An heregulin-EGFR-HER3 autocrine signaling axis can mediate acquired lapatinib resistance in HER2+ breast cancer models.
ABSTRACT: INTRODUCTION: The human epidermal growth factor receptor 2 (HER2) receptor tyrosine kinase (RTK) oncogene is an attractive therapeutic target for the treatment of HER2-addicted tumors. Although lapatinib, an FDA-approved small-molecule HER2 and epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), represents a significant therapeutic advancement in the treatment of HER2+ breast cancers, responses to lapatinib have not been durable. Consequently, elucidation of mechanisms of acquired therapeutic resistance to HER-directed therapies is of critical importance. METHODS: Using a functional protein-pathway activation mapping strategy, along with targeted genomic knockdowns applied to a series of isogenic-matched pairs of lapatinib-sensitive and resistant cell lines, we now report an unexpected mechanism of acquired resistance to lapatinib and similar TKIs. RESULTS: The signaling analysis revealed that whereas HER2 was appropriately inhibited in lapatinib-resistant cells, EGFR tyrosine phosphorylation was incompletely inhibited. Using a targeted molecular knockdown approach to interrogate the causal molecular underpinnings of EGFR-persistent activation, we found that lapatinib-resistant cells were no longer oncogene addicted to HER2-HER3-PI3K signaling, as seen in the parental lapatinib-sensitive cell lines, but instead were dependent on a heregulin (HRG)-driven HER3-EGFR-PI3K-PDK1 signaling axis. Two FDA-approved EGFR TKIs could not overcome HRG-HER3-mediated activation of EGFR, or reverse lapatinib resistance. The ability to overcome EGFR-mediated acquired therapeutic resistance to lapatinib was demonstrated through molecular knockdown of EGFR and treatment with the irreversible pan-HER TKI neratinib, which blocked HRG-dependent phosphorylation of HER3 and EGFR, resulting in apoptosis of resistant cells. In addition, whereas HRG reversed lapatinib-mediated antitumor effects in parental HER2+ breast cancer cells, neratinib was comparatively resistant to the effects of HRG in parental cells. Finally, we showed that HRG expression is an independent negative predictor of clinical outcome in HER2+ breast cancers, providing potential clinical relevance to our findings. CONCLUSIONS: Molecular analysis of acquired therapeutic resistance to lapatinib identified a new resistance mechanism based on incomplete and "leaky" inhibition of EGFR by lapatinib. The selective pressure applied by incomplete inhibition of the EGFR drug target resulted in selection of ligand-driven feedback that sustained EGFR activation in the face of constant exposure to the drug. Inadequate target inhibition driven by a ligand-mediated autocrine feedback loop may represent a broader mechanism of therapeutic resistance to HER TKIs and suggests adopting a different strategy for selecting more effective TKIs to advance into the clinic.
Project description:Extrinsic signals are implicated in breast cancer resistance to HER2-targeted tyrosine kinase inhibitors (TKIs). To examine how microenvironmental signals influence resistance, we monitored TKI-treated breast cancer cell lines grown on microenvironment microarrays composed of printed extracellular matrix proteins supplemented with soluble proteins. We tested ?2,500 combinations of 56 soluble and 46 matrix microenvironmental proteins on basal-like HER2+ (HER2E) or luminal-like HER2+ (L-HER2+) cells treated with the TKIs lapatinib or neratinib. In HER2E cells, hepatocyte growth factor, a ligand for MET, induced resistance that could be reversed with crizotinib, an inhibitor of MET. In L-HER2+ cells, neuregulin1-?1 (NRG1?), a ligand for HER3, induced resistance that could be reversed with pertuzumab, an inhibitor of HER2-HER3 heterodimerization. The subtype-specific responses were also observed in 3D cultures and murine xenografts. These results, along with bioinformatic pathway analysis and siRNA knockdown experiments, suggest different mechanisms of resistance specific to each HER2+ subtype: MET signaling for HER2E and HER2-HER3 heterodimerization for L-HER2+ cells.
