Dual Targeting Of HER2 Research Articles

The Hippo Pathway Transducers YAP1/TEAD Induce Acquired Resistance to Trastuzumab in HER2-Positive Breast Cancer.

Trastuzumab is the first-line targeted therapeutic drug for HER2-positive breast cancer, leading to improved overall survival. However, acquired resistance inevitably occurs. We aimed to identify, quantify, and assess the mechanisms of acquired resistance to trastuzumab. We established an acquired trastuzumab-resistant model in vitro from BT-474, a trastuzumab-sensitive, HER2-amplified breast-cancer cell line. A multi-omic strategy was implemented to obtain gene, proteome, and phosphoproteome signatures associated with acquired resistance to trastuzumab in HER2-positive breast cancer, followed by validation in human clinical samples. YAP1 dephosphorylation and TEAD2 overexpression were detected as significant alterations in the Hippo pathway in trastuzumab-resistant breast cancer. Because of the emerging role of these proteins as mediators of normal growth and tumorigenesis, we assessed the exogenous modulation of their activity, either by in vitro gene silencing or by pharmacological inhibition of the YAP1/TEAD complexes, both in vitro and in vivo. Moreover, we identified increased signaling through the Hippo pathway in human samples after progression following trastuzumab treatment. Finally, YAP1/TAZ nuclear accumulation in malignant cells in HER2 breast tumor was significantly associated with worse progression-free and overall survival in metastatic HER2-positive breast-cancer patients. Our results suggest the involvement of Hippo signaling in acquired trastuzumab resistance in breast cancer. Additionally, we provide novel evidence for a potential breast-cancer treatment strategy based on dual targeting of HER2 and Hippo pathway effectors, which may improve the antitumor activity of trastuzumab and help overcome resistance.

Abstract OT2-01-01: Randomized, double-blind, phase 3 study of tucatinib or placebo in combination with T-DM1 for subjects with unresectable locally-advanced or metastatic HER2+ breast cancer

Abstract Background: Tucatinib is an orally-available, reversible HER2 small molecule tyrosine kinase inhibitor (TKI) being developed as a novel treatment for patients with HER2+ metastatic breast cancer (mBC), including patients with brain metastases. Two key features of tucatinib are its potency and selectivity for HER2, compared to the epidermal growth factor receptor (EGFR). Ado-trastuzumab emtansine (T-DM1) is approved for the treatment of patients with HER2+ mBC after prior treatment with trastuzumab and a taxane. While treatment with T-DM1 has led to significant improvements in progression-free survival (PFS) and overall survival, further improvements in therapy are needed, especially for patients with active brain metastases. Based upon evidence that dual targeting of HER2 may lead to further improvements in efficacy in mBC, a phase 1b trial enrolled subjects with HER2+ mBC, previously treated with trastuzumab and taxane, to receive tucatinib (300 mg orally [PO] twice a day [BID]) with T-DM1 (Borges et al, 2018). Forty-six percent of these subjects had received prior pertuzumab. The combination of tucatinib with T-DM1 demonstrated a median PFS of 8.2 months (95% CI, 4.8-10.3) and an objective response rate of 47% in subjects with measurable disease. Sixty percent of subjects treated with this combination had baseline brain metastases and showed a brain-specific response rate (using modified RECIST v1.1 criteria) of 36% in subjects with measurable central nervous system disease. Tucatinib with T-DM1 was found to have a tolerable safety profile, the most common adverse events were nausea (72%), diarrhea (60%), and fatigue (56%), with the majority of events being grade 1 or 2. This encouraging clinical activity, including in subjects with brain metastases, provides rationale for a randomized trial to further evaluate this combination. Study Design: This is a randomized, double-blind, placebo-controlled, international, multicenter, phase 3 study designed to evaluate the efficacy and safety of tucatinib in combination with T-DM1 in subjects with unresectable locally-advanced or metastatic HER2+ breast cancer. Subjects must have had prior treatment with a taxane and trastuzumab in any setting (adjuvant, neoadjuvant, or metastatic). The primary objective of the study is to compare PFS between the treatment arms per investigator assessment, with overall survival as a key secondary endpoint. In this study, subjects must be ≥18 years, with an ECOG of ≤1 and have histologically confirmed HER2+ mBC. Prior treatment with any investigational anti-HER2 or anti-EGFR agent or HER2 TKI agent is not permitted. Prior pertuzumab therapy is allowed, but not required. Subjects with stable, progressing, or untreated brain metastases not requiring immediate local therapy, are eligible for inclusion in the trial. Approximately 460 subjects will be randomized 1:1 to receive 21-day cycles of either tucatinib (300 mg PO BID) or placebo in combination with T-DM1 (3.6 mg/kg intravenously). Disease response and progression will be assessed using RECIST v1.1. While on study treatment, radiographic disease evaluations will be performed every 6 weeks for the first 24 weeks, and every 9 weeks thereafter. A subset of subjects will participate in a pharmacokinetic substudy. Citation Format: Sara Hurvitz, Linda Vahdat, Nadia Harbeck, Antonio C Wolff, Sara M Tolaney, Sherene Loi, Norikazu Masuda, Cassie Dong, Luke Walker, Evelyn Rustia, Virginia Borges. Randomized, double-blind, phase 3 study of tucatinib or placebo in combination with T-DM1 for subjects with unresectable locally-advanced or metastatic HER2+ breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr OT2-01-01.

