce HER3 phosphorylation, although the effect of the latter is more marked (Fig 5A, 5B, Fig. S4). Both AKTi-1/2 and Lapatinib inhibit AKT phosphorylation and induce HER3 expression. In contrast, rapamycin does not induce expression, only phosphorylation. Thus, AKT and mTORC1 both regulate the feedback inactivation of HER kinase signaling. However, mTORC1 affects HER3 phosphorylation but not expression. AKT but not mTORC1 is responsible for the feedback regulation of HER3 expression.
We compared the effects of Rapamycin and AKTi-1/2 on the phosphorylation of receptor tyrosine kinases in BT474 (Fig. 5B). After 24 hours of treatment, the effects of the two drugs were markedly different. Rapamycin induced HER3 phosphorylation more than 4-fold (2.5 fold-less than the AKTi-1/2) and had no effect on Insulin receptor or IGF1-R phosphorylation. Several other receptors (FLT-3, HER4, MSPR, EphA1, and EphA7) whose phosphorylation was induced by AKT inhibition were either less affected or unaffected by Rapamycin. In contrast, rapamycin induced the phosphorylation of two receptors (MER and MuSK) more potently than AKT inhibition did. These findings suggest that inhibition of AKT and mTORC1 relieve different aspects of PI3K-induced feedback.
As levels of HER3, IGF-1R, and Insulin receptor protein increase after AKT inhibition, we determined whether the mRNAs encoding these receptors were affected as well. We compared the effects of AKT inhibition and mTORC1 inhibition on HER2, HER3, IGF-1R, Insulin Receptor, RET, and MET mRNA levels. Insulin Receptor, HER3, and IGF-1R mRNA were all induced between 2.5 and 5 fold after exposure of cells to either the AKT inhibitor or Lapatinib but not to Rapamycin (Fig. 6A). Rapamycin induced only minor changes in mRNA expression. Expression of HER2, RET, and MET mRNAs was not induced by any of the inhibitors. The results confirm that relief of feedback inhibition of RTK signaling in response to AKT inhibition cannot be explained by inhibition of mTORC1 alone. Feedback inhibition of mitogenic signaling in response to AKT activation must be mediated by other targets as well.
FOXO transcription factors regulate a number of genes involved in cell survival and longevity and are inhibited when phosphorylated by AKT (Kenyon et al., 1993; Kops et al., 1999; Myatt and Lam, 2007). The Drosophila Insulin Receptor has been demonstrated to be dependent upon the activity of the FOXO transcription factor for its expression (Ayala et al., 1999; Puig and Tjian, 2005). As inhibition of the PI3K-AKT pathway causes dephosphorylation of FOXO proteins and prevents their nuclear translocation, we investigated whether mammalian FOXO transcription factors may be involved in the induction of the RTKs by the AKT inhibitor (Myatt and Lam, 2007). FOXO1, 3 and 4 were depleted individually and in combination using specific, small interfering RNAs and the effect on basal and induced levels of receptors was examined (Fig. 6B). In BT-474 cells, knockdown of these factors alone or in combination had little effect on steady state levels of HER3, IGF-1R, or Insulin receptor mRNA expression. However, the induction of receptor mRNA by the AKT inhibitor was markedly diminished by FOXO knockdown. Of the three isoforms, inhibition of FOXO3 expression had the most potent effect, diminishing HER3, IGF1R, and insulin receptor induction by 64, 90, and 69%. Knocking down FOXO1 or FOXO4 had less effect, diminishing HER3 and Insulin Receptor inductions by <20%. However, combined knockdown of all three isoforms was most potent, diminishing HER3, IGF1R, and insulin receptor induction by 73, 91, and 80% of that in the control cells. Knockdown of FOXO isoforms had comparable effects on receptor protein expression in BT-474 cells and the non-transformed MCF10A cell line (Fig. 6D and Fig. S5). Moreover, the induction of phosphorylation of HER3, IGF-1R, and Insulin Receptor was significantly reduced in cells in which FOXOs were silenced (Fig. S5). Induction of P-HER3 was reduced nearly three-fold whereas inductions of P-IGF-1R and P-Insulin receptor did not occur. The incomplete suppression of induction of HER3 phosphorylation in cells in which FOXO expression was knocked down is consistent with the modest induction of HER3 phosphorylation by Rapamycin (Fig. 5). This suggests that the increase in RTK phosphorylation in response to AKT inhibition is due both to a FOXO-dependent increase in receptor expression and to increased phosphorylation in response to TORC1 inhibition. These data show that activation of FOXOs in response to AKT inhibition is required for the upregulation of RTK expression.
