Introduction
The HER2 oncogene is amplified in nearly 25% of all primary breast cancers [
1] and has been a focus for targeted therapy development for decades. HER2 is a member of a family of four single-pass transmembrane receptor tyrosine kinases (EGFR (HER1), HER2, HER3, and HER4) that form hetero- and homodimers and activate diverse signaling pathways including phosphatidylinositol 3-kinase (PI3-kinase; reviewed in [
2]). The growth and survival of HER2-amplified tumors is dependent on HER2 function, so disruption of HER2 signaling is detrimental to these cells [
3,
4]. Several drugs have now been approved that specifically target HER2 signaling, including antibodies such as trastuzumab [
5], pertuzumab [
6], and small molecules that target the tyrosine kinase activity such as lapatinib [
7], tucatinib [
8], and neratinib [
9]. Ado-trastuzumab emtansine has also been approved, but has a distinct mechanism of action by delivering a cytotoxic agent conjugated to trastuzumab to specifically target HER2-positive cells [
10].
HER2-mediated PI3-kinase activation occurs through the recruitment of a 110-kDa catalytic subunit (encoded by PIK3CA) via a p85α regulatory subunit (encoded by PIK3R1) to the cell membrane. This complex activates signaling via phosphorylation of the membrane lipid PIP
2 (phosphatidylinositol (4,5) bisphosphate) at the 3′ position resulting in phosphatidylinositol (3,4,5) triphosphate (PIP
3). PIP
3 serves as a docking site for proteins that contain pleckstrin homology (PH) domains, such as the AKT family of proteins [
11]. AKT proteins influence a variety of processes inside the cell involving cell growth, regulation of apoptosis, glucose metabolism, and others [reviewed in [
12]].
Importantly, responses to HER2-targeted drugs are thought to be affected adversely by the co-occurrence of mutations that influence downstream PI3-kinase signaling [
13,
14]. PIK3CA is the most frequently mutated gene in this pathway with mutations occurring in about 20% of HER2-amplified breast cancers [
15]. PIK3CA mutations occur preferentially in two “hotspot” regions of the protein. One hotspot region lies in the kinase domain (H1047R) and the other in the helical domain of
PIK3CA (E542K and E545K) that binds the p85 regulatory subunit of PI3K [
16]. Helical and kinase domain mutations account for about 85% of all mutations seen in
PIK3CA in breast cancer [
17]. These mutations are sometimes combined in clinical outcome association studies [
18,
19] based in part on laboratory studies showing that overexpression of the two mutation types in biological models produces similar response phenotypes [
20,
21]. However, they are functionally distinct with crystallographic studies of p110α showing conformational differences between the two hotspot mutations [
16,
22].
We sought to better understand the impact of helical (E545K) and kinase (1047R) domain mutations on the therapeutic response of HER2+ cells to targeted inhibitors. We have previously reported generation of cell lines in which
PIK3CA mutations are overexpressed by conventional transgene expression and that
PIK3CA mutant cell lines show increased resistance to lapatinib [
21]. These cells required combination therapy using lapatinib plus an AKT inhibitor to restore sensitivity to the same levels as control cells. However, this methodology is limited because transgenic cells retain two endogenous wildtype copies of
PIK3CA as well as an unknown number of transgenic mutant alleles. We report here the generation of isogenic knockin mutants of each
PIK3CA hotspot mutation, which has the major advantage of maintaining
PIK3CA gene expression under control of the endogenous promoter, resulting in physiological levels of expression of the mutants. This is distinct from the majority of studies that have introduced mutations via transfection or transduction, which result in overexpression of the mutants. We report below our findings of the substantial differences of the impact of kinase versus helical domain mutations of PIK3CA on signaling and therapeutic responses to HER2-targeted receptor tyrosine kinase inhibitors in the HER2-amplified cell line, SK-BR-3.
