Sensitivity to targeted therapy differs between HER2-amplified breast cancer cells harboring kinase and helical domain mutations in PIK3CA

Abstract

Background: HER2-amplified breast cancer is a clinically defined subtype of breast cancer for which there are multiple viable targeted therapies. Resistance to these targeted therapies is a common problem, but the mechanisms by which resistance occurs remain incompletely defined. One mechanism that has been proposed is through mutation of genes in the PI3-kinase pathway. Intracellular signaling from the HER2 pathway can occur through PI3-kinase, and mutations of the encoding gene PIK3CA are known to be oncogenic. Mutations in PIK3CA co-occur with HER2-amplification in ~ 20% of cases within the HER2-amplified subtype.

Methods: We generated isogenic knockin mutants of each PIK3CA hotspot mutation in HER2-amplified breast cancer cells using adeno-associated virus-mediated gene targeting. Isogenic clones were analyzed using a combinatorial drug screen to determine differential responses to HER2-targeted therapy. Western blot analysis and immunofluorescence uncovered unique intracellular signaling dynamics in cells resistant to HER2-targeted therapy. Subsequent combinatorial drug screens were used to explore neuregulin-1-mediated resistance to HER2-targeted therapy. Finally, results from in vitro experiments were extrapolated to publicly available datasets.

Results: Treatment with HER2-targeted therapy reveals that mutations in the kinase domain (H1047R) but not the helical domain (E545K) increase resistance to lapatinib. Mechanistically, sustained AKT signaling drives lapatinib resistance in cells with the kinase domain mutation, as demonstrated by staining for the intracellular product of PI3-kinase, PIP₃. This resistance can be overcome by co-treatment with an inhibitor to the downstream kinase AKT. Additionally, knockout of the PIP₃ phosphatase, PTEN, phenocopies this result. We also show that neuregulin-1, a ligand for HER-family receptors, confers resistance to cells harboring either hotspot mutation and modulates response to combinatorial therapy. Finally, we show clinical evidence that the hotspot mutations have distinct expression profiles related to therapeutic resistance through analysis of TCGA and METABRIC data cohorts.

Conclusion: Our results demonstrate unique intracellular signaling differences depending on which mutation in PIK3CA the cell harbors. Only mutations in the kinase domain fully activate the PI3-kinase signaling pathway and maintain downstream signaling in the presence of HER2 inhibition. Moreover, we show there is potentially clinical importance in understanding both the PIK3CA mutational status and levels of neuregulin-1 expression in patients with HER2-amplified breast cancer treated with targeted therapy and that these problems warrant further pre-clinical and clinical testing.

Fig. 1

Gene targeting validation in SKBR3 cells and response of knockin derivative cells to lapatinib treatment. A Targeted Sanger sequencing of E545K locus (left) and H1047R locus (right) on both genomic DNA (top) and cDNA (bottom) shows correct targeting in two clones for each mutation (arrows). B Drug response to lapatinib after 72 h shows that overexpression of PIK3CA mutations (top panel) makes cells less sensitive to lapatinib treatment regardless of which mutation is introduced. Site-specific genome editing reveals that while the E545K mutation does not confer reduced sensitivity to lapatinib treatment (middle panel) whereas the H1047R mutation does (bottom panel). Error bars represent standard deviation of triplicate assays. C GR50 calculations for SK-BR-3 parental, control, and mutant clones shows that overexpression clones and H1047R mutant knockin clones have significantly higher GR50 values than parental and control cells, but that E545K knockin mutant clones do not. Error bars represent standard deviation of triplicate measurements. * denotes significant difference from control or parental cells by t-test (p < 0.05)

Fig. 2

Analysis of signaling pathways showing H1047R knockin cells maintain intracellular signaling in presence of lapatinib. A Whole cell lysates collected after 48 h of exposure to 500 nM lapatinib or vehicle were separated by gel electrophoresis and analyzed for the indicated proteins. H1047R knockin cells retain high levels of phospho-AKT and downstream substrates phospho-PRAS40 and phospho-S6 (red boxes) whereas E545K knockin cells do not. B Cells were treated with 500 nM lapatinib for a timecourse of 72 h. Whole cell lysates were collected at the indicated times and separated by gel electrophoresis and analyzed for the indicated proteins. H1047R knockin cells show recovery of phospho-AKT levels and persistent phosphorylation of S6 which is not seen in E545K knockin cells