FOXO protein binding to the HER3, IGF-1R, and Insulin receptor promoters and whether binding is altered in response to AKT inhibition was examined by chromatin immunoprecipitation assays (Fig. 6C). BT-474 cells were treated with AKT inhibitor, fixed with formalin, and the total chromatin was collected. Immunoprecipitation with a combination of antibodies against FOXO1, FOXO3 and FOXO4 resulted in enhanced amplification of the RTK 5’UTR over input chromatin as measured by RT-PCR. Exposure of cells to the AKT inhibitor resulted in an enhancement of RTK amplification (HER3 – 4.3 fold, IGF-1R – 2.4 fold, INSR – 2.3 fold), whereas inhibition of FOXO protein expression with siRNA abolished this effect. These data suggest that activation of FOXO proteins is directly responsible for induction of RTK transcription.
The prevalence of AKT activation in human tumors has led to a widespread effort to develop inhibitors of the pathway. Relief of feedback inhibition of signaling by these inhibitors may limit their therapeutic effects. The induction of HER3 expression in response to the AKTi-1/2/3 in BT-474 cells is associated with increased HER3 binding to PI3K and to HER2 as shown by co-immunoprecipitation and cross-linking experiments in Fig. S1.
We used HER kinase inhibitors to ask whether blocking induction of HER family kinase activity enhances the antitumor efficacy of AKT inhibitors in vivo. We investigated the effects of the combination in two models in which AKT inhibition induces HER3: BT474, in which HER2 is amplified (Fig. 7A) and H292 non-small cell lung cancer in which wild type EGFR is overexpressed (Fig. 7B). In both models, AKT inhibition induces HER3 and IGF1R/Insulin receptor phosphorylation (7C, 7D).
In the BT474 model, chronic administration of the AKT inhibitor completely inhibits tumor growth, but does not cause regression ((7A).7A). In H292, the AKT inhibitor causes a marginal decrease in growth rate ((7B).7B). In order to determine whether inhibition of feedback induction of HER kinases increases the therapeutic effects of the AKT inhibitor, we used low doses of HER kinase inhibitors that affect baseline HER3 phosphorylation only minimally, but do prevent induction of HER3/IGF1R/Insulin receptor phosphorylation in response to the AKT inhibitor (7C,D). Although Lapatinib given at a more frequent schedule has significant antitumor effects in this model, at this schedule the Lapatinib had little effect. However, it significantly altered the response to the AKT inhibitor and the combination caused partial tumor regression. Maximal doses of the AKT inhibitor alone cause growth arrest without regression in this model.
In the H292 model, the AKT inhibitor did not significantly block tumor growth. However, the addition of the EGFR kinase inhibitor to the AKTi-1/2/3 caused significant and persistent tumor regression ((7B).7B). Iressa by itself did significantly inhibit tumor growth but, as previously reported, the effects were transient and significantly less than the combination (Buck et al., 2008). Thus, doses of HER kinase inhibitor sufficient to prevent relief of feedback inhibition of HER3 by the AKT inhibitor sensitize tumors to this drug. In keeping with these data, we find that prevention of receptor tyrosine kinase induction with an Hsp90 inhibitor also sensitizes tumors to AKT inhibitors and causes profound tumor regression in the BT474 model (Fig. S6).
It is increasingly clear that dysregulation of mitogenic signaling by constitutively activated oncoproteins in cancer cells drives high levels of feedback inhibition of the signaling network (Courtois-Cox et al., 2006; O’Reilly et al., 2006; Pratilas et al., 2009). This may have important phenotypic consequences in the transformed cell. Hyperactivation of signaling by the oncoprotein may depend on its relative insensitivity to negative feedback or to other mutations that inactivate elements of the feedback machinery. Anticancer drugs that inhibit oncoprotein function will relieve this negative feedback and thus reactivate multiple signaling pathways that limit the extent and duration of the anticancer effects.
The PI3K-AKT signaling pathway is a central downstream effector of growth factor receptors and is often dysregulated in cancer. The Insulin and IGF1 receptors exert many of their physiologic effects by activating PI3K, but mutation or overexpression of other receptors, such as HER2 in breast cancer, commonly dysregulate the pathway in tumors (Holbro et al., 2003; Kooijman et al., 1995). Activation of PI3K-AKT leads to activation of many downstream targets that together account for the proliferative, antiapoptotic and metabolic effects of the pathway. Of these, the mTOR kinase has attracted much attention because of its central function in integrating nutrient and energy availability and growth signals in the regulation of cell proliferation and size. mTOR functions in two multiprotein complexes, mTORC1 and mTORC2 (Guertin and Sabatini, 2007). The natural product rapamycin is a specific inhibitor of mTORC1 and leads to dephosphorylation of its two most well characterized substrates S6 kinase and 4EBP1(Brunn et al., 1997; von Manteuffel et al., 1997). It thereby inhibits cap-dependent translation and cell proliferation.
