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Resistance mechanisms to HER2-targeted therapy in gastroesophageal adenocarcinoma: A systematic review

  • Author Footnotes
    1 DB and CIS contributed equally to this work.
    Dionne Blangé
    Correspondence
    Corresponding authors at: Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands.
    Footnotes
    1 DB and CIS contributed equally to this work.
    Affiliations
    Amsterdam UMC Location University of Amsterdam, Center for Experimental and Molecular Medicine, Laboratory of Experimental Oncology and Radiobiology, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands

    Amsterdam UMC Location University of Amsterdam, Department of Medical Oncology, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands

    Cancer Center Amsterdam, Cancer Biology, Amsterdam, The Netherlands
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  • Author Footnotes
    1 DB and CIS contributed equally to this work.
    Charlotte I. Stroes
    Footnotes
    1 DB and CIS contributed equally to this work.
    Affiliations
    Amsterdam UMC Location University of Amsterdam, Center for Experimental and Molecular Medicine, Laboratory of Experimental Oncology and Radiobiology, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands

    Amsterdam UMC Location University of Amsterdam, Department of Medical Oncology, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands

    Cancer Center Amsterdam, Cancer Biology, Amsterdam, The Netherlands
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  • Sarah Derks
    Affiliations
    Cancer Center Amsterdam, Cancer Biology, Amsterdam, The Netherlands

    Amsterdam UMC Location Vrije Universiteit Amsterdam, Department of Medical Oncology, De Boelelaan 1117-1118, 1081 HV Amsterdam, The Netherlands

    Oncode Institute, Amsterdam, The Netherlands
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  • Maarten F. Bijlsma
    Affiliations
    Amsterdam UMC Location University of Amsterdam, Center for Experimental and Molecular Medicine, Laboratory of Experimental Oncology and Radiobiology, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands

    Cancer Center Amsterdam, Cancer Biology, Amsterdam, The Netherlands

    Oncode Institute, Amsterdam, The Netherlands
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  • Hanneke W.M. van Laarhoven
    Correspondence
    Corresponding authors at: Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands.
    Affiliations
    Amsterdam UMC Location University of Amsterdam, Department of Medical Oncology, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands

    Cancer Center Amsterdam, Cancer Biology, Amsterdam, The Netherlands
    Search for articles by this author
  • Author Footnotes
    1 DB and CIS contributed equally to this work.
Open AccessPublished:May 30, 2022DOI:https://doi.org/10.1016/j.ctrv.2022.102418

      Highlights

      • Resistance to anti-HER2 therapy in esophagogastric adenocarcinoma hampers efficacy.
      • Different mechanisms of resistance are discussed in this review.
      • HER2 receptor changes and activation of alternative receptors and downstream signaling are main mechanisms of resistance to anti-HER2.
      • More research in the clinical setting is needed to identify relevant resistance mechanisms.

      Abstract

      Introduction

      Despite promising results following targeted treatment with human epidermal growth factor receptor 2 (HER2)-inhibitors in HER2-positive gastric and esophageal adenocarcinoma (GEA), prognosis remains dismal. Many patients ultimately demonstrate progression following treatment due to resistance to HER2-targeted therapy. Here, we describe the potential primary and secondary resistance mechanisms to HER2-targeted therapy in GEA.

      Methods

      We systematically searched PubMed/MEDLINE, EMBASE, and CENTRAL for eligible studies describing changes that were associated with drug resistance. Study quality was assessed using an adjusted version of the OHAT risk of bias tool. Quality of proposed resistance mechanisms was assessed using predefined criteria.

      Results

      In total, 913 records were screened, of which 73 were included that investigated mechanisms of resistance against anti-HER2 treatment in cell lines, xenograft models, patient tissue samples, and publicly available datasets. HER2-targeted therapy resistance was found to be caused by HER2 receptor changes, upregulation of compensatory receptors, (re)activation of downstream signaling pathways like PI3K/AKT and MAPK, epithelial-to-mesenchymal transition, acquirement of stem cell-like properties, alterations in cell cycle related genes, cellular metabolism, and drug pharmacokinetics.

      Discussion

      Several different mechanisms can contribute to drug resistance to anti-HER2 treatment in GEA, mainly through loss of or mutations in the HER2 receptor and upregulation of alternative receptors such as MET, HER3, and FGFRs. Despite these preclinical results, methods to overcome the proposed resistance mechanisms in the clinical setting are lacking. Therefore, further investigation of therapy resistance in GEA patients treated with HER2 targeted therapy is essential to overcome resistance and improve treatment outcome of these patients.

      Keywords

      Abbreviations:

      ADCC (Antibody dependent cellular cytotoxicity), AKT (Serine/threonine-protein kinase), EGFR (Epidermal growth factor receptor), EMT (Epithelial-to-mesenchymal transition), FGFR (Fibroblast growth factor receptor), GEA (Gastroesophageal adenocarcinoma), HER (Human epidermal growth factor receptor), MAPK (Mitogen-activated protein kinase), MUC (Membrane type mucin), OS (Overall survival), PFS (Progression free survival), PI3K (Phosphoinositide-3-kinase), PTEN (Phosphatase and tensin homolog), STAT3 (Signal transducer and activator of transcription 3), ST6Gal1 (β-galactoside α2,6-sialyltransferase 1), TGFβ (Transforming growth factor β), TME (Tumor Microenvironment)

      Introduction

      Over one million individuals worldwide are diagnosed with esophageal or gastric cancer annually. Both cancer types are major causes of mortality, ranking sixth and third as most common causes of cancer-related deaths worldwide, respectively [
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      ]. According to the ESMO guidelines, it is currently recommended to treat patients with locoregional resectable esophageal adenocarcinoma with perioperative chemotherapy or neoadjuvant chemoradiotherapy, whereas perioperative chemotherapy or adjuvant chemo(radio)therapy is recommended in patients with resectable gastric cancer [
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      ]. Despite multimodality treatment, the prognosis of gastroesophageal adenocarcinoma (GEA) remains dismal [

      SEER Cancer Stat Facts: Esophageal Cancer. Available from: https://seer.cancer.gov/statfacts/html/esoph.html.