Dual targeting of HER2-positive breast cancer with trastuzumab emtansine and pertuzumab: understanding clinical trial results.

Targeting of HER2-positive tumors with trastuzumab has shown to improve survival in early stage and advanced breast cancer. The addition of pertuzumab, another anti-HER2 antibody, to trastuzumab-containing regimens has demonstrated a modest increase in disease-free survival in the adjuvant setting. Unexpectedly, when pertuzumab was explored in combination with the antibody-drug conjugate TDM1 in the metastatic setting, no additional benefit was observed compared with dual targeting of HER2 with pertuzumab and trastuzumab, together with chemotherapy. Similar results were observed when exploring pathologic complete response in the neoadjuvant setting. In this article, we discuss basic science and translational data that may explain the limited efficacy observed with the combination of TDM1 and pertuzumab, including tumor heterogeneity, clonal selection, bystander effect or downregulation of the receptor by competitive binding. In addition, we review ongoing studies that could help to understand these findings.

Dual EGFR/HER2 inhibitors and apoptosis inducers: New benzo[g]quinazoline derivatives bearing benzenesulfonamide as anticancer and radiosensitizers

Dual targeting of EGFR and HER2 is a proven anticancer strategy for the treatment of solid tumors. An array of new N-substituted-2-(4-oxo-3-(4-sulfamoylphenyl)-3,4-dihydrobenzo[g]quinazolin-2-ylthio) acetamides 5–18 were designed and synthesized from the starting compound 4-(2-mercapto-4-oxobenzo[g]quinazolin-3(4H)-yl) benzenesulfonamide 4. The targeted compounds were screened for their cytotoxic activity against MDA-MB-231 breast cancer cell line. The IC50 of all the compounds were in the range of 0.36–40.90 µM. The percentage inhibition towards EGFR was measured and found to be in the range of 63.00–16.90 %. The most potent compounds 5, 9, 15, 17 and 18 were further screened for their activity against both EGFR and HER2 receptors. The compounds showed IC50 in the range of 0.64–1.81 µM for EGFR and 1.13–2.21 µM for HER2, in comparison to erlotinib, the reference drug. Compound 17, the most potent towards EGFR in this series, undergoes cell cycle analysis and was found to arrest the cycle at the G2/M phase. Measurement of the cytotoxicity of compound 17 against normal breast cell line showed mild cytotoxic activity. The most potent compounds were subjected to a single dose of 8 Gy of γ-radiation and the cytotoxicity of the tested compounds was found to increase after irradiation, thus proving the synergistic effect of γ-irradiation. Molecular docking was adopted for all the synthesized compounds to confirm their mechanism of action.

Cooperation of neurotrophin receptor TrkB and Her2 in breast cancer cells facilitates brain metastases

BackgroundPatients with primary breast cancer that is positive for human epidermal growth factor receptor 2 (Her2+) have a high risk of developing metastases in the brain. Despite gains with systemic control of Her2+ disease using molecular therapies, brain metastases remain recalcitrant to therapeutic discovery. The clinical predilection of Her2+ breast cancer cells to colonize the brain likely relies on paracrine mechanisms. The neural niche poses unique selection pressures, and neoplastic cells that utilize the brain microenvironment may have a survival advantage.MethodsTropomyosin-related kinase B (TrkB), Her2, and downstream targets were analyzed in primary breast cancer, breast-to-brain metastasis (BBM) tissues, and tumor-derived cell lines using quantitative real-time PCR, western blot, and immunohistochemical assessment. TrkB function on BBM was confirmed with intracranial, intracardiac, or mammary fat pad xenografts in non-obese diabetic/severe combined immunodeficiency mice. The function of brain-derived neurotrophic factor (BDNF) on cell proliferation and TrkB/Her2 signaling and interactions were confirmed using selective shRNA knockdown and selective inhibitors. The physical interaction of Her2-TrkB was analyzed using electron microscopy, co-immunoprecipitation, and in silico analysis. Dual targeting of Her2 and TrkB was analyzed using clinically utilized treatments.ResultsWe observed that patient tissues and cell lines derived from Her2+ human BBM displayed increased activation of TrkB, a neurotrophin receptor. BDNF, an extracellular neurotrophin, with roles in neuronal maturation and homeostasis, specifically binds to TrkB. TrkB knockdown in breast cancer cells led to decreased frequency and growth of brain metastasis in animal models, suggesting that circulating breast cancer cells entering the brain may take advantage of paracrine BDNF-TrkB signaling for colonization. In addition, we investigated a possible interaction between TrkB and Her2 receptors on brain metastatic breast cancer cells, and found that BDNF phosphorylated both its cognate TrkB receptor and the Her2 receptor in brain metastatic breast cancer cells.ConclusionCollectively, our findings suggest that heterodimerization of Her2 and TrkB receptors gives breast cancer cells a survival advantage in the brain and that dual inhibition of these receptors may hold therapeutic potential.