Experiments with rapamycin first revealed the extent and clinical implications of oncogene induced feedback. Insulin signaling is feedback regulated in part, by an mTOR/S6K-dependent phosphorylation and downregulation of the major insulin receptor substrate IRS1 (Haruta et al., 2000). Inhibition of mTOR with rapamycin relieves this feedback, activates insulin and IGF signaling, and thereby activates PI3K and ERK signaling (O’Reilly et al., 2006). This occurs in vivo in patients and likely decreases the therapeutic efficacy of the drug (Mellinghoff et al., 2005; O’Reilly et al., 2006).
PI3K and AKT regulate many processes besides mTORC1 activity. We reasoned that, in tumor cells, mutational activation of the PI3K-AKT pathway would induce mTOR independent feedback pathway as well. We used a selective, allosteric inhibitor of AKT to assess AKT-dependent feedback in breast tumor cells in which the pathway is driven by amplification of HER2. We found that inhibition of AKT in these cells induced the expression of HER3. There was a concomitant induction of HER3-HER2 heterodimers and a marked induction of HER3 phosphorylation.
The results are consistent with the idea that AKT activation causes feedback inhibition of HER kinase expression, especially of HER3, which, when phosphorylated docks with and activates PI3K. The induction of HER3 in response to AKT inhibition is associated with an increase in HER2-HER3 heterodimers and leads to increased HER3 phosphorylation. HER3 phosphorylation is blocked by the HER1/2 kinase inhibitor Lapatinib, but the increase in HER3 expression is not. This finding suggests that the increase in HER3 expression is in large part responsible for the observed increase in phosphorylation.
HER3 expression is induced by inhibitors of PI3K or AKT or by knockdown of AKT. That induction of HER3 expression and phosphorylation in response to AKT inhibition represents release of AKT-dependent feedback inhibition of the pathway is supported by the downregulation of HER3 expression that occurs when the AKT inhibitor is washed out of cells. We used phospho-receptor tyrosine kinase arrays to ask whether AKT-induced negative feedback was confined to HER3 or involved other receptors as well. We found that, although HER3 induction was very prominent, the phosphorylation of multiple other receptors was induced as well. Induction of receptor phosphorylation was not confined to HER2-dependent breast cancers, it occurred in tumor cells derived from all lineages tested (breast, prostate, ovary, lung, melanoma). We identified a set of nine RTKs whose phosphorylation is commonly induced after AKT inhibition. Four of these (HER3, IGF1R, Insulin receptor, and EphA7) responded in almost all cells tested.
Phosphorylated HER3 has a high capacity and affinity for PI3K, docking it to the membrane. The most obvious physiologic role of PI3K-AKT signaling is mediating the effects of the Insulin and IGF1 receptors. It seems from our data that these three receptors are coordinately feedback downregulated by AKT when the pathway is activated. AKT inhibition induces the expression as well as the phosphorylation of HER3, IGF1R, and Insulin receptors. Induction of other kinases such as RET and HER4 is confined to phosphorylation; expression is typically unaffected. HER2 is the dominant activated kinase in breast cancers in which it is amplified and, in these tumors, the induction of phosphorylation of the other RTKs is HER2 dependent. Lapatinib blocks their phosphorylation, but not the induction of expression of IGF1R and IR. Previous work by other labs has demonstrated that IGF1R and Insulin receptor kinases are not antagonized by Lapatinib at doses as high as 3μM and the lack of effect of Lapatinib upon IGF1R and Insulin receptor in non-HER2 driven models like H1975 (Fig. S3) supports that the activity seen here is not due to direct inhibition of those kinases. Whether the HER-kinase dependence of induction of IGF-1R/insulin receptor phosphorylation represents transphosphorylation of these kinases by HER2 or a HER2 dependent activation of autophosphorylation is under investigation. Activation of IGF1-R and Insulin receptor by AKT inhibition does involve both induction of expression and HER-kinase dependent phosphorylation of these kinases. In the non-small cell lung cancer model H292, the induction of phosphorylation of some RTKs like HER3 is HER kinase inhibitor sensitive. Others, such as FGFR and IGF1R are insensitive. It is clear that activation of AKT in tumors induces a complex and broad pattern of feedback inhibition of RTKs that is relieved by inhibition of AKT.
