ABSTRACT
INTRODUCTION: Despite multiple advances in the treatment of HER2+ breast cancers, resistance develops even to combinations of HER2 targeting agents. Inhibition of PI3K pathway signaling is critical for the efficacy of HER2 inhibitors. Activating mutations in PIK3CA can overlap with HER2 amplification and have been shown to confer resistance to HER2 inhibitors in preclinical studies. METHODS: Lapatinib-resistant cells were profiled for mutations in the PI3K pathway with the SNaPshot assay. Hotspot PIK3CA mutations were retrovirally transduced into HER2-amplified cells. The impact of PIK3CA mutations on the effect of HER2 and PI3K inhibitors was assayed by immunoblot, proliferation and apoptosis assays. Uncoupling of PI3K signaling from HER2 was investigated by ELISA for phosphoproteins in the HER2-PI3K signaling cascade. The combination of HER2 inhibitors with PI3K inhibition was studied in HER2-amplified xenograft models with wild-type or mutant PIK3CA. RESULTS: Here we describe the acquisition of a hotspot PIK3CA mutation in cells selected for resistance to the HER2 tyrosine kinase inhibitor lapatinib. We also show that the gain of function conferred by these PIK3CA mutations partially uncouples PI3K signaling from the HER2 receptor upstream. Drug resistance conferred by this uncoupling was overcome by blockade of PI3K with the pan-p110 inhibitor BKM120. In mice bearing HER2-amplified wild-type PIK3CA xenografts, dual HER2 targeting with trastuzumab and lapatinib resulted in tumor regression. The addition of a PI3K inhibitor further improved tumor regression and decreased tumor relapse after discontinuation of treatment. In a PIK3CA-mutant HER2+ xenograft, PI3K inhibition with BKM120 in combination with lapatinib and trastuzumab was required to achieve tumor regression. CONCLUSION: These results suggest that the combination of PI3K inhibition with dual HER2 blockade is necessary to circumvent the resistance to HER2 inhibitors conferred by PIK3CA mutation and also provides benefit to HER2+ tumors with wild-type PIK3CA tumors.
Subject(s)
Breast Neoplasms/drug therapy , Drug Resistance, Neoplasm/drug effects , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors/pharmacology , Receptor, ErbB-2/antagonists & inhibitors , Aminopyridines/pharmacology , Animals , Antibodies, Monoclonal, Humanized/pharmacology , Antineoplastic Agents/pharmacology , Biomarkers, Tumor , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Class I Phosphatidylinositol 3-Kinases , Female , Humans , Lapatinib , Mice , Mice, Nude , Morpholines/pharmacology , Mutation , Phosphatidylinositol 3-Kinases/genetics , Quinazolines/pharmacology , Receptor, ErbB-2/metabolism , Trastuzumab , Xenograft Model Antitumor AssaysABSTRACT
Most estrogen receptor α (ER)-positive breast cancers initially respond to antiestrogens, but many eventually become estrogen-independent and recur. We identified an estrogen-independent role for ER and the CDK4/Rb/E2F transcriptional axis in the hormone-independent growth of breast cancer cells. ER downregulation with fulvestrant or small interfering RNA (siRNA) inhibited estrogen-independent growth. Chromatin immunoprecipitation identified ER genomic binding activity in estrogen-deprived cells and primary breast tumors treated with aromatase inhibitors. Gene expression profiling revealed an estrogen-independent, ER/E2F-directed transcriptional program. An E2F activation gene signature correlated with a lesser response to aromatase inhibitors in patients' tumors. siRNA screening showed that CDK4, an activator of E2F, is required for estrogen-independent cell growth. Long-term estrogen-deprived cells hyperactivate phosphatidylinositol 3-kinase (PI3K) independently of ER/E2F. Fulvestrant combined with the pan-PI3K inhibitor BKM120 induced regression of ER(+) xenografts. These data support further development of ER downregulators and CDK4 inhibitors, and their combination with PI3K inhibitors for treatment of antiestrogen-resistant breast cancers.