521P - Association between HER2 amplification and cetuximab efficacy in patients with RAS wild-type metastatic colorectal cancer

Cetuximab has shown clinical benefit in patients with metastatic colorectal cancer (mCRC) harbouring wild-type RAS. HER2 amplification has been suggested as one of the resistant mechanisms of cetuximab treatment. We evaluated association between HER2 amplification and cetuximab efficacy in mCRC patients harbouring extended RAS wild-type. Between December 2003 and June 2013, we found 253 mCRC patients whose tumor harboured wild type in exon 2/3/4 both of KRAS and NRAS by high throughput sequencing (OncoMap version 4.0) and were treated with cetuximab as 2nd or later-line. We finally included 243 mCRC cases whose HER2 status could be determined by both immunohistochemical (IHC) scoring according to HERACLES criteria and silver in-situ hybridisation (SISH). Progression-free survival (PFS) and overall survival (OS) were analysed in homogenous group (n = 149) who were progressed after oxaliplatin, irinotecan and fluoropyrimidines. The median age was 55 years (range 19–76 years); 168 patients (69.1%) were male. Of the 243 RAS wild-type tumor, we observed 11 cases (4.5%) of HER2 amplification (table). After the median follow-up of 13.5 months (range, 0.4–78.1), median PFS was statistically different according to the HER2 status: 3.1 months in patients harbouring HER2 amplification vs 5.7 months in those harbouring non-amplified HER2 (p = 0.013). OS was not statistically different according to the HER2 status although there was a tendency towards shorter OS in patients harbouring HER2 amplification (10.1 vs 13.5 months, p = 0.408).Tabled 1HER2 IHC (HERACLES criteria)SISHNegativeEquivocalPositiveTotalAmplification1†HER2/CEP17 ratio 3.31HER2/CEP17 ratio 2.79†HER2/CEP17 ratio >5 (all cases)11No amplification22930232Total23049243† HER2/CEP17 ratio 3.3†† HER2/CEP17 ratio 2.7††† HER2/CEP17 ratio >5 (all cases) Open table in a new tab HER2 amplification is associated with shorter PFS after cetuximab in mCRC patients harbouring extended RAS wild-type. Recent study of dual targeting of EGFR and HER2 demonstrated promising result; further study is warranted for this population.

Antiproliferative and apoptotic effects of a specific anti-insulin-like growth factor I receptor single chain antibody on breast cancer cells.

Insulin-like growth factor I receptor (IGF-IR) is expressed on breast cancer cells and involves in metastasis, survival, and proliferation. Currently, application of IGF-IR-targeting monoclonal antibodies (mAbs), alone or in combination with other drugs, is a promising strategy for breast cancer therapy. Single-chain fragment variable (scFv) antibodies have been introduced as appropriate tools for tumor-targeting purposes because of their advantages over whole antibodies. In the present study, we employed a naïve phage library and isolated scFvs against a specific epitope from extracellular domain of IGF-IR by panning process. The selected scFvs were further characterized using polyclonal and monoclonal phage ELISA, soluble monoclonal ELISA, and colony PCR and sequencing. Antiproliferative and apoptotic effects of selected scFv antibodies on breast cancer cell lines were also evaluated by MTT and Annexin V/PI assays. The results of ELISA indicated specific reactions of the isolated scFvs against the IGF-IR peptide, and analyses of PCR product and sequencing confirmed the presence of full length VH and Vκ inserts. Treatment of MCF7 and SKBR3 cells with anti-IGF-IR scFv led to a significant growth inhibition. The results also showed that scFv treatment significantly augmented trastuzumab growth inhibitory effects on SKBR3 cells. The percentage of the apoptotic MCF7 and SKBR3 cells after 24-h treatment with scFv was 39 and 30.70 %, respectively. Twenty-four-hour treatment with scFv in combination with trastuzumab resulted in 44.75 % apoptosis of SKBR3 cells. Taken together, our results demonstrate that the targeting of IGF-IR by scFv can be an effective strategy in the treatment of breast cancer and provide further evidence for effectiveness of dual targeting of HER2 and IGF-IR in breast cancer therapy.