Project description:An increasing number of tumors, including breast cancer, overexpress proteins of the epidermal growth factor receptor (EGFR) family. The interaction between family members activates signaling pathways that promote tumor progression and resistance to treatment. Human epidermal growth factor receptor type II (HER2) positive breast cancer represents a clinical challenge for current therapy. It has motivated the development of novel and more effective therapeutic EGFR family target drugs, such as tyrosine kinase inhibitors (TKIs). This review focuses on the effects of three TKIs mostly studied in HER2- positive breast cancer, lapatinib, gefitinib and neratinib. Herein, we discuss the mechanism of action, therapeutic advantages and clinical applications of these TKIs. To date, TKIs seem to be promising therapeutic agents for the treatment of HER2-overexpressing breast tumors, either as monotherapy or combined with other pharmacological agents.
Project description:ERBB family members including epidermal growth factor receptor (EGFR) also known as HER1, ERBB2/HER2/Neu, ERBB3/HER3 and ERBB4/HER4 are aberrantly activated in multiple cancers and hence serve as drug targets and biomarkers in modern precision therapy. The therapeutic potential of HER3 has long been underappreciated, due to impaired kinase activity and relatively low expression in tumors. However, HER3 has received attention in recent years as it is a crucial heterodimeric partner for other EGFR family members and has the potential to regulate EGFR/HER2-mediated resistance. Upregulation of HER3 is associated with several malignancies where it fosters tumor progression via interaction with different receptor tyrosine kinases (RTKs). Studies also implicate HER3 contributing significantly to treatment failure, mostly through the activation of PI3K/AKT, MAPK/ERK and JAK/STAT pathways. Moreover, activating mutations in HER3 have highlighted the role of HER3 as a direct therapeutic target. Therapeutic targeting of HER3 includes abrogating its dimerization partners' kinase activity using small molecule inhibitors (lapatinib, erlotinib, gefitinib, afatinib, neratinib) or direct targeting of its extracellular domain. In this review, we focus on HER3-mediated signaling, its role in drug resistance and discuss the latest advances to overcome resistance by targeting HER3 using mono- and bispecific antibodies and small molecule inhibitors.
Project description:Non-small-cell lung cancer harboring epidermal growth factor receptor (EGFR) mutations attains a meaningful response to EGFR-tyrosine kinase inhibitors (TKIs). However, acquired resistance to EGFR-TKIs could affect long-term outcome in almost all patients. To identify the potential mechanisms of resistance, we established cell lines resistant to EGFR-TKIs from the human lung cancer cell lines PC9 and11-18, which harbored activating EGFR mutations. One erlotinib-resistant cell line from PC9 and two erlotinib-resistant cell lines and two gefitinib-resistant cell lines from 11-18 were independently established. Almost complete loss of mutant delE746-A750 EGFR gene was observed in the erlotinib-resistant cells isolated from PC9, and partial loss of the mutant L858R EGFR gene copy was specifically observed in the erlotinib- and gefitinib-resistant cells from 11-18. However, constitutive activation of EGFR downstream signaling, PI3K/Akt, was observed even after loss of the mutated EGFR gene in all resistant cell lines even in the presence of the drug. In the erlotinib-resistant cells from PC9, constitutive PI3K/Akt activation was effectively inhibited by lapatinib (a dual TKI of EGFR and HER2) or BIBW2992 (pan-TKI of EGFR family proteins). Furthermore, erlotinib with either HER2 or HER3 knockdown by their cognate siRNAs also inhibited PI3K/Akt activation. Transfection of activating mutant EGFR complementary DNA restored drug sensitivity in the erlotinib-resistant cell line. Our study indicates that loss of addiction to mutant EGFR resulted in gain of addiction to both HER2/HER3 and PI3K/Akt signaling to acquire EGFR-TKI resistance.