      ,

      SEER Cancer Stat Facts: Stomach Cancer. Available from: https://seer.cancer.gov/statfacts/html/stomach.html.

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      ]. Moreover, half of the patients demonstrate progression of disease following treatment with curative intent. Although chemotherapy in the metastatic setting has demonstrated significant survival benefit whilst maintaining the patients’ quality of life, median survival is less than one year [
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      ]. Therefore, there is an unmet need to improve current therapies.
      About 15–43% of esophageal and 7–34% of gastric adenocarcinomas demonstrate overexpression or gene amplification of the ErbB receptor human epidermal growth factor receptor 2 (HER2), enabling HER2 as a target for therapy [
      • Bang Y.-J.
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      Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastro-oesophageal junction cancer (ToGA): a phase 3, open-label, randomised controlled trial.
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      HER2/neu (ERBB2) expression and gene amplification correlates with better survival in esophageal adenocarcinoma.
      ]. Activation of HER2 through homo- or heterodimerization with for instance HER3 promotes cell proliferation and signaling [
      • Gravalos C.
      • Jimeno A.
      HER2 in gastric cancer: a new prognostic factor and a novel therapeutic target.
      ,
      • Yarden Y.
      • Sliwkowski M.X.
      Untangling the ErbB signalling network.
      ]. Nevertheless, the relation between HER2 overexpression and clinical outcome remain contradictory [
      • Plum P.S.
      • Gebauer F.
      • Krämer M.
      • Alakus H.
      • Berlth F.
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      HER2/neu (ERBB2) expression and gene amplification correlates with better survival in esophageal adenocarcinoma.
      ,
      • Slamon D.J.
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      • McGuire W.L.
      Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene.
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      • Jibodh-Mulder R.A.
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      The dynamics of HER2 status in esophageal adenocarcinoma.
      ]. To date, several HER2-targeted treatments have been investigated for the treatment of GEA. Humanized monoclonal antibodies (trastuzumab, pertuzumab) directly target HER2, preventing dimerization and activation of pro-oncogenic signaling, and inducing antibody dependent cytotoxicity (ADCC) [
      • Hsu J.L.
      • Hung M.-C.
      The role of HER2, EGFR, and other receptor tyrosine kinases in breast cancer.
      ,
      • Cooley S.
      • Burns L.J.
      • Repka T.
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      Natural killer cell cytotoxicity of breast cancer targets is enhanced by two distinct mechanisms of antibody-dependent cellular cytotoxicity against LFA-3 and HER2/neu.
      ]. Tyrosine kinase inhibitors (lapatinib, afatinib) exert inhibitory effects by binding the intracellular tyrosine kinase domain of HER2 [
      • Schlam I.
      • Swain S.M.
      HER2-positive breast cancer and tyrosine kinase inhibitors: the time is now.
      ]. Antibody-drug conjugates (ado-trastuzumab emtansine (T-DM1), trastuzumab deruxtecan (DS-8201)) combine the anti-tumor effects of trastuzumab and the cytotoxicity of a microtubule-modulating compound and a topoisomerase I inhibitor, respectively [
      • Barok M.
      • Joensuu H.
      • Isola J.
      Trastuzumab emtansine: mechanisms of action and drug resistance.
      ,
      • Shitara K.
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      • Sugimoto N.
      • Ryu M.-H.
      • Sakai D.
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      Trastuzumab deruxtecan in previously treated HER2-positive gastric cancer.
      ].
      The addition of trastuzumab to first-line chemotherapy in patients with advanced GEA significantly improved outcome in the ToGA study [
      • Bang Y.-J.
      • Van Cutsem E.
      • Feyereislova A.
      • Chung H.C.
      • Shen L.
      • Sawaki A.
      • et al.
      Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastro-oesophageal junction cancer (ToGA): a phase 3, open-label, randomised controlled trial.
      ]. However, it is being debated whether patients can benefit from the addition of HER2 targeting in earlier stages of disease [
      • Stroes C.I.
      • et al.
      Phase II feasibility and biomarker study of neoadjuvant trastuzumab and pertuzumab with chemoradiotherapy for resectable human epidermal growth factor receptor 2-positive esophageal adenocarcinoma: TRAP study.
      ]. A considerable number of patients lack durable response to treatment with HER2 inhibition, potentially due to resistance [
      • Bang Y.-J.
      • Van Cutsem E.
      • Feyereislova A.
      • Chung H.C.
      • Shen L.
      • Sawaki A.
      • et al.
      Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastro-oesophageal junction cancer (ToGA): a phase 3, open-label, randomised controlled trial.
      ,
      • Stroes C.I.
      • et al.
      Phase II feasibility and biomarker study of neoadjuvant trastuzumab and pertuzumab with chemoradiotherapy for resectable human epidermal growth factor receptor 2-positive esophageal adenocarcinoma: TRAP study.
      ,
      • Tabernero J.
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      Pertuzumab plus trastuzumab and chemotherapy for HER2-positive metastatic gastric or gastro-oesophageal junction cancer (JACOB): final analysis of a double-blind, randomised, placebo-controlled phase 3 study.
      ,
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      ,
      • Safran H.P.
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      • DiPetrillo T.
      • Haddock M.G.
      • et al.
      Trastuzumab with trimodality treatment for oesophageal adenocarcinoma with HER2 overexpression (NRG Oncology/RTOG 1010): a multicentre, randomised, phase 3 trial.
      ]. Whereas some patients exhibit intrinsic resistance (primary resistance), others acquire resistance upon treatment (secondary resistance) in a short period of time. Resistance mechanisms to HER2-targeted therapies have been investigated in breast cancer [
      • Vernieri C.
      • Milano M.
      • Brambilla M.
      • Mennitto A.
      • Maggi C.
      • Cona M.S.
      • et al.
      Resistance mechanisms to anti-HER2 therapies in HER2-positive breast cancer: current knowledge, new research directions and therapeutic perspectives.
      ], but it is unclear if similar mechanisms contribute to resistance of HER2-positive GEA. It has been suggested that downregulation or changes of HER2, or upregulation of other receptor tyrosine kinase receptors and downstream signaling pathways such as phosphoinositide-3-kinase (PI3K)/serine/threonine-protein kinase (AKT) and/or mitogen-activated protein kinase (MAPK) contribute to therapy resistance [
      • Shimoyama S.
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      ]. Our limited understanding of resistance mechanisms hampers further development and improvement of HER2 targeted therapies. Therefore, we aimed to systematically review the literature regarding resistance mechanisms to HER2-targeted therapy in GEA.