Protein tyrosine phosphatase SHP-1 sensitizes EGFR/HER-2 positive breast cancer cells to trastuzumab through modulating phosphorylation of EGFR and HER-2.

BackgroundTrastuzumab resistance in HER-2 positive breast cancer cells is closely related to overexpression of both epidermal growth factor receptor (EGFR) and human epidermal receptor (HER-2). SHP-1 has been demonstrated to downregulate tyrosine kinase activity including EGFR via its phosphatase function, but its effect on HER-2 activity is still unknown. Here, we examined the hypothesis that SHP-1 enhances the anticancer efficacy of trastuzumab in EGFR/HER-2 positive breast cancer cells through combining dual inhibition of EGFR and HER-2.MethodsTrastuzumab-resistant breast cancer SKBr-3 cells were generated by long-term in vitro culture of SKBr-3cells in the presence of trastuzumab. The SHP-1 was ectopically expressed by stable transfection. The activity and expression of EGFR, HER-2, and downstream signaling pathways were tested by Western blot. Cell viability was examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and apoptosis was examined by flow cytometry. The binding between SHP-1 and EGFR/HER-2 was evaluated by immunoprecipitation assay and bimolecular fluorescence complementation. The effects of SHP-1 on tumorigenicity and trastuzumab sensitivity were confirmed via in vivo xenograft model.ResultsTrastuzumab-resistant SKBr-3 cells showed aberrant co-expression of EGFR and HER-2. Introduction of wild-type SHP-1 inhibited cell proliferation, clone formation, and promoted the apoptosis induced by trastuzumab. Meanwhile, SHP-1 overexpression reduced phosphorylation levels of EGFR and HER-2 both in parental and trastuzumab-resistant SKBr-3 cells. In vivo study showed an increased antitumor effect of trastuzumab in SHP-1 overexpressed xenografts. At last, we discovered that SHP-1 can make complexes with both EGFR and HER-2, and both phospho-EGFR and phosphor-HER-2 levels in wild-type SHP-1 immunoprecipitates were less than those in phosphatase-inactive SHP-1 (C453S) immunoprecipitates, indicating that EGFR and HER-2 are potential substrates of SHP-1.ConclusionTaken together, we have demonstrated that the SHP-1 is a negative regulatory factor of the tyrosine kinase activity of HER-2 and EGFR through inhibiting phosphorylation. Dual targeting of EGFR and HER-2, by combining trastuzumab with SHP-1 overexpression, may improve response in HER-2 overexpressing breast cancer cells that also express high levels of EGFR.

Abstract P5-19-04: A phase I/II study of neratinib plus temsirolimus in HER2+ metastatic breast cancer reveals ongoing HER2 pathway dependence in many patients despite several lines of HER2 targeted therapy

