Selective inhibitors of Abl tyrosine kinase (TK) are effective in putting nearly all patients with bcr-abl driven leukemia in chronic phase into complete remission 11
. This proof of concept sheds new hope for the treatment of other TK-driven cancers. Another important TK family is the human epidermal growth factor receptor (HER) family consisting of EGFR, HER2, HER3, and HER4. A subset of breast cancers are driven by overactive EGFR or HER2 TKs and an abundance of data from in vitro
and mouse models suggests that continued activity of these TKs drives cancer progression 12–14
. While the complexities of this multi-member TK family are not yet fully understood, their oncogenic signaling functions should, in theory, be amenable to silencing by TK inhibitors (TKIs). Several orally bioavailable HER family selective TKIs are in preclinical and clinical development. Although in in vitro
biochemical assays these agents differ in their relative activities against individual HER kinase family members, in cell-based assays they are effective at inhibiting both EGFR and HER2 and equally effective at suppressing the growth of EGFR and HER2 driven tumor cells 15–19
. They are also effective at inhibiting EGFR and HER2 phosphorylation in patients tissues and tumors 5–8
. But these agents show very limited clinical anti-tumor activity 1–5
. Their clinical development to this point has been driven largely by the detection of modest delays in tumor progression. The failure to reverse cancer progression despite an apparent inhibition of HER kinase function has created an enigma in the concept of TKI therapy of cancer that we have been exploring.
It is through heterodimerization and transphosphorylation that the HER family performs its signaling functions. Importantly, downstream PI3K/Akt pathway signaling is predominantly mediated through the transphosphorylation of the kinase-inactive member HER3 9,10
. We have previously reported that sensitivity to HER family TKI therapy correlates with the inhibition of PI3K/Akt pathway signaling 15,20
. We and others have also reported that failure to inhibit PI3K/Akt signaling leads to TK inhibitor resistance 20–22
. In contrast to reports from in vitro
models, Akt activity is not inhibited in most patients on HER TKI therapy 5,6,8
. This discordancy has led us to look more closely at the inhibition of PI3K/Akt signaling.
To investigate this discrepancy, we studied the durability of Akt inhibition by TKI with surprising results. Although as previously reported, gefitinib inhibits Akt signaling in HER2-driven cancer cells, this inhibition is not durable. Akt signaling resumes after a transient inhibition despite continued drug therapy (). In light of this finding, we looked at the broader HER family signaling activities over a period of 96 hours following continuous exposure of BT474 breast cancer cells to gefitinib at concentrations that nonselectively inhibit EGFR and HER2. TKI treatment effects a sustained inhibition of EGFR and HER2 phosphorylation and a durable inhibition of downstream MAPK and JNK pathway signaling (). However phosphorylation of the kinase-inactive family member HER3 is merely transient. HER3 signaling resumes and persists despite continued drug exposure and effective suppression of EGFR and HER2 (). The reactivation of HER3 signaling explains the reactivation of Akt signaling since HER3 is the principal HER family member that binds PI3K and drives Akt signaling 9,10
. TKI-refractory Akt signaling remains sensitive to PI3K inhibitors as expected (not shown). These time-dependent findings are not due to drug degradation since the drug is replenished daily in these studies and HER3/Akt signaling resumes despite repeatedly refreshing drug supply up to and beyond the point of resumption of Akt signaling (not shown). There is no significant expression of HER4 before or after drug treatment in these cells (data not shown). These findings are not unique to BT474 and SkBr3 cells and have been confirmed in other HER2 overexpressing breast cancer cells including MDA-453, AU565, MDA-361, HCC1954 (supplementary figure 1
). These findings are not unique to gefitinib and are seen with other HER TKIs including agents with in vitro
selectivity profiles favoring EGFR or HER2, such as erlotinib or AG825 (). These findings are not artifacts of the in vitro
models either. Treatment of mice bearing various HER2-driven xenograft tumors with gefitinib similarly fails to durably supress HER3 and Akt signaling, despite a transient suppression (, and supplementary figure 2
). This is not due to ineffective drug biodistribution, since in these models gefitinib was dosed three times higher than doses known to achieve sustained xenograft tumor concentrations above 2–4uM and averaging 6–10uM 23
. Since we had previously established that inactivation of PI3K/Akt signaling is mechanistically linked to HER family TK inhibitor sensitivity in HER family driven cancers, we felt that the failure of these drugs to durably inactivate PI3K/Akt signaling is entirely consistent with their limited clinical activities. Therefore we set out to study the molecular basis by which HER3 evades TKI therapy.