      Methods

      Search strategy and eligibility criteria

      In line with the guidelines of Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA), a systematic search was performed of PubMed/MEDLINE, EMBASE, and CENTRAL from November 2021 and updated in February 2022 for articles investigating resistance mechanisms to HER2-targeted therapies in GEA. Both preclinical and clinical articles were included to collect all available data. The search included terms for the type of cancer (gastric or esophageal adenocarcinoma), HER2 treatment (HER2-neu receptor and anti-HER2 treatments), and resistance mechanisms (Supplementary Tables 1-3). Studies should describe fundamental research investigating a resistance mechanism, defined as changes in expression of pathways, genes, or immunohistochemistry upon treatment with anti-HER2 agents. Exclusion criteria were articles in non-English/Dutch, reviews, and case reports. The search strategy for PubMed/MEDLINE was rewritten for the search in EMBASE and CENTRAL. Inconsistency between conference abstracts and final papers were evaluated, and final papers were included upon availability. Two authors (DB, CIS) independently performed the data screening. Disagreements that could not be resolved by discussion, were investigated with a third arbiter (HvL).

      Study quality and risk of bias assessment

      To evaluate the quality of the included studies, an adjusted version of the OHAT risk of bias tool was used [

      Handbook for conducting a literature-based health assessment using OHAT approach for systematic review and evidence integration. 2019.

      ]. The studies were assessed on reporting bias, information bias based on (statistical) methods and the use of negative controls, selection bias based on cell or mouse population and replicated experiments, incomplete outcome bias, and other bias. The studies were graded ‘high risk’, ‘low risk’, or ‘unclear risk’, for each type of bias (Supplementary Fig. 1). In addition, the quality of the proposed resistance mechanism was assessed using predefined criteria: the presence of a control group, the use of > 2 methods and models to investigate resistance, identification, and analysis of upstream or downstream participants, correlation with resistance outcome, and restoring of sensitivity through removal of the proposed mechanism (Supplementary Fig. 2).

      Data extraction and synthesis

      Data from eligible studies were extracted using a predefined extraction sheet, including type of study, type of malignancy, type of resistance, HER2-targeting treatment, the study population (cells, xenograft models, patient tissue, datasets), the control group, the method of creating a resistant model, the method(s) of confirming resistance, and the proposed resistance mechanism. The data extraction and quality assessment of included articles were performed by two researchers (DB, CIS). Furthermore, each author checked three random studies that were analyzed by the other author. All data were obtained from the published studies or conference abstracts. No meta-analysis was performed since studies presented highly heterogeneous outcome measures.