Discussion
We generated isogenic knockin mutants of each PIK3CA mutation while maintaining the expression of PIK3CA under endogenous promoter control to study the impact of mutation on therapeutic response. While our analyses of these cell lines confirm that PIK3CA mutations can confer resistance to HER2-targeted kinase inhibitors such as lapatinib and neratinib, we find significant differences in drug response phenotype between the two mutation classes. Specifically, mutation in the helical domain does not confer resistance to lapatinib while mutation in the kinase domain does when PIK3CA is expressed at physiological levels. Our time course analysis of lapatinib-treated cells shows striking differences in AKT phosphorylation levels between the different mutants. Lapatinib does not durably suppress PI3K-AKT signaling in H1047R knockin mutant cells for much more than 12 h, but does suppress pAKT in E545K knockin mutants for at least 72 h. As a result, AKT signaling is maintained in cells carrying H1047R mutations, allowing the cell to escape apoptosis via canonical signaling through mTORC and ribosomal protein S6. Thus, the kinase domain mutation confers resistance by causing sustained AKT signaling and associated high levels of PIP3 in the presence of HER2 inhibitors. These kinase domain mutant cells respond synergistically to treatment with lapatinib and the pan-AKT inhibitor GSK690693. Cells carrying E545K mutations are not able to maintain PIP3 signaling in the presence of lapatinib and remain sensitive to lapatinib, and treatment with GSK690693 does not increase efficacy. Importantly, these results suggest that H1047R mutants are capable of maintaining signaling independent of signals from the receptor, whereas E545K mutants are not.
These results are quantitatively different than those obtained by overexpressing the PIK3CA mutations, which showed that both mutations increased resistance to lapatinib. The increased resistance to lapatinib observed in both of the overexpression clones suggests that introducing oncogenic mutations via overexpression may mask the true biology seen in cells in which a single mutant allele is driven from the endogenous promoter—the genotype that is most commonly seen in mutations of oncogenes. However, as we found that if we overexpressed the E545K mutant using retroviral vectors, we could achieve the same levels of resistance as observed in the H1047R mutants; it suggests that if the E545K mutation is also overexpressed in a tumor, it would likely cause resistance to therapy. Thus, our findings support the notion that if cells express the E545K mutants at physiological levels, then they are unlikely to be resistant to therapy, but overexpression of the same mutant will result in resistance. In contrast, H1047R mutants will result in resistance regardless of the level of expression.
Cells engineered to carry the H1047R kinase domain mutation upregulated PIP
3 during treatment with high doses of HER2-targeted therapy such as lapatinib. High levels of PIP
3 maintained high levels of phospho-AKT and hence activation of ribosomal protein S6. Active S6 combines with other proteins to form the 40S ribosomal subunit that then carries out translation initiation [
42]. Cells carrying the E545K helical domain mutation did not upregulate high PIP
3 levels during lapatinib treatment and remained sensitive to the drug. Although the mechanism by which this occurs remains unknown, we speculate that the kinase domain mutation decouples the PI3K pathway from the receptor, allowing it to function independently if growth signals from HER2 are removed. Thus, when HER2 is inhibited, the H1047R mutant of PIK3CA is able to compensate through increased activity, resulting in increased PIP
3 levels. In contrast, the E545K mutant still relies on signal from HER2 for activity, and thus is unable to increase PIP
3 to maintain signaling in the same manner. We showed that PI3-kinase is the signaling axis utilized in these cells since inhibition of the Ras-Raf-MEK-ERK axis had no effect on cell growth. Changes in other downstream targets of phospho-AKT signaling such as phosphorylation levels of 4E-BP-1 were not observed. This is not surprising because phosphorylation of 4E- BP-1 at Thr37/46 is insensitive to rapamycin [
43] and so it is possible that it is also insensitive to other perturbations of mTORC1 signaling investigated in this study.