HER TK inhibitors fail to induce sustained inhibition of HER3 signaling in HER2-driven breast cancer cells
TKI-refractory HER3 phosphorylation is due to HER2, since it can be suppressed by anti-HER2 siRNA transfection (). Although cross talk between receptor families can occur, we have found no evidence for non-HER family TKs mediating TKI refractory HER3 phosphorylation. The reactivation of HER3 signaling is not associated with the induction of any new TKs and remains resistant to the broad spectrum kinase inhibitor staurosporine (not shown). This apparent desensitization of HER3 signaling to TKIs is due to a forward shift in the equilibrium of the HER3 phosphorylation-dephosphorylation reactions establishing a new steady state HER3 phosphorylation despite significant inhibition of HER2 kinase and autophosphorylation activity by TKIs. This forward shift becomes clearly evident in the form of HER3 and Akt superphosphorylation when drug inhibition is withdrawn during the new steady state (). HER3 phosphorylation remains suppressible by TKI in the new steady state, but much higher concentrations are required to completely dephosphorylate HER3 since the uninhibited HER3 phosphorylation state is significantly higher in the new steady state (, compare left and right). Therefore drug-refractory HER3 phosphorylation is due to resistance at the level of the substrate HER3, and is driven by residual HER2 kinase activity. Similar characteristics apply to the more potent irreversible TKIs 24
. The irreversible pan-HER family TKI PD168393, when used at partially or near-maximal inhibitory concentrations, induces a similar desensitization of HER3 to continued drug therapy (, 0.1–0.2uM doses). However, at fully inactivating concentrations both reversible (, 40uM dose) and irreversible TKI () can durably suppress HER3 and Akt signaling.
Forward shift in HER3 phosphorylation-dephosphorylation equilibrium following extended HER TKI treatment
The biological consequence of drug-refractory HER3 and Akt signaling is tumor cell survival. In fact the anti-proliferative activity of TKIs is reversible and tumor cells resume proliferative growth after drug withdrawal. If drug-refractory HER3 signaling is averted by anti-HER3 siRNA, TKI treatment of HER2-driven cancer cells leads to apoptotic cell death (, and supplemental figure 4
). This is the expected outcome of effective oncoprotein inactivation and recapitulates the apoptotic fate of oncogene withdrawal seen in reversible transgenic models of HER2 tumorigenesis 14
. Sustained inhibition of HER3 signaling using TKIs at their fully inactivating doses (from ) also leads to apoptotic tumor cell death not seen with doses that allow HER3 escape ().
The TKI-induced forward shift in the HER3 phosphorylation-dephosphorylation steady state is due to increased HER3 substrate concentration driving the forward reaction, and decreased phosphatase activity impeding the reverse reaction. Increased HER3 substrate concentration occurs through a significant increase in HER3 expression at the plasma membrane where the phosphorylation reaction occurs (). Unlike HER2 which is predominantly localized to the plasma membrane, the HER3 pool is largely within intracellular compartments with some membrane expression 25
. The TKI-induced forward shift in HER3 steady state phosphorylation is driven by HER3 relocalization to the plasma membrane and can be suppressed by inhibitors of vesicular trafficking (). The HER3 dephosphorylation rate is also slowed after 48 hours of TKI exposure (). The retarded HER3 dephosphorylation rate may be due to reduced access to cytosolic protein tyrosine phosphatases (PTPs) due to altered endocytic trafficking, or it may be due to inhibition of PTPs. In support of the latter, TKI therapy leads to increased cellular reactive oxygen species (ROS) () which are known to inhibit PTPs and are emerging as an important regulator of PTP activity 26,27
. Consistent with this, drug-refractory HER3 signaling can be suppressed by concomitant treatment with certain anti-oxidants ().
Mechanism of HER3 reactivation following extended HER TKI treatment
The changes in steady state HER3 signaling that evolve with TKI treatment are driven by the loss of Akt signaling and likely involve Akt-mediated negative feedback signaling. Consistent with this, HER3 signaling does not escape TKI treatment when a constitutively active Akt is transfected (). Conversely, inhibition of Akt signaling by a PI3K inhibitor leads to a compensatory increase in HER3 phosphorylation (). Complete inactivation of HER2 kinase with high doses of TKIs induces the maximum feedback signaling and HER3 redistribution, however due to the complete inactivation of HER2 kinase HER3 signaling cannot be restored and the feedback loop fails to rescue Akt activity.
Akt regulates HER3 signaling via negative feedback signaling