      Results

      Search

      A total of 913 studies were found using our search strategy in the PubMed/MEDLINE, EMBASE, and CENTRAL databases (Fig. 1). From the identified records, 73 studies were included. From the included studies, 57 investigated gastric cancer, six investigated esophageal adenocarcinoma, and 10 investigated both combined (Table 1).
      Figure thumbnail gr1
      Fig. 1Flow diagram of included studies according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA).
      Table 1All studies included in this review. Studies are organized per category of resistance mechanism and could therefore be mentioned in multiple categories if more than one resistance mechanism was proposed.
      Author, YearStudy DesignType of CancerType of Cell LineAnti-HER2 AgentsType of ResistanceFactors Involved
      Activation of downstream signaling
      Deguchi, 2017
      • Deguchi Y.
      • Okabe H.
      • Oshima N.
      • Hisamori S.
      • Minamiguchi S.
      • Muto M.
      • et al.
      PTEN loss is associated with a poor response to trastuzumab in HER2-overexpressing gastroesophageal adenocarcinoma.
      In vitro + in vivo + PDMGECAGS + KATO-II + MKN-1 + MKN-7 + MKN-45 + MKN-74 + NCI-N87 + OE19 + OE33 + SNU-1TrastuzumabSecondaryPTEN loss
      Eto, 2014
      • Eto K.
      • Iwatsuki M.
      • Watanabe M.
      • Ida S.
      • Ishimoto T.
      • Iwagami S.
      • et al.
      The microRNA-21/PTEN pathway regulates the sensitivity of HER2-positive gastric cancer cells to trastuzumab.
      In vitroGCNCI-N87 + NUGC4TrastuzumabSecondaryPTEN inhibition
      Gambardella, 2017b
      • Gambardella V.
      • Sampera A.
      • Castillo J.
      • Sánchez-Martín F.
      • Gimeno-Valiente F.
      • Tarazona N.
      • et al.
      42 - SRC-S6 axis as a potential mechanism of resistance to anti HER2 treatment in gastric cancer (GC) cell lines.
      In vitroGECNCI-N87 + OE19Lapatinib, TrastuzumabSecondaryUpregulation of PI3K/AKT and MAPK
      Gambardella, 2019
      • Gambardella V.
      • Gimeno-Valiente F.
      • Tarazona N.
      • Ciarpaglini C.M.
      • Roda D.
      • Fleitas T.
      • et al.
      NRF2 through RPS6 activation is related to anti-HER2 drug resistance in HER2-amplified gastric cancer.
      In vitro + in vivo + PDMGECNCI-N87 + OE19Lapatinib, TrastuzumabSecondaryUpregulation of PI3K/AKT
      Hong, 2014
      • Hong Y.S.
      • Kim J.
      • Pectasides E.
      • Fox C.
      • Hong S.-W.
      • Ma Q.
      • et al.
      Src mutation induces acquired lapatinib resistance in ERBB2-amplified human gastroesophageal adenocarcinoma models.
      In vitroECOE19 + OE33LapatinibSecondarySrc activation
      Jin, 2017
      • Jin M.H.
      • Nam A.-R.
      • Park J.E.
      • Bang J.-H.
      • Bang Y.-J.
      • Oh D.-Y.
      Resistance mechanism against trastuzumab in HER2-positive cancer cells and its negation by Src inhibition.
      In vitro + in vivoGCNCI-N87 + SNU-216TrastuzumabSecondarySrc activation
      Kim J, 2014
      • Kim J.
      • Fox C.
      • Peng S.
      • Pusung M.
      • Pectasides E.
      • Matthee E.
      • et al.
      Preexisting oncogenic events impact trastuzumab sensitivity in ERBB2-amplified gastroesophageal adenocarcinoma.
      In vitro + PDMGECESO26 + OE19 + OE33 + MKN-7 + NCI-N87Lapatinib, TrastuzumabPrimaryPI3K mutations
      Kim, 2013
      • Kim H.S.
      • Zhang X.
      • Park K.H.
      • Park J.S.
      • Kim K.H.
      • Chung H.C.
      • et al.
      PI3K pathway as a major determinant of resistance to HER2-targeted therapy in advanced gastric cancer.
      PDMGCNALapatinib, TrastuzumabSecondaryPTEN loss
      Kim, 2017
      • Kim C.
      • Lee C.-K.
      • Chon H.J.
      • Kim J.H.
      • Park H.S.
      • Heo S.J.
      • et al.
      PTEN loss and level of HER2 amplification is associated with trastuzumab resistance and prognosis in HER2-positive gastric cancer.
      PDMGCNATrastuzumabSecondaryPTEN loss
      Liu, 2016
      • Liu J.
      • Pan C.
      • Guo L.
      • Wu M.
      • Guo J.
      • Peng S.
      • et al.
      A new mechanism of trastuzumab resistance in gastric cancer: MACC1 promotes the Warburg effect via activation of the PI3K/AKT signaling pathway.
      In vitro + in vivoGCMKN-45 + NCI-N87 + SGC-7901TrastuzumabSecondaryUpregulation of PI3K/AKT
      Liu, 2017
      • Liu W.
      • Chang J.
      • Liu M.
      • Yuan J.
      • Zhang J.
      • Qin J.
      • et al.
      Quantitative proteomics profiling reveals activation of mTOR pathway in trastuzumab resistance.
      In vitroGCNCI-N87TrastuzumabSecondaryUpregulation of PI3K/AKT
      Ma, 2016
      • Ma L.
      • Zhu W.
      • Wang Q.
      • Yang F.
      • Qian J.
      • Xu T.
      • et al.
      JWA down-regulates HER2 expression via c-Cbl and induces lapatinib resistance in human gastric cancer cells.
      In vitroGCBGC-823 + HGC-27 + NCI-N87 + SGC-7901LapatinibPrimaryJWA-induced MAPK activation
      Ning, 2021
      • Ning G.
      • Zhu Q.
      • Kang W.
      • Lee H.
      • Maher L.
      • Suh Y.-S.
      • et al.
      A novel treatment strategy for lapatinib resistance in a subset of HER2-amplified gastric cancer.
      In vitro + in vivoGECNCI-N87 + OE19LapatinibSecondaryPTEN loss
      Sampera, 2016
      • Sampera A.
      • Gelabert-Baldrich M.
      • Sánchez-Martín F.J.
      • Dalmases A.
      • Arpi O.
      • Iglesias M.
      • et al.
      Identification of molecular mechanisms of acquired resistance to trastuzumab in gastric cancer.
      In vitroGCNCI-N87TrastuzumabSecondarySrc activation
      Sampera, 2019
      • Sampera A.
      • Sánchez-Martín F.J.
      • Arpí O.
      • Visa L.
      • Iglesias M.
      • Menéndez S.
      • et al.
      HER-family ligands promote acquired resistance to trastuzumab in gastric cancer.
      In vitro + in vivoGCNCI-N87 + OE19TrastuzumabSecondarySrc-driven activation of PI3K/AKT and MAPK
      Shi, 2021
      • Shi W.
      • Zhang G.
      • Ma Z.
      • Li L.
      • Liu M.
      • Qin L.
      • et al.
      Hyperactivation of HER2-SHCBP1-PLK1 axis promotes tumor cell mitosis and impairs trastuzumab sensitivity to gastric cancer.
      In vitro + in vivo + PDM + datasetsGCNCI-N87 + SNU-216TrastuzumabPrimaryShc1-mediated activation of PI3K/AKT and MAPK
      Tang, 2017
      • Tang L.
      • Long Z.
      • Zhao N.a.
      • Feng G.
      • Guo X.
      • Yu M.
      NES1/KLK10 promotes trastuzumab resistance via activation of PI3K/AKT signaling pathway in gastric cancer.
      In vitro + in vivoGCBGC-823 + SGC-7901TrastuzumabSecondaryUpregulation of PI3K/AKT
      Wang, 2019
      • Wang D.-S.
      • Liu Z.-X.
      • Lu Y.-X.
      • Bao H.
      • Wu X.
      • Zeng Z.-L.
      • et al.
      Liquid biopsies to track trastuzumab resistance in metastatic HER2-positive gastric cancer.
      In vitro + in vivo + PDMGCNCI-N87 + SNU-216TrastuzumabPrimary + SecondaryPI3K mutations
      Yang, 2015
      • Yang Z.
      • Guo L.
      • Liu D.
      • Sun L.
      • Chen H.
      • Deng Q.
      • et al.
      Acquisition of resistance to trastuzumab in gastric cancer cells is associated with activation of IL-6/STAT3/Jagged-1/Notch positive feedback loop.