Abstract Background: Targeting a single node in the HER2/PI3K pathway has been associated with benefit but frequently results in acquired resistance in the metastatic setting. Direct inhibition of HER2 even with combinations may be limited by mutational activation of the downstream PI3K pathway. Conversely, inhibition downstream results in feedback upregulation of receptor tyrosine kinase signaling which can diminish efficacy. We hypothesize that dual targeting of HER2 and mTOR will be tolerable while overcoming these limitations. We conducted a phase I/II trial examining the tolerability and efficacy of temsirolimus (T), an mTOR inhibitor, and neratinib (N), a HER1/2 kinase inhibitor, in patients with HER2+ metastatic breast cancer (MBC). Tumor biopsies were obtained to ascertain if PI3K pathway activation is frequent and to identify biomarkers of response. Methods: The phase I study utilized a 3+3 dose escalation design to determine the maximum tolerated dose (MTD) of T (IV weekly) with N (fixed dose 240 mg oral daily) in patients with HER2+ trastuzumab-refractory MBC. Loperamide prophylaxis (4 mg daily) was initiated at Day 1 and then left to patient/physician discretion. The phase II study utilized a Simon two-stage design to assess the overall response rate by RECIST in HER2+ trastuzumab-refractory MBC. An expansion cohort has been subsequently initiated to investigate the benefit of more aggressive loperamide prophylaxis (16 mg daily) and temsirolimus dose escalation. All patients on the initial Ph I/II study underwent biopsy of metastatic disease for biomarker assessment. Activating mutations in the PI3K pathway were assayed using the Sequenom MassARRAY system or as part of Next Generation Sequencing of panel of ∼250 cancer related genes along with PTEN immunohistochemistry. Results: Eight patients enrolled in the phase I trial and the MTD was determined to be T at 8 mg IV weekly with neratinib at 240 mg daily with grade 3 diarrhea as the dose limiting toxicity. The phase II trial enrolled 34 patients. Seventy percent of patients on the Ph I/II studies had progression of disease on prior lapatinib, pertuzumab, or T-DM1. The most frequent treatment-related grade 2/3 events at the MTD (n=40) were diarrhea (gr 2 35%/gr 3 25%), mucositis (23%/10%), and leukopenia (28%/5%). Thirty-five patients treated at the MTD are evaluable for response; 13 patients had PR (9 cPRs) and 2 patients had SD ≥6 months. Most of the 15 responders had prior T-DM1 (6), pertuzumab (2), or lapatinib (8). Molecular analyses of pretreatment biopsies is ongoing and thus far has revealed PI3K pathway activation (PIK3CA or AKT mutation or PTEN low) in >70% of the analyzed tumors (24/33). Responses were frequent in this cohort but not in a group of tumors that had lost HER2 overexpression. Conclusions: Temsirolimus and neratinib has clinical activity in the setting of HER2+ MBC with mutational activation of PI3K pathway and among patients exposed to multiple prior HER2 targeted agents. Final results of the efficacy, safety, and biomarker data will be presented along with preliminary data from the ongoing expansion study. Citation Format: Devika Gajria, Shanu Modi, Cristina Saura, Rita Sakr, Karl Solano, Helen Won, Harpreet Pannu, Sujata Patil, Diana Lake, Tiffany Traina, Tari King, Michael Berger, Jose Baselga, Neal Rosen, Cliff Hudis, Sarat Chandarlapaty. A phase I/II study of neratinib plus temsirolimus in HER2+ metastatic breast cancer reveals ongoing HER2 pathway dependence in many patients despite several lines of HER2 targeted therapy [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-19-04.

Insulin-like growth factor-1 receptor signaling increases the invasive potential of human epidermal growth factor receptor 2-overexpressing breast cancer cells via Src-focal adhesion kinase and forkhead box protein M1.

Resistance to the human epidermal growth factor receptor (HER2)-targeted antibody trastuzumab is a major clinical concern in the treatment of HER2-positive metastatic breast cancer. Increased expression or signaling from the insulin-like growth factor-1 receptor (IGF-1R) has been reported to be associated with trastuzumab resistance. However, the specific molecular and biologic mechanisms through which IGF-1R promotes resistance or disease progression remain poorly defined. In this study, we found that the major biologic effect promoted by IGF-1R was invasion, which was mediated by both Src-focal adhesion kinase (FAK) signaling and Forkhead box protein M1 (FoxM1). Cotargeting IGF-1R and HER2 using either IGF-1R antibodies or IGF-1R short hairpin RNA in combination with trastuzumab resulted in significant but modest growth inhibition. Reduced invasion was the most significant biologic effect achieved by cotargeting IGF-1R and HER2 in trastuzumab-resistant cells. Constitutively active Src blocked the anti-invasive effect of IGF-1R/HER2 cotargeted therapy. Furthermore, knockdown of FoxM1 blocked IGF-1-mediated invasion, and dual targeting of IGF-1R and HER2 reduced expression of FoxM1. Re-expression of FoxM1 restored the invasive potential of IGF-1R knockdown cells treated with trastuzumab. Overall, our results strongly indicate that therapeutic combinations that cotarget IGF-1R and HER2 may reduce the invasive potential of cancer cells that are resistant to trastuzumab through mechanisms that depend in part on Src and FoxM1.

Dual targeting of HER2 with lapatinib and trastuzumab

Dual targeting of HER2 with lapatinib and trastuzumab

Dual Targeting of HER2-Positive Cancer with Trastuzumab Emtansine and Pertuzumab: Critical Role for Neuregulin Blockade in Antitumor Response to Combination Therapy

Targeting HER2 with multiple HER2-directed therapies represents a promising area of treatment for HER2-positive cancers. We investigated combining the HER2-directed antibody-drug conjugate trastuzumab emtansine (T-DM1) with the HER2 dimerization inhibitor pertuzumab (Perjeta). Drug combination studies with T-DM1 and pertuzumab were performed on cultured tumor cells and in mouse xenograft models of HER2-amplified cancer. In patients with HER2-positive locally advanced or metastatic breast cancer (mBC), T-DM1 was dose-escalated with a fixed standard pertuzumab dose in a 3+3 phase Ib/II study design. Treatment of HER2-overexpressing tumor cells in vitro with T-DM1 plus pertuzumab resulted in synergistic inhibition of cell proliferation and induction of apoptotic cell death. The presence of the HER3 ligand, heregulin (NRG-1β), reduced the cytotoxic activity of T-DM1 in a subset of breast cancer lines; this effect was reversed by the addition of pertuzumab. Results from mouse xenograft models showed enhanced antitumor efficacy with T-DM1 and pertuzumab resulting from the unique antitumor activities of each agent. In patients with mBC previously treated with trastuzumab, lapatinib, and chemotherapy, T-DM1 could be dosed at the maximum tolerated dose (MTD; 3.6 mg/kg every 3 weeks) with standard dose pertuzumab. Adverse events were mostly grade 1 and 2, with indications of clinical activity. Dual targeting of HER2 with the combination of T-DM1 and pertuzumab in cell culture and mouse xenograft models resulted in enhanced antitumor activity. In patients, this combination showed an encouraging safety and tolerability profile with preliminary evidence of efficacy.