Fig. 3

Response of knockin derivative cells to AKT inhibition alone or in combination with lapatinib. Drug response after 72 h shows minimal differential sensitivity to AKT inhibition with GSK690693 (red) among all cell lines. Equimolar combination of both GSK690693 and lapatinib (green) shows synergistic growth inhibition in cells overexpressing PIK3CA mutations or with knockin of the H1047R mutation but not the E545K mutation. Points at which significant synergistic interactions as measured by the combination index between lapatinib and GSK690693 are indicated with an *. Lapatinib monotherapy data from Fig. 1B is repeated here in each graph for comparison (blue)

Fig. 4

Characterization of PTEN null clones reveals these cells closely phenocopy cells with H1047R mutation. A Combinatorial drug screen using lapatinib (blue), AKT inhibitor GSK690693 (red), or equimolar combination of both drugs (green) shows that knockout of PTEN makes cells less sensitive to lapatinib treatment, similar to H1047R knockin cells. B Whole cell lysates collected after 48 h of exposure to 500 nM lapatinib or vehicle were separated by gel electrophoresis and analyzed for the indicated proteins. C Cells were treated with 500 nM lapatinib for a timecourse of 72 h. Whole cell lysates were collected at the indicated times and separated by gel electrophoresis and analyzed for the indicated proteins

Fig. 5

PIP₃ signaling remains high in H1047R knockin but not E545K knockin cells after lapatinib treatment. A Confocal imaging of cells after 36 h of treatment with DMSO or lapatinib (500 nM). PIP3 staining can appear in clusters (yellow arrows, top left image). B Quantification of PIP₃ staining in cells using CellProfiler. Cell boundary was determined by phalloidin stain (not shown) and PIP₃ stain overlapping with phalloidin was calculated. Sum of pixel intensity of PIP₃ divided by the area overlapping with phalloidin was calculated for every cell per image. Average values across 15 images are shown in the graph. Error bars represent standard deviation. * and ** indicates PIP₃ intensity values for lapatinib-treated cells are significantly higher than untreated matched samples (* indicates p < 0.005 and ** p < 0.001)

Fig. 6

H1047R and E545K knockin cells are hyper-responsive to neuregulin-1 beta 1 (NRG1β) treatment. A Parental SK-BR-3 cells treated with NRG1β and lapatinib show rescue from lapatinib at high concentrations of NRG1β, but only partial rescue when treated with low doses of NRG1β (2 ng/mL). B The H1047R cells show complete rescue from lapatinib with all doses of NRG1β, even at the highest dose of lapatinib, suggesting that the mutant cells are more responsive to ligand stimulation than their wildtype counterparts. C The E545K cells also show complete rescue from lapatinib inhibition by all doses of NRG1β. Value shown are median cell counts relative to untreated controls. Error bars are ± standard deviation

Fig. 7

Addition of NRG1β de-sensitizes cells to lapatinib, co-treatment with AKT inhibitor or pertuzumab overcomes this effect. A Parental SKBR3 cells or PIK3CA mutant clones treated with lapatinib, AKT inhibitor GSK690693, or combination of the two drugs at a fixed molar ratio in the absence or presence of NRG1β for 72 h. Only H1047R knockin mutant cells show decreased sensitivity to lapatinib at baseline, consistent with results shown in Fig. 1. Treatment with NRG1β makes cells more resistant to lapatinib, and response is partially restored by co-treatment with GSK690693. The efficacy of the combination is greatest in the H1047R mutant cells as measured by the GR_max response. Significant synergistic interactions are marked with an *. B Parental SKBR3 cells or PIK3CA mutant clones treated with lapatinib, pertuzumab, or combination of the two drugs at a fixed molar ratio in the absence or presence of NRG1β for 72 h. Combination of drugs resulted in synergistic growth inhibition at low doses in all three cell lines, but offered no benefit over lapatinib alone at higher doses. Addition of NRG1β-induced resistance to lapatinib in all three cell lines was abrogated with the addition of pertuzumab. Note that synergy could not be accurately calculated by the combination index method for these curves since it requires fitting dose-response curves and pertuzumab did not have any effect on the growth of cells as a monotherapy

Fig. 8

Kinase domain mutant or PTEN-loss samples have significantly lower expression of ERBB2 and GRB7, by Mann-Whitney analysis. A Boxplots comparing expression levels of ERBB2 mRNA (top) or GRB7 mRNA (bottom) between wildtype PIK3CA samples and either mutant group in RNAseq data from TCGA. ***P value < 0.0001. B Boxplots of RPPA data for amount of HER2 protein in wildtype PIK3CA samples and either mutant group. *P value < 0.001. C Boxplots comparing expression levels of ERBB2 (top) or GRB7 (bottom) between wildtype PIK3CA samples and either mutant group in RNAseq data from METABRIC. *P value < 0.01