Subject(s)
Breast Neoplasms/genetics , E2F Transcription Factors/genetics , E2F Transcription Factors/metabolism , Estrogen Receptor alpha/genetics , Estrogen Receptor alpha/metabolism , Estrogens/deficiency , Animals , Breast Neoplasms/metabolism , Breast Neoplasms/therapy , Cell Line, Tumor , Cyclin-Dependent Kinase 4/genetics , Cyclin-Dependent Kinase 4/metabolism , Down-Regulation , Drug Resistance, Neoplasm , Estrogen Receptor Modulators/pharmacology , Estrogens/metabolism , Estrogens/pharmacology , Female , Gene Expression , Humans , Mice , Mice, Nude , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Transcription, GeneticABSTRACT
The cardiac potassium channel encoded by the human ether-à-go-go related gene (HERG) is blocked by a diverse array of common therapeutic compounds. Even transient exposure to such agents may provoke the life-threatening cardiac arrhythmia torsades de pointes in some, but not all, individuals. Although the molecular and genetic factors predicting such wide variability in drug response remain unclear, known sequence variations within the coding region of HERG do not explain the adverse drug response in many cases. Although other proteins can modulate HERG function, no studies have identified protein partners capable of limiting the pharmacological sensitivity of HERG. Here we show that KCR1, a protein identified previously in rat cerebellum, is a plasma membrane-associated protein expressed at the RNA level in the human heart and can be immunoprecipitated with HERG. Functionally, KCR1 reduces the sensitivity of HERG to classic proarrhythmic HERG blockers (sotalol, quinidine, dofetilide) in both cardiac and noncardiac cell lines. We propose that KCR1, when coupled to HERG, may limit the sensitivity of HERG to proarrhythmic drug blockade and may be a rational target for modifying the proarrhythmic effects of otherwise clinically useful compounds.
Subject(s)
Anti-Arrhythmia Agents/pharmacology , Cation Transport Proteins , DNA-Binding Proteins , Myocytes, Cardiac/physiology , Nerve Tissue Proteins/physiology , Potassium Channel Blockers/pharmacology , Potassium Channels, Voltage-Gated , Potassium Channels/physiology , Trans-Activators , Animals , Anti-Arrhythmia Agents/antagonists & inhibitors , CHO Cells , Cell Line , Cricetinae , ERG1 Potassium Channel , Electric Conductivity , Ether-A-Go-Go Potassium Channels , Glucosyltransferases/metabolism , Humans , Ion Channel Gating , Membrane Transport Proteins/metabolism , Models, Biological , Myocardium/metabolism , Myocytes, Cardiac/drug effects , Nerve Tissue Proteins/genetics , Potassium Channel Blockers/antagonists & inhibitors , Potassium Channels/genetics , Transcriptional Regulator ERG , TransfectionABSTRACT
Mutations in the human Ether-à-go-go-Related gene (HERG), encoding the protein underlying the cardiac K(+) current, I(Kr), cause chromosome 7-linked long QT syndrome (LQT2). In this study, we show that deletion of the C-terminal 147 amino acids (HERG(Delta147)) abolished I(Kr), whereas a larger, 159-amino acid deletion (HERG(Delta159)) identified in an LQT2 kindred did generate I(Kr), albeit with reduced amplitude compared with the wild type. The 12 amino acids present in HERG(Delta147) and absent in HERG(Delta159) include a potential endoplasmic reticulum (ER) retention signal, RGR, which when mutated to LGL (HERG(Delta147-LGL)) restored I(Kr). Streptavidin selection of biotin-labeled surface proteins showed good expression of wild-type and HERG(Delta159) at the cell surface and low expression of HERG(Delta147-LGL) and HERG(Delta147). Additionally, a 100-amino acid peptide spanning the RGR triplet can rescue the defect in HERG(Delta147) when co-expressed as an ER-targeted minigene. Failure of HERG trafficking is known to cause LQT2, and this identified a molecular mechanism underlying this defect. Further, our data indicate that a key function of the C-terminal 104 amino acids is to mask the RGR ER retention signal, which becomes exposed when mutations truncate the HERG C terminus.