Project description:Data from 8 breast cancer genome-sequencing projects identified 25 patients with HER2 somatic mutations in cancers lacking HER2 gene amplification. To determine the phenotype of these mutations, we functionally characterized 13 HER2 mutations using in vitro kinase assays, protein structure analysis, cell culture, and xenograft experiments. Seven of these mutations are activating mutations, including G309A, D769H, D769Y, V777L, P780ins, V842I, and R896C. HER2 in-frame deletion 755-759, which is homologous to EGF receptor (EGFR) exon 19 in-frame deletions, had a neomorphic phenotype with increased phosphorylation of EGFR or HER3. L755S produced lapatinib resistance, but was not an activating mutation in our experimental systems. All of these mutations were sensitive to the irreversible kinase inhibitor, neratinib. These findings show that HER2 somatic mutation is an alternative mechanism to activate HER2 in breast cancer and they validate HER2 somatic mutations as drug targets for breast cancer treatment.We show that the majority of HER2 somatic mutations in breast cancer patients are activating mutations that likely drive tumorigenesis. Several patients had mutations that are resistant to the reversible HER2 inhibitor lapatinib, but are sensitive to the irreversible HER2 inhibitor, neratinib. Our results suggest that patients with HER2 mutation–positive breast cancers could benefit from existing HER2-targeted drugs.
Project description:The incidence of breast cancer ranks first among female malignant tumors that affect women's health. Epidermal growth factor receptor (EGFR) family overexpression, especially human epidermal receptor2 (HER2), features prominently in breast cancer with a significant relation to poor prognosis. Currently, specific monoclonal antibodies and tyrosine kinase inhibitors (TKIs) are the two HER2 targeting strategies that have successfully improved the prognosis of patients with HER2-positive breast cancer. This paper focuses on three officially approved TKIs for HER2 breast cancer, namely, lapatinib, neratinib and pyrotinib, and systematically reviews the mechanism, safety, efficacy and resistance of these TKIs.
Project description:The response rate to EGFR tyrosine kinase inhibitors (TKIs) may be poor and unpredictable in cancer patients with EGFR expression itself being an inadequate response indicator. There is limited understanding of the mechanisms underlying this resistance. Furthermore, although TKIs suppress the growth of HER2-overexpressing breast tumor cells, they do not fully inhibit HER2 oncogenic function at physiological doses.Here we have provided a molecular mechanism of how HER2 oncogenic function escapes TKIs' inhibition via alternative HER receptor activation as a result of autocrine ligand release. Using both Förster Resonance Energy Transfer (FRET) which monitors in situ HER receptor phosphorylation as well as classical biochemical analysis, we have shown that the specific tyrosine kinase inhibitors (TKIs) of EGFR, AG1478 and Iressa (Gefitinib) decreased EGFR and HER3 phosphorylation through the inhibition of EGFR/HER3 dimerization. Consequent to this, we demonstrate that cleavage of HER4 and dimerization of HER4/HER2 occur together with reactivation of HER3 via HER2/HER3, leading to persistent HER2 phosphorylation in the now resistant, surviving cells. These drug treatment-induced processes were found to be mediated by the release of ligands including heregulin and betacellulin that activate HER3 and HER4 via HER2. Whereas an anti-betacellulin antibody in combination with Iressa increased the anti-proliferative effect in resistant cells, ligands such as heregulin and betacellulin rendered sensitive SKBR3 cells resistant to Iressa.These results demonstrate the role of drug-induced autocrine events leading to the activation of alternative HER receptors in maintaining HER2 phosphorylation and in mediating resistance to EGFR tyrosine kinase inhibitors (TKIs) in breast cancer cells, and hence specify treatment opportunities to overcome resistance in patients.