We and others have shown previously that NRG1β interacts with the HER2/3 heterodimer to induce resistance to lapatinib and neratinib [
36]. We show now that the E545K and H1047R knockin cells became hypersensitized to NRG1β-mediated resistance to lapatinib, as lower levels of NRG1β were capable of conferring resistance to lapatinib than in wildtype cells. This potentially has important implications for patients, since the kinase domain mutation could impact lapatinib response on multiple levels—first by making cells more resistant at baseline, and second by making them more susceptible to NRG1β-mediated changes in HER2/3 heterodimer conformation that reduces the binding efficacy of lapatinib and neratinib. We previously reported that serum levels of NRG1β equivalent to the doses used in these studies can be found in patients [
36], suggesting that these effects are operational in the clinic. Indeed, high levels of NRG1β in all HER2+ breast cancer patients were reported to be correlated with higher rates of recurrence [
44], which may be exacerbated by the presence of PIK3CA mutations.
The differences in response phenotype between the kinase and helical domain mutants have not been observed before because the vast majority of previous studies have used traditional transgene overexpression to study the function of mutations of
PIK3CA. Our results support previous reports showing the value of knockin techniques to uncover subtle phenotypes that might otherwise be obfuscated by traditional transgene overexpression [
23,
30]. Slight differences in p110α kinase activity have been previously reported for kinase and helical domain mutations [
20]. However, our report is the first to show differential response to drug because of the proposed lower catalytic activity of the E545K mutation compared to H1047R mutation when both are expressed at physiological levels. Additionally, our results provide a functional definition for the term synergy in this particular case of drug combinations; specifically, in a lapatinib-resistant cell line, co-treatment with an AKT inhibitor and lapatinib restores the effect of drug to what is seen with lapatinib alone seen in wildtype cells.
Additionally, we show here that unique gene expression patterns exist depending on the
PIK3CA mutation. We speculated that because the H1047R mutants appear to be less reliant on signaling from the receptor, there might be less selective pressure to maintain high levels of HER2. Indeed, we found that the kinase domain and PTEN mutants had lower levels of expression of both HER2 and GRB7 in two large independent public data sets, whereas the helical domain mutants did not show this difference. Kinase domain mutant cells may not respond well to HER2-targeted therapy as there is significant downregulation of the gene
ERBB2 in these clinical samples and concomitant lower protein expression of HER2 not seen in helical mutant samples in addition to the PI3K mutation; this hypothesis is bolstered by published evidence that high levels of HER2 expression are predictive of response to lapatinib and trastuzumab [
45]. While it is impossible to know the sequence of genomic alteration in these tumors—that is, whether HER2 became amplified first or
PIK3CA acquired its mutation first—these findings may suggest a mechanism by which patients acquire resistance even prior to therapy. Specifically, the genomic locus of HER2 is amplified which is detected clinically at the DNA level and informs the clinician to start HER2-targeted therapy, but
PIK3CA also acquires a mutation in the kinase domain; this kinase mutation renders the protein constitutively active which circumvents the need for continuous signaling from HER2, causing downregulation of HER2 expression at the mRNA level and the protein level, thereby making the tumor refractory to HER2-targeted therapy. While this mechanism is speculative, the data we present here clearly show that the two hotspot mutations of
PIK3CA respond differently to HER2-targeted therapy in vitro and that clinical samples of HER2-amplified tumors with
PIK3CA mutation have unique gene expression patterns which support our results.
Ultimately, the phenotypic disparity between helical and kinase domain mutations of
PIK3CA may have important clinical implications. It is important to note that publicly available data [
46,
47] demonstrates that approximately 21% of HER2-amplified cases of breast cancer also harbor a mutation at one of the hotspots of
PIK3CA. Our results suggest that HER2+ tumors with helical domain mutations of
PIK3CA will respond to tyrosine kinase inhibitors such as lapatinib and neratinib whereas HER2+ tumors with H1047R mutations will not, although both a hypersensitive to resistance mediated by NRG1β. Our data and previous studies also suggest that adding pertuzumab to counter NRG1β-mediated resistance to lapatinib and neratinib will be important in HER2+ tumors carrying
PIK3CA mutations of either type. Altogether, we show that important phenotypic disparities exist between the two hotspot mutations of
PIK3CA. These data raise the intriguing possibility that patients may one day benefit from differential treatments based on PIK3CA mutational status. Our study sets the stage for the necessary pre-clinical animal studies and follow-up clinical investigations to validate these findings.
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