417 A Mechanistic Logic for Dual Targeting of HER2 and PI3K/AKT/mTOR Signaling in HER2 Amplified Breast Cancer

S. Chandarlapaty, V. Rodrik-Outmezguine, M. Maurizio Scaltriti, R. Sakr, M. Will, D. Giri, C. Hudis, J. Baselga, T. King, N. Rosen. Memorial Sloan-Kettering Cancer Center, Human Oncology and Pathogenesis Program, New York, USA; Memorial Sloan-Kettering Cancer Center, Molecular Pharmacology, New York, USA; Massachusetts General Hospital Cancer Center, Medicine, Boston, USA; Memorial Sloan-Kettering Cancer Center, Surgery, New York, USA; Memorial Sloan-Kettering Cancer Center, Pathology, New York, USA; Memorial Sloan-Kettering Cancer Center, Medicine, New York, USA

Abstract 5584: Disregulation of phosphorelated Amplified In Breast Cancer3 (AIB3) contributes to poor prognosis and mediates trastuzumab efficacy in breast cancer patients

Abstract Trastuzumab (Herceptin) is the first line HER2 (ErbB2)/neu-directed therapy for the treatment of patients with metastatic breast cancer. Although the efficacy of trastuzumab depends on the HER2/neu status of the tumour, the single-agent response rate ranges from 12 to 30% and ew patients respond to trastuzumab monotherapy largely due to various resistance mechanisms. Amplifeid In Breast Cancer3 (AIB3) is a transcriptional coactivator. AIB3 plays an important role in embryonic development, adipocyte differentiation, metabolism. AIB3 was initially found to be amplified in subgroup of breast cancer patients. Our previous data using knockout mouse mode combined cell culture indicate that AIB3 plays important role in breast tumorigenesis. Here we report that AIB3 can be phosphorylated by MAPK at S884 site and phosphorylation at S884 of AIB3 dramatically reduced ligand-dependent AIB3 interactions with estrogen receptors when compared with the wild-type unphosphorylated form, although there was a significant increase of interactions with the unliganded receptor. Misexpression of phosphorylated AIB3 was observed in human breast cancer tissues and disregulation of phosphorylated AIB3 was associated with patients’ poor prognosis. Breast cancer is a heterogeneous disease and intrinsic subtypes play an extremely important role in improve on current standards for breast cancer prognosis and prediction of chemotherapy. Noticeably, phosphorylated AIB3 was highly expressed in Her2-enriched subtype of human breast cancer patients, as well as in the mammary tumor of MMTV-Her2 transgenic mice. Mechanism studies reveal that phosphorylated AIB3 alters trastuzumab efficacy in cultured breast cancer cells. The association between pAIB3 and important clinical outcomes confers pAIB3 a significant prognostic marker in breast cancer. Phosphorylated AIB3 as a modulator in ErbB2-MAPK regulating loop represents a potentially novel mechanism for trastuzumab (herceptin) efficacy. The implied interaction between pAIB3 and Her2 suggests the potential of dual targeting of Her2 and pAIB3 as a promising therapeutic approach for personalized breast cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5584. doi:1538-7445.AM2012-5584

Inhibition of IGF1R activity enhances response to trastuzumab in HER-2-positive breast cancer cells