      In vitro + in vivoGCMKN-45 + NCI-N87TrastuzumabSecondaryIL-6/STAT3/Jagged-1/Notch activation
      Yokoyama, 2021
      • Yokoyama D.
      • Hisamori S.
      • Deguchi Y.
      • Nishigori T.
      • Okabe H.
      • Kanaya S.
      • et al.
      PTEN is a predictive biomarker of trastuzumab resistance and prognostic factor in HER2-overexpressing gastroesophageal adenocarcinoma.
      In vitro + PDMGECNCI-N87 + OE19TrastuzumabSecondaryPTEN loss
      Yoshioka, 2019
      • Yoshioka T.
      • et al.
      Acquired resistance mechanisms to afatinib in HER2-amplified gastric cancer cells.
      In vitro + in vivoGCNCI-N87 + SNU-216AfatinibSecondarySrc activation
      Zuo, 2015
      • Zuo Q.
      • et al.
      Development of trastuzumab-resistant human gastric carcinoma cell lines and mechanisms of drug resistance.
      In vitroGCNCI-N87TrastuzumabSecondaryPTEN loss
      Alterations in cell adhesion signaling
      Sauveur, 2018
      • Sauveur J.
      • Matera E.-L.
      • Chettab K.
      • Valet P.
      • Guitton J.
      • Savina A.
      • et al.
      Esophageal cancer cells resistant to T-DM1 display alterations in cell adhesion and the prostaglandin pathway.
      In vitroGECOE19T-DM1SecondaryReduced cell adhesion
      Yuan, 2020
      • Yuan Q.-H.
      • Liu G.
      • Hu Q.
      • Wang J.
      • Leng K.
      Identification of lapatinib sensitivity-related genes by integrative functional module analysis.
      In vitro + datasetsGCSNU-216LapatinibSecondaryRap1 signaling activation
      Alternative receptor signaling
      Chen, 2012
      • Chen C.-T.
      • Kim H.
      • Liska D.
      • Gao S.
      • Christensen J.G.
      • Weiser M.R.
      MET activation mediates resistance to lapatinib inhibition of HER2-amplified gastric cancer cells.
      In vitroGCNCI-N87 + SNU-16 + SNU-216LapatinibSecondaryMET activation
      Chen, 2019
      • Chen Z.
      • Liu Z.
      • Zhang M.
      • Huang W.
      • Li Z.
      • Wang S.
      • et al.
      EPHA2 blockade reverses acquired resistance to afatinib induced by EPHA2-mediated MAPK pathway activation in gastric cancer cells and avatar mice.
      In vitro + in vivoGCHGC-27 + MKN-45 + NCI-N87 + NUGC-4 + SNU-216AfatinibSecondaryEPHA2 activation
      Ebbing, 2016
      • Ebbing E.A.
      • Medema J.P.
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      Kim HP, 2014
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      Sampera, 2019
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      Heregulin induces resistance to lapatinib-mediated growth inhibition of HER2-amplified cancer cells.
      In vitroGCESO26 + KYAE-1 + NCI-N87 + OE19 + SK-GT-2LapatinibSecondaryHER3 activation
      Shi, 2013
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      Catecholamine-Induced β2-adrenergic receptor activation mediates desensitization of gastric cancer cells to trastuzumab by upregulating MUC4 expression.
      In vitro + in vivoGCBGC-823 + HGC-27 + MGC-803 + NCI-N87TrastuzumabSecondaryβ2-AR activation
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      In vitro + in vivoGCMKN-45 + NCI-N87TrastuzumabSecondaryHER4 activation
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      Combining trastuzumab and cetuximab combats trastuzumab-resistant gastric cancer by effective inhibition of EGFR/ErbB2 heterodimerization and signaling.
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      In vitroGCNCI-N87TrastuzumabSecondaryIGF1R activation
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      Jin, 2021
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      In vitro + in vivoGCNCI-N87 + SNU-216TrastuzumabSecondaryInhibited DNA damage response
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      Kashiwada, 2018
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      PDMGCNATrastuzumabSecondaryLow HER2 amplification
      Li, 2014
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      • Fan K.
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      In vitro + in vivoGCNCI-N87TrastuzumabSecondaryHER2 coverage by mucins
      Liu N, 2018
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      In vitroGCSGC-7901TrastuzumabSecondaryHER2 glycosylation
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      In vitroGCBGC-823 + HGC-27 + NCI-N87 + SGC-7901LapatinibPrimaryHER2 downregulation
      Piro, 2016
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      • Carbone C.
      • Cataldo I.
      • Di Nicolantonio F.
      • Giacopuzzi S.
      • Aprile G.
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      An FGFR3 autocrine loop sustains acquired resistance to trastuzumab in gastric cancer patients.
      In vitro + in vivoGCNCI-N87TrastuzumabSecondaryHER2 downregulation
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      In vitro + in vivoGCBGC-823 + HGC-27 + MGC-803 + NCI-N87TrastuzumabSecondaryHER2 coverage by mucins
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      Hassan, 2020
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      Pharmacokinetics
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      41 - Gene expression changes responsible for lapatinib acquired resistance in HER2 positive gastric cancer cell lines: a microarray analysis.
      In vitroGECOE19LapatinibSecondaryUpregulation of multidrug resistance associated proteins
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      DS-8201a, a new HER2-targeting antibody-drug conjugate incorporating a novel DNA topoisomerase I inhibitor, overcomes HER2-positive gastric cancer T-DM1 resistance.
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      Wang, 2017
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      Stemness
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      datasetsGCNCI-N87TrastuzumabSecondaryCD44 expression
      β2-AR: β2-adrenergic receptor. AKT: serine/threonine-protein kinase. CMIP: C-Maf-inducing protein. EC: esophageal cancer. EGFR: epidermal growth factor receptor. EMT: epithelial-to-mesenchymal transition. EPHA2: erythropoietin-producing hepatocellular receptor A2. FGFR1/2/3: fibroblast growth factor receptor 1/2/3. GC: gastric cancer. GEC: gastroesophageal cancer. GEJ: gastroesophageal junction cancer. GSE1: Gse1 coiled-coil protein. HER2/3/4: human epidermal growth factor receptor 2/3/4. IGF1R: insulin-like growth factor 1 receptor. MAPK: mitogen-activated protein kinase. NA: not applicable. PDM: patient derived material. PI3K: Phosphoinositide-3-kinase. PTEN: Phosphatase and tensin homolog. STAT3: signal transducer and activator of transcription 3. T-DM1: trastuzumab emtansine. TGFβ: Transforming growth factor β. YAP1: yes-associated protein.
      Most articles investigated resistance to trastuzumab (n = 43), lapatinib (n = 14), or a combination (n = 4). Other compounds investigated were afatinib (n = 4), pertuzumab (n = 2), pyrotinib (n = 2), and trastuzumab emtansine (T-DM1) (n = 5). The most commonly reported causes of HER2-targeted therapy resistance were HER2 receptor changes (n = 19), signaling via an alternative receptor (n = 24), and aberrant activation of downstream signaling (n = 23) (Fig. 2).