Project description:Human MAP3K4 (MTK1) functions upstream of mitogen activated protein kinases (MAPKs). In this study we show MTK1 is required for human epidermal growth factor receptor 2/3 (HER2/HER3)-heregulin beta1 (HRG) induced cell migration in MCF-7 breast cancer cells. We demonstrate that HRG stimulation leads to association of MTK1 with activated HER3 in MCF-7 and T-47D breast cancer cells. Activated HER3 association with MTK1 is dependent on HER2 activation and is decreased by pre-treatment with the HER2 inhibitor, lapatinib. Moreover, we also identify the actin interacting region (AIR) on MTK1. Disruption of actin cytoskeletal polymerization with cytochalasin D inhibited HRG induced MTK1/HER3 association. Additionally, HRG stimulation leads to extracellular acidification that is independent of cellular proliferation. HRG induced extracellular acidification is significantly inhibited when MTK1 is knocked down in MCF-7 cells. Similarly, pre-treatment with lapatinib significantly decreased HRG induced extracellular acidification. Extracellular acidification is linked with cancer cell migration. We performed scratch assays that show HRG induced cell migration in MCF-7 cells. Knockdown of MTK1 significantly inhibited HRG induced cell migration. Furthermore, pre-treatment with lapatinib also significantly decreased cell migration. Cell migration is required for cancer cell metastasis, which is the major cause of cancer patient mortality. We identify MTK1 in the HER2/HER3-HRG mediated extracellular acidification and cell migration pathway in breast cancer cells.
Project description:Prolonged treatment of HER2 positive breast cancer cells with tyrosine kinase inhibitors (TKIs) leads to the emergence of acquired resistance. However, the effects of continuous TKI exposure on cell fate, and the steps leading to the acquisition of a resistant phenotype are poorly understood. To explore this, we exposed five HER2 positive cells lines to HER2 targeted therapies for periods of up to 4 weeks and examined senescence associated ?-galactosidase (SA-?-gal) activity together with additional markers of senescence. We found that lapatinib treatment resulted in phenotypic alterations consistent with a senescent phenotype and strong SA-?-gal activity in HER2-positive cell lines. Lapatinib-induced senescence was associated with elevated levels of p15 and p27 but was not dependent on the expression of p16 or p21. Restoring wild type p53 activity either by transfection or by treatment with APR-246, a molecule which reactivates mutant p53, blocked lapatinib-induced senescence and caused increased cell death. In contrast to lapatinib, SA-?-gal activity was not induced by exposing the cells to trastuzumab as a single agent but co-administration of lapatinib and trastuzumab induced senescence, as did treatment of the cells with the irreversible HER2 TKIs neratinib and afatinib. Neratinib- and afatinib-induced senescence was not reversed by removing the drug whereas lapatinib-induced senescence was reversible. In summary, therapy-induced senescence represents a novel mechanism of action of HER2 targeting agents and may be a potential pathway for the emergence of resistance.
Project description:The use of antibodies in therapy and diagnosis has undergone an unprecedented expansion during the past two decades. This is due in part to innovations in antibody engineering that now offer opportunities for the production of "second generation" antibodies with multiple specificities or altered valencies. The targeting of individual components of the human epidermal growth factor receptor (HER)3-PI3K signaling axis, including the preferred heterodimerization partner HER2, is known to have limited anti-tumor effects. The efficacy of antibodies or small molecule tyrosine kinase inhibitors (TKIs) in targeting this axis is further reduced by the presence of the HER3 ligand, heregulin. To address these shortcomings, we performed a comparative analysis of two distinct approaches toward reducing the proliferation and signaling in HER2 overexpressing tumor cells in the presence of heregulin. These strategies both involve the use of engineered antibodies in combination with the epidermal growth factor receptor (EGFR)/HER2 specific TKI, lapatinib. In the first approach, we generated a bispecific anti-HER2/HER3 antibody that, in the presence of lapatinib, is designed to sequester HER3 into inactive HER2-HER3 dimers that restrain HER3 interactions with other possible dimerization partners. The second approach involves the use of a tetravalent anti-HER3 antibody with the goal of inducing efficient HER3 internalization and degradation. In combination with lapatinib, we demonstrate that although the multivalent HER3 antibody is more effective than its bivalent counterpart in reducing heregulin-mediated signaling and growth, the bispecific HER2/HER3 antibody has increased inhibitory activity. Collectively, these observations provide support for the therapeutic use of bispecifics in combination with TKIs to recruit HER3 into complexes that are functionally inert.