TORC1 inhibition by rapamycin has also been shown to activate signaling and less selective PI3K inhibitors that target both mTORC1 and PI3K have been shown to induce HER3 expression (Amin et al., 2010; Sergina et al., 2007). We asked whether AKT inhibition activated signaling via inhibition of mTORC1. Rapamycin partially reproduced the effects of AKT inhibition, inducing the phosphorylation of HER3 along with several other RTKs. However, induction of HER3 was considerably weaker than that observed with AKT inhibition and the phosphorylation of most of the RTKs induced by AKT inhibition was unaffected by rapamycin. The differences between the effects of the AKT inhibitor and rapamycin suggest that there are AKT-regulated feedback pathways that are not mediated by TORC1.
A clue to the nature of these pathways came from studies on the mechanism of induction of expression of HER3, IGF1R and insulin receptor. AKT inhibition results in marked induction of the mRNAs encoding these receptors, whereas rapamycin has either no or marginal effects. AKT has been shown to phosphorylate the FOXO family of transcription factors and thereby prevent their nuclear translocation, thus inhibiting their function (Brunet et al., 1999). We show that AKT inhibition recruits FOXO proteins to the HER3 promoter and that FOXO1/3/4 knockdown with siRNA suppresses the induction of IGF1-R/IR/HER3 expression and phosphorylation. We note that the knockdown of FOXO proteins has little effect on the basal expression of the RTKs. We postulate that in these cells with activated PI3K/AKT signaling, FOXOs are effectively inhibited and expression of HER3, IGF1R and IR are dependent on other factors in this state. However, AKT inhibition results in activation of these transcription factors enabling them to promote RTK expression. We thus conclude that AKT regulates the expression of these receptors by inhibiting FOXO-dependent transcription.
We propose the following model based on our current understanding to explain the regulation of PI3K-AKT signaling by negative feedback in tumors and how it is affected by targeted drugs (Fig. 8). Receptor activation of PI3K-AKT causes AKT-dependent phosphorylation of FOXO proteins which downregulate the expression of some of the receptors that are tightly coupled to PI3K, including HER3, IGF1R and insulin receptor. In addition, AKT activation leads to activation of TORC1 and S6K which feedback inhibits IRS1 expression and other undefined regulators of receptor signaling. The result is downmodulation of the signal.
Therapeutic inhibition of different components of the pathway reactivates feedback, but by mechanisms specific to the inhibited target (Fig. 8). Thus, AKT inhibition will result in activation of FOXO-dependent transcription of receptors and inhibition of S6K-dependent inhibition of signaling with resultant activation of multiple receptors. The downstream effects of AKT will be suppressed, but other RTK driven signaling pathways will be activated. In contrast, TORC1 inhibition blocks S6K-dependent feedback, activates IGF and HER kinases but not their expression and thus activates both AKT and ERK signaling.
These findings have important basic and therapeutic implications. The enhancement of signaling by autocrine activation or mutation of RTKs that activate PI3K-AKT signaling would be expected to be limited by negative feedback. Selection of oncogenes that encode proteins that overcome or are unresponsive to feedback would be favored (e.g. activating mutation in PI3K or loss of PTEN). All drugs that inhibit components of dysregulated mitogenic signaling pathways would be expected to relieve feedback inhibition of other components of the signaling network. This may reduce the antitumor effects of the drug, but also ameliorate toxicity. Combined inhibition of the oncoprotein and key pathways reactivated by inhibition of negative feedback should have enhanced antitumor activity. This is consistent with our finding that the AKT inhibitor causes tumor regressions when combined with low doses of HER kinase or HSP90 inhibitors that prevent or attenuate induction of receptor phosphorylation. Whether effective inhibition of both PI3K-AKT signaling and feedback reactivated pathways will have an enhanced therapeutic index will have to be evaluated in clinical trials.
AKTi-1/2 and AKTi-1/2/3 (MK-2206) were from Merck; both drugs were dissolved in DMSO for in vitro studies. For in vivo studies, AKTi-1/2 was dissolved in 25% hydroxypropyl β-cyclodextrin (pH 4–5), and administered subcutaneously while AKTi-1/2/3 was dissolved in 30% Capitisol and administered by oral gavage. A detailed list of other reagents utilized is provided in supplemental procedures.