      Figure thumbnail gr2
      Fig. 2Graphic overview of reported resistance mechanisms against HER2-targeted therapies in GEA. (A) Overexpression, downregulation, modification, or degradation of the HER2 receptor reduce anti-HER2 treatment sensitivity, such as trastuzumab, pertuzumab, lapatinib, and T-DM1 [
      • Arienti C.
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      • Carloni S.
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      Preclinical evidence of multiple mechanisms underlying trastuzumab resistance in gastric cancer.
      ,
      • Shi J.
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      • Yao X.
      • Mou T.
      • Xu Z.
      • Han Z.
      • et al.
      The HER4-YAP1 axis promotes trastuzumab resistance in HER2-positive gastric cancer by inducing epithelial and mesenchymal transition.
      ,
      • Kashiwada T.
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      • Iwatsuki M.
      • Narita Y.
      • et al.
      Multicenter observational study on re-evaluation of HER2 status in patients with HER2-positive advanced or recurrent gastric cancer refractory to trastuzumab.
      ,
      • Shibata T.
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      • Kawahara A.
      • et al.
      Y-box binding protein-1 contributes to both HER2/ErbB2 expression and lapatinib sensitivity in human gastric cancer cells.
      ,
      • Yang G.
      • et al.
      Bioinformatics analysis of potential key genes in trastuzumab-resistant gastric cancer.
      ,
      • Kim C.
      • Lee C.-K.
      • Chon H.J.
      • Kim J.H.
      • Park H.S.
      • Heo S.J.
      • et al.
      PTEN loss and level of HER2 amplification is associated with trastuzumab resistance and prognosis in HER2-positive gastric cancer.
      ,
      • Ma L.
      • Zhu W.
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      • Yang F.
      • Qian J.
      • Xu T.
      • et al.
      JWA down-regulates HER2 expression via c-Cbl and induces lapatinib resistance in human gastric cancer cells.
      ,
      • Piro G.
      • Carbone C.
      • Cataldo I.
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      • Giacopuzzi S.
      • Aprile G.
      • et al.
      An FGFR3 autocrine loop sustains acquired resistance to trastuzumab in gastric cancer patients.
      ,
      • Hong J.
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      Regulation of ERBB2 receptor by t-DARPP mediates trastuzumab resistance in human esophageal adenocarcinoma.
      ,
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      • et al.
      ST6Gal1 targets the ectodomain of ErbB2 in a site-specific manner and regulates gastric cancer cell sensitivity to trastuzumab.
      ,
      • Liu N.
      • et al.
      Increasing HER2 α2,6 sialylation facilitates gastric cancer progression and resistance via the Akt and ERK pathways.
      ]. (B) Upon HER2 downregulation, upregulation of other receptors activate the PI3K/AKT and MAPK pathways via Src kinase [
      • Shi J.
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      • Mou T.
      • Xu Z.
      • Han Z.
      • et al.
      The HER4-YAP1 axis promotes trastuzumab resistance in HER2-positive gastric cancer by inducing epithelial and mesenchymal transition.
      ,
      • Piro G.
      • Carbone C.
      • Cataldo I.
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      • et al.
      An FGFR3 autocrine loop sustains acquired resistance to trastuzumab in gastric cancer patients.
      ,
      • Shi M.
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      • Liu D.
      • Hu Y.
      • Qian L.
      • et al.
      Catecholamine-Induced β2-adrenergic receptor activation mediates desensitization of gastric cancer cells to trastuzumab by upregulating MUC4 expression.
      ,
      • Li G.
      • Zhao L.
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      • Fan K.
      • Qian W.
      • Hou S.
      • et al.
      Feedback activation of STAT3 mediates trastuzumab resistance via upregulation of MUC1 and MUC4 expression.
      ,
      • Sanchez-Vega F.
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      EGFR and MET amplifications determine response to HER2 Inhibition in ERBB2-amplified esophagogastric cancer.
      ,
      • Kim J.
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      • Matthee E.
      • et al.
      Preexisting oncogenic events impact trastuzumab sensitivity in ERBB2-amplified gastroesophageal adenocarcinoma.
      ,
      • Hassan M.S.
      • et al.
      MET activation mediates lapatinib resistance in experimental esophageal adenocarcinoma.
      ,
      • Kang K.H.
      • et al.
      Resistance mechanisms to HER2-targeting treatment in HER2-positive gastric cancer.
      ,
      • Chen C.-T.
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      MET activation mediates resistance to lapatinib inhibition of HER2-amplified gastric cancer cells.
      ,
      • Lee Y.Y.
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      • et al.
      Phosphoproteomic analysis identifies activated MET-axis PI3K/AKT and MAPK/ERK in lapatinib-resistant cancer cell line.
      ,
      • Zhang Z.
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      • et al.
      Functional genetic approach identifies MET, HER3, IGF1R, INSR pathways as determinants of lapatinib unresponsiveness in HER2-positive gastric cancer.
      ,
      • Yoshioka T.
      • et al.
      Acquired resistance mechanisms to afatinib in HER2-amplified gastric cancer cells.
      ,
      • Park J.
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      • et al.
      FOXO1 Suppression is a determinant of acquired lapatinib-resistance in HER2-positive gastric cancer cells through MET upregulation.
      ,
      • Kim H.-P.
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      • Jeong E.-G.
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      • Hwang D.
      • et al.
      Testican-1-mediated epithelial-mesenchymal transition signaling confers acquired resistance to lapatinib in HER2-positive gastric cancer.
      ,
      • Zheng L.
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      Combining trastuzumab and cetuximab combats trastuzumab-resistant gastric cancer by effective inhibition of EGFR/ErbB2 heterodimerization and signaling.
      ,
      • Sato Y.
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      Heregulin induces resistance to lapatinib-mediated growth inhibition of HER2-amplified cancer cells.
      ,
      • Sampera A.
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      HER-family ligands promote acquired resistance to trastuzumab in gastric cancer.
      ,
      • Gambardella V.
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      42 - SRC-S6 axis as a potential mechanism of resistance to anti HER2 treatment in gastric cancer (GC) cell lines.
      ,
      • Guo J.
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      miR-301a-3p induced by endoplasmic reticulum stress mediates the occurrence and transmission of trastuzumab resistance in HER2-positive gastric cancer.