Background: Although trastuzumab has improved the prognosis for HER-2-positive breast cancer patients, not all HER-2-positive breast tumours respond to trastuzumab treatment and those that initially respond frequently develop resistance. Insulin-like growth factor-1 receptor (IGF1R) signalling has been previously implicated in trastuzumab resistance. We tested IGF1R inhibition to determine if dual targeting of HER-2 and IGF1R improves response in cell line models of acquired trastuzumab resistance.Materials and methods: HER-2, IGF1R, phospho-HER-2, and phospho-IGF1R levels were measured by enzyme-linked immunosorbent assays in parental and trastuzumab-resistant SKBR3 and BT474 cells. IGF1R signalling was targeted in these cells using both small interfering RNA (siRNA) and the tyrosine kinase inhibitor, NVP-AEW541.Results: IGF1R levels were significantly increased in the trastuzumab-resistant model, SKBR3/Tr, compared with the parental SKBR3 cell line. In both the SKBR3/Tr and BT474/Tr cell lines, inhibition of IGF1R expression with siRNA or inhibition of tyrosine kinase activity by NVP-AEW541 significantly increased response to trastuzumab. The dual targeting approach also improved response in the parental SKBR3 cells but not in the BT474 parental cells.Conclusions: Our results confirm that IGF1R inhibition improves response to trastuzumab in HER-2-positive breast cancer cells and suggest that dual targeting of IGF1R and HER-2 may improve response in HER-2-positive tumours.

Simultaneous targeting of estrogen receptor and HER2 in breast cancer

Approximately 50% of HER2-positive breast cancers express estrogen receptor (ER) and these tumors are characterized by short-lived responses to hormonal agents. Preclinical models have shown that dual targeting of ER and HER2 could reverse and delay the development of drug resistance. Two studies (TAnDEM & EGF3008) have recently been published addressing the combined use of an aromatase inhibitor (AI) and an anti-HER2-targeted agent. Both studies showed that the combined approach is associated with improvement in response rate and progression-free survival compared with an AI alone with an acceptable toxicity profile. These results would indeed extend the treatment options for patients with ER/HER2-positive metastatic breast cancer. In this article, we discuss how the improved understanding of the complex cross-talk between ER and HER2 has resulted in better clinical outcomes. We analyze clinical evidence regarding the combined use of AIs and anti-HER2-targeted agents. We also touch on possible mechanisms of resistance and ways to improve research in this field.

Anti-invasion, anti-proliferation and anoikis-sensitization activities of lapatinib in nasopharyngeal carcinoma cells

Nasopharyngeal cancer (NPC) is a highly prevalent and invasive head and neck cancer in Asia. Disease recurrence and distant metastasis account for major NPC deaths. Therefore, more effective therapy is needed. Lapatinib, a dual tyrosine kinase inhibitor (TKI) against both EGFR and HER-2, has been known to exert potent antitumor activity against several cancer models. Given that both EGFR and HER-2 are co-expressed in NPC, we hypothesized that dual targeting of EGFR and HER-2 by this small molecule EGFR/HER-2 TKI would elicit anti-tumor activity in NPC. Using in vitro models of NPC, we demonstrated that lapatinib was able to efficiently inhibit the phosphorylation of both EGFR and HER-2. This was accompanied by significant growth inhibition of NPC cells (with maximal growth inhibition >90%). For the most lapatinib-sensitive cell line (HK1-LMP1, with IC(50) ∼ 600 nM), which harbored the highest levels of both EGFR and HER-2, inhibition of cell growth was associated G(0)/G(1) cell cycle arrest, marked PARP cleavage, caspase-3 cleavage, as well as significant downregulation of several important survival proteins (e.g. survivin, Mcl-1 and cyclin D1). NPC cells are intrinsically invasive. We found that lapatinib was able to inhibit cellular invasion of both HK1-LMP1 and HONE-1 cells. Furthermore, our data demonstrated for the first time that lapatinib harbored potent anoikis-sensitization activity (i.e. sensitizing cancer cells to detachment-induced apoptosis) in human cancer cells overexpressing both EGFR and HER-2 (HK1-LMP1 and HK1). Taken together, our findings suggest that lapatinib is a promising anti-cancer agent for NPC with anti-invasion and anoikis-sensitization activities.

Abstract 5607: Dual targeting of HER2: Enhanced antitumor efficacy of trastuzumab-DM1 combined with pertuzumab