Lysates from cells in culture were prepared by washing twice in cold PBS followed by lysis with either SDS-lysis buffer (50mM Tris-HCl, (pH7.4) 2% SDS) or RIPA-lysis buffer supplemented with protease and phosphatase inhibitors (Pierce Chemical). For immunoprecipitations, cells were lysed with NP40-lysis buffer ([50 mmol/L Tris (pH 7.4), 1% NP40, 150 mmol/L NaCl, 40 mmol/L NaF) supplemented with protease and phosphatase inhibitors (Pierce Chemical). For lysis in SDS, lysates were boiled for 5 minutes followed by brief sonication. Lysates were cleared by centrifugation at 14,000×g (10min) and the supernatant was collected. Protein concentration of each sample was determined using the BCA kit (Pierce) per manufacturer’s instructions. 25 or 50 μg protein was loaded onto 6 or 10% SDS-PAGE minigels for immunoblotting. Further details on immunoprecipitation and cross-linking provided in supplemental procedures.
Taqman reactions were done using an ABI 7500 real-time quantitative PCR system. For data analysis, raw counts were normalized to housekeeping gene average for the same time point and condition (ΔCt). Counts are reported as fold change relative to the untreated control (2–ΔΔCt). A list of probes and reagents used are provided in the supplemental procedures.
Chromatin immunoprecipitation (ChIP) assays were performed according to the manufacturer’s instructions (MagnaChIP assay kit; Upstate Biotechnology). Approximately 1 × 107 cells were used for each immunoprecipitation. DNA was amplified by PCR using primer pairs against the HER3 5’ UTR (GPH003183(-)01A from SA Biosciences), IGF-1R 5’ UTR (GPH004604(-)02A), and Insulin receptor 5’ UTR (GPH019975(-)01A) and SYBR Green reaction mix (SA Biosciences) and product was quantified using an ABI 7500 real time quantitative PCR system. Reactions were performed in triplicate and the mean normalized to input chromatin and reported as relative fold enrichment ± SEM.
BT-474, SKBr-3, MDA-MB-453, MDA-MB-361, LNCAP, NCI-H292, MDA-MB-231, HCC-1806, NCI-H441, MCF10A, UACC-893, and NCI-H1975 were obtained from the American Type Culture Collection. H3255 cells were a gift from B. Johnson and P. Janne (Dana Farber Cancer Institute) and IGROV-1 cells were a gift from D. Spriggs (MSKCC). Details on medium conditions and use of cell lines in xenograft studies are found in supplemental methods.
4-6 week old nu/nu athymic BALB/c female mice were obtained from the NCI-Frederick Cancer Center and maintained in pressurized ventilated caging. All studies were performed in compliance with institutional guidelines under an IACUC approved protocol (MSKCC#09-05-009). For efficacy studies, mice with well-established tumors were selected and randomized approximately fourteen days post-implantation (Size > 200mm3); BT-474 xenograft tumors were established in nude mice by subcutaneously implanting 0.72 mg sustained release 17β-estradiol pellets with a 10g trocar into one flank followed by injecting 1 × 107 cells suspended 1:1 (volume) with reconstituted basement membrane (Matrigel, Collaborative Research) on the opposite side 3 days afterwards. Mice were treated with AKTi-1/2, AKTi-1/2/3, Gefitinib, SNX5422, or Lapatinib with the indicated doses. Tumor dimensions were measured with vernier calipers and tumor volumes calculated (π/6 × larger diameter × (smaller diameter)2).
Human Phospho-RTK arrays were utilized according to manufacturer’s instructions. Briefly, cells were washed with cold PBS and lysed in NP40 lysis buffer and 50-250μg of lysates incubated with blocked membranes overnight. Membranes were subsequently washed and exposed to chemiluminescent reagent and exposed to x-ray film. Quantification of was pixels was performed by densitometry using Adobe CS2 and Fuji Film Multi Gauge software.
Constitutive feedback inhibition of upstream signaling pathways is a fundamental feature of tumors with activated oncoproteins. AKT activation leads to feedback by inhibiting the nuclear localization of FOXO and other transcription factors and activating mTOR kinase. The antitumor effects of inhibitors of oncoproteins may be attenuated by relief of feedback. In tumors with activation of PI3K-AKT signaling, AKT inhibitors activate receptor signaling and expression by activating FOXO and inhibiting mTORC1. In contrast, rapamycin inhibits mTORC1, but activates AKT. Thus, inhibitors of different components of the activated pathway will relieve different aspects of negative feedback. Effective therapy may require combining signaling inhibitors with inhibitors of the specific pathways they induce.
This work is supported by the National Institute of Health Program Grant: P01-CA094060, the Breast Cancer Research Foundation, the European Research Council (AdG09 250244), and the generous support of Arlene Taub. S. Chandarlapaty is supported by a National Institutes of Health Mentored Clinical Scientist Award: K08-CA134833.
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