      ,
      • Yu Y.
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      miR-494 inhibits cancer-initiating cell phenotypes and reverses resistance to lapatinib by downregulating FGFR2 in HER2-positive gastric cancer.
      ,
      • Zuo Q.
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      Development of trastuzumab-resistant human gastric carcinoma cell lines and mechanisms of drug resistance.
      ,
      • Su B.
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      Apatinib exhibits synergistic effect with pyrotinib and reverses acquired pyrotinib resistance in HER2-positive gastric cancer via stem cell factor/c-kit signaling and its downstream pathways.
      ,
      • Chen Z.
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      EPHA2 blockade reverses acquired resistance to afatinib induced by EPHA2-mediated MAPK pathway activation in gastric cancer cells and avatar mice.
      ,
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      Esophageal adenocarcinoma cells and xenograft tumors exposed to Erb-b2 receptor tyrosine kinase 2 and 3 inhibitors activate transforming growth factor beta signaling, which induces epithelial to mesenchymal transition.
      ]. (C) Upregulation of EGFR and subsequent STAT3 activation result in mucin upregulation, preventing compound binding to the HER2 receptor [
      • Shi M.
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      Catecholamine-Induced β2-adrenergic receptor activation mediates desensitization of gastric cancer cells to trastuzumab by upregulating MUC4 expression.
      ,
      • Li G.
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      Feedback activation of STAT3 mediates trastuzumab resistance via upregulation of MUC1 and MUC4 expression.
      ,
      • Deng M.
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      Effect of MUC1 siRNA on drug resistance of gastric cancer cells to trastuzumab.
      ]. (D) Mutations in the receptors’ compound binding/kinase domain impair compound binding and drug efficacy [
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      Clinical implications of plasma ctDNA features and dynamics in gastric cancer treated with HER2-targeted therapies.
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      Liquid biopsies to track trastuzumab resistance in metastatic HER2-positive gastric cancer.
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      Oncogenic HER2 fusions in gastric cancer.
      ,
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      Systematic molecular profiling of inhibitor response to the clinical missense mutations of ErbB family kinases in human gastric cancer.
      ,
      • Zhang Y.
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      Identification of an activating mutation in the extracellular domain of HER2 conferring resistance to pertuzumab.
      ]. (E) Activation of ligands, such as heregulin (Her) confer resistance
      [
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      ADAM10-mediated release of heregulin confers resistance to trastuzumab by activating HER3.
      ]
      . (F) PI3K/AKT and MAPK signaling become activated by overexpression of regulating proteins and mutations in PTEN, PI3K, or Src, maintaining downstream signaling [
      • Kim C.
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      PTEN loss and level of HER2 amplification is associated with trastuzumab resistance and prognosis in HER2-positive gastric cancer.
      ,
      • Ma L.
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      JWA down-regulates HER2 expression via c-Cbl and induces lapatinib resistance in human gastric cancer cells.
      ,
      • Shi M.
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      • Hu M.
      • Liu D.
      • Hu Y.
      • Qian L.
      • et al.
      Catecholamine-Induced β2-adrenergic receptor activation mediates desensitization of gastric cancer cells to trastuzumab by upregulating MUC4 expression.
      ,
      • Wang D.-S.
      • Liu Z.-X.
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      Liquid biopsies to track trastuzumab resistance in metastatic HER2-positive gastric cancer.
      ,
      • Kim J.
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      • Matthee E.
      • et al.
      Preexisting oncogenic events impact trastuzumab sensitivity in ERBB2-amplified gastroesophageal adenocarcinoma.
      ,
      • Yoshioka T.
      • et al.
      Acquired resistance mechanisms to afatinib in HER2-amplified gastric cancer cells.
      ,
      • Sampera A.
      • Sánchez-Martín F.J.
      • Arpí O.
      • Visa L.
      • Iglesias M.
      • Menéndez S.
      • et al.
      HER-family ligands promote acquired resistance to trastuzumab in gastric cancer.
      ,
      • Gambardella V.
      • Sampera A.
      • Castillo J.
      • Sánchez-Martín F.
      • Gimeno-Valiente F.
      • Tarazona N.
      • et al.
      42 - SRC-S6 axis as a potential mechanism of resistance to anti HER2 treatment in gastric cancer (GC) cell lines.
      ,
      • Zuo Q.
      • et al.
      Development of trastuzumab-resistant human gastric carcinoma cell lines and mechanisms of drug resistance.
      ,
      • Liu W.
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      Quantitative proteomics profiling reveals activation of mTOR pathway in trastuzumab resistance.
      ,
      • Shi W.
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      • Liu M.
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      Hyperactivation of HER2-SHCBP1-PLK1 axis promotes tumor cell mitosis and impairs trastuzumab sensitivity to gastric cancer.
      ,
      • Gambardella V.
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      NRF2 through RPS6 activation is related to anti-HER2 drug resistance in HER2-amplified gastric cancer.
      ,
      • Tang L.
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      NES1/KLK10 promotes trastuzumab resistance via activation of PI3K/AKT signaling pathway in gastric cancer.
      ,
      • Liu J.
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      A new mechanism of trastuzumab resistance in gastric cancer: MACC1 promotes the Warburg effect via activation of the PI3K/AKT signaling pathway.
      ,
      • Yang Z.
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      Acquisition of resistance to trastuzumab in gastric cancer cells is associated with activation of IL-6/STAT3/Jagged-1/Notch positive feedback loop.
      ,
      • Ning G.
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      A novel treatment strategy for lapatinib resistance in a subset of HER2-amplified gastric cancer.
      ,
      • Eto K.
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      The microRNA-21/PTEN pathway regulates the sensitivity of HER2-positive gastric cancer cells to trastuzumab.
      ,
      • Kim H.S.
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      PI3K pathway as a major determinant of resistance to HER2-targeted therapy in advanced gastric cancer.
      ,
      • Yokoyama D.