Abstract The EGFR family of transmembrane receptors consists of HER1 (ErbB1/EGFR), HER2 (ErbB2), HER3 (ErbB3) and HER4 (ErbB4). These receptors are often dysregulated in human solid tumors. For example, amplification and overexpression of HER2 occur in approximately 20 percent of human breast cancers and are predictive of poor clinical outcome. A principal means by which these receptors exert their growth-stimulatory effect is through homo- and hetero-dimerization which can occur in either a ligand-dependent or -independent manner and subsequently promotes receptor tyrosine kinase activity, leading to autophosphorylation and activation of downstream signal transduction pathways. The humanized HER2 antibody, trastuzumab (Herceptin®), is approved for use in adjuvant and metastatic HER2-positive breast cancer and has shown significant clinical benefit. Trastuzumab-DM1 (T-DM1, TMAb-mcc-DM1, trastuzumab emtansine) is an antibody-cytotoxic drug conjugate composed of the maytansine derivative DM1 directly coupled, through a thioether SMCC linker, to trastuzumab. We previously reported the potent in vitro and in vivo efficacy of T-DM1 in trastuzumab-sensitive and -refractory breast tumor models. Both trastuzumab and T-DM1 bind domain IV of the HER2 extracellular domain (ECD) and inhibit HER2 function in a ligand-independent manner. T-DM1 provides additional efficacy through selective delivery of the cytotoxic agent DM1 to HER2-overexpressing cancer cells. Pertuzumab is a humanized HER2 antibody that binds domain II of the HER2 ECD and thereby inhibits ligand-induced activation of HER2 by blocking dimerization of HER2 with other HER family members. Pertuzumab in combination with trastuzumab has shown impressive preclinical as well as clinical activity in HER2-positive breast cancer. The purpose of our studies was to evaluate the in vitro and in vivo efficacy of T-DM1 combined with pertuzumab. Our in vitro results show that combination treatment resulted in synergistic inhibition of cell proliferation in human breast and lung cancer cells that over-express HER2. Drug combination effects were analyzed by the Chou and Talalay method for determining combination index values. Similarly, we observed enhanced in vivo efficacy when administering T-DM1 in combination with pertuzumab, as compared with either agent alone, in breast and lung cancer xenograft models. Treatment of cells with T-DM1 combined with pertuzumab also resulted in a synergistic increase in apoptosis as measured by increased activity of the apoptotic enzymes, caspases 3 and 7, and increased levels of the apoptotic population in cell cycle experiments. Together, these studies support the hypothesis that T-DM1 in combination with pertuzumab for HER2-amplified cancer may offer an additional therapeutic approach for patients whose disease progresses on trastuzumab and lapatinib-based therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5607.

Synergistic interaction between trastuzumab and EGFR/HER-2 tyrosine kinase inhibitors in HER-2 positive breast cancer cells

Overexpression of HER-2 in breast cancer is frequently associated with expression of EGFR, and EGFR expression influences response to HER-2 inhibition. The aim of this study was to examine the effects of combining dual inhibition of EGFR and HER-2, using trastuzumab, gefitinib and lapatinib, in HER-2 overexpressing breast cancer cells. Combination proliferation assays were performed in two HER-2 positive breast cancer cell lines, SKBR-3 and BT-474. Trastuzumab combined with lapatinib was also tested in BT-474 xenografts. In proliferation assays, dual targeting with trastuzumab and gefitinib or lapatinib showed synergy or additivity in both SKBR-3 and BT-474 cells. Trastuzumab (10 nM) or gefitinib (5 µM) alone did not induce significant apoptosis, whereas lapatinib (0.75 µM) induced significant apoptosis in SKBR-3 cells. Trastuzumab combined with lapatinib further enhanced apoptosis induction. Trastuzumab (10 nM) and gefitinib (5 µM) induced apoptosis comparable to lapatinib alone (0.75 µM), suggesting that inhibition of both EGFR and HER-2 may be required to induce apoptosis in these cells. Pre-treatment with trastuzumab and gefitinib or lapatinib enhanced response to chemotherapy in vitro. The combination of trastuzumab and lapatinib also effectively blocked tumour growth in vivo. Dual targeting of EGFR and HER-2, by combining trastuzumab with EGFR/HER-2 tyrosine kinase inhibitors, may improve response in HER-2 overexpressing breast cancer cells that also express EGFR.

Dual targeting of EGFR and HER-2 in colon cancer cell lines

A number of studies have revealed that coexpression of EGFR and HER-2 has been found in a subset of colon cancers and may cooperatively promote tumor cell growth and survival. In the present work, two tyrosine kinase inhibitors, gefitinib and lapatinib, together with trastuzumab, raised a monoclonal antibody against HER-2 were evaluated in two colon cancer cell lines, DLD-1 and Caco-2. The aim of the study was to investigate their effect on tumor cell proliferation and apoptosis. Cell proliferation was assessed using the MTT assay and apoptosis was evaluated by DNA fragmentation and the Annexin V binding assay. EGFR and HER-2 protein and mRNA levels were evaluated by immunoblotting and quantitative RT-PCR, respectively. Treatment of cells with each agent alone resulted in inhibition of cell proliferation after 48 h in a dose-dependent manner except for trastuzumab, which did not alter cell proliferation of DLD-1. Apoptosis increased in DLD-1 cells, after 24 h treatment with gefitinib. None of the tested agents altered apoptosis in Caco-2 cells. HER-2 and EGFR protein levels did not follow the changes of mRNA levels after treatment with the tested agents. Tauhe inhibitory effect of these agents on cell proliferation and the induction of apoptosis differ for the two colon cancer cell lines under consideration. Further studies are necessary to investigate the way they exert their antitumor effect.