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      PTEN is a predictive biomarker of trastuzumab resistance and prognostic factor in HER2-overexpressing gastroesophageal adenocarcinoma.
      ,
      • Deguchi Y.
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      PTEN loss is associated with a poor response to trastuzumab in HER2-overexpressing gastroesophageal adenocarcinoma.
      ,
      • Sampera A.
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      Identification of molecular mechanisms of acquired resistance to trastuzumab in gastric cancer.
      ,
      • Jin M.H.
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      Resistance mechanism against trastuzumab in HER2-positive cancer cells and its negation by Src inhibition.
      ,
      • Hong Y.S.
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      Src mutation induces acquired lapatinib resistance in ERBB2-amplified human gastroesophageal adenocarcinoma models.
      ]. (G) Induction of EMT by Wnt signaling and TGFβ [
      • Shi J.
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      • Mou T.
      • Xu Z.
      • Han Z.
      • et al.
      The HER4-YAP1 axis promotes trastuzumab resistance in HER2-positive gastric cancer by inducing epithelial and mesenchymal transition.
      ,
      • Yang G.
      • et al.
      Bioinformatics analysis of potential key genes in trastuzumab-resistant gastric cancer.
      ,
      • Piro G.
      • Carbone C.
      • Cataldo I.
      • Di Nicolantonio F.
      • Giacopuzzi S.
      • Aprile G.
      • et al.
      An FGFR3 autocrine loop sustains acquired resistance to trastuzumab in gastric cancer patients.
      ,
      • Zhang Z.
      • Wang J.
      • Ji D.
      • Wang C.
      • Liu R.
      • Wu Z.
      • et al.
      Functional genetic approach identifies MET, HER3, IGF1R, INSR pathways as determinants of lapatinib unresponsiveness in HER2-positive gastric cancer.
      ,
      • Kim H.-P.
      • Han S.-W.
      • Song S.-H.
      • Jeong E.-G.
      • Lee M.-Y.
      • Hwang D.
      • et al.
      Testican-1-mediated epithelial-mesenchymal transition signaling confers acquired resistance to lapatinib in HER2-positive gastric cancer.
      ,
      • Yang Z.
      • Guo L.
      • Liu D.
      • Sun L.
      • Chen H.
      • Deng Q.
      • et al.
      Acquisition of resistance to trastuzumab in gastric cancer cells is associated with activation of IL-6/STAT3/Jagged-1/Notch positive feedback loop.
      ,
      • Ebbing E.A.
      • Steins A.
      • Fessler E.
      • Stathi P.
      • Lesterhuis W.J.
      • Krishnadath K.K.
      • et al.
      Esophageal adenocarcinoma cells and xenograft tumors exposed to Erb-b2 receptor tyrosine kinase 2 and 3 inhibitors activate transforming growth factor beta signaling, which induces epithelial to mesenchymal transition.
      ,
      • Zhou X.
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      miR-200c inhibits TGF-β-induced-EMT to restore trastuzumab sensitivity by targeting ZEB1 and ZEB2 in gastric cancer.
      ,
      • Liu W.
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      Label-free quantitative proteomics combined with biological validation reveals activation of Wnt/β-catenin pathway contributing to trastuzumab resistance in gastric cancer.
      ], altered cell cycle regulation [
      • Liu N.
      • et al.
      Increasing HER2 α2,6 sialylation facilitates gastric cancer progression and resistance via the Akt and ERK pathways.
      ,
      • Kim J.
      • Fox C.
      • Peng S.
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      • Matthee E.
      • et al.
      Preexisting oncogenic events impact trastuzumab sensitivity in ERBB2-amplified gastroesophageal adenocarcinoma.
      ,
      • Shi W.
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      • Li L.
      • Liu M.
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      Hyperactivation of HER2-SHCBP1-PLK1 axis promotes tumor cell mitosis and impairs trastuzumab sensitivity to gastric cancer.
      ,
      • Shu S.
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      Abstract 2691: role of trastuzumab in the combination treatment for a HER2-positive trastuzumab-resistant gastric cancer xenograft model.
      ,
      • Jin M.-H.
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      WEE1 inhibition reverses trastuzumab resistance in HER2-positive cancers.
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      The sensitivity of gastric cancer to trastuzumab is regulated by the miR-223/FBXW7 pathway.
      ], stemness [
      • Yang Z.
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      Acquisition of resistance to trastuzumab in gastric cancer cells is associated with activation of IL-6/STAT3/Jagged-1/Notch positive feedback loop.
      ,
      • Gao X.
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      ARPP-19 mediates herceptin resistance via regulation of CD44 in gastric cancer.
      ,
      • Xiang R.u.
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      • Yu C.
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      Prediction of key genes and pathways involved in trastuzumab-resistant gastric cancer.
      ,
      • Mori H.
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      Overexpression of CD44V9 in gastric cancer cells confers resistance to trastuzumab by inducing antioxidant enzymes.
      ,
      • Wang W.
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      Overexpression of GSE1 related to trastuzumab resistance in gastric cancer cells.
      ], metabolic reprogramming [
      • Liu J.
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      A new mechanism of trastuzumab resistance in gastric cancer: MACC1 promotes the Warburg effect via activation of the PI3K/AKT signaling pathway.
      ,
      • Mori H.
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      Overexpression of CD44V9 in gastric cancer cells confers resistance to trastuzumab by inducing antioxidant enzymes.
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      • Hassan M.S.
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      Abstract 1916: targeting Warburg effect to overcome lapatinib resistance in esophageal adenocarcinoma.
      ,
      • Ye H.
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      Autophagy flux inhibition augments gastric cancer resistance to the anti-human epidermal growth factor receptor 2 antibody trastuzumab.
      ], and decreased production and increased efflux of DM1 [
      • Gambardella V.
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      41 - Gene expression changes responsible for lapatinib acquired resistance in HER2 positive gastric cancer cell lines: a microarray analysis.
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      DS-8201a, a new HER2-targeting antibody-drug conjugate incorporating a novel DNA topoisomerase I inhibitor, overcomes HER2-positive gastric cancer T-DM1 resistance.
      ,
      • Wang H.
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      • Yang Y.
      • Chen X.
      • Quan H.
      • et al.
      Aberrant intracellular metabolism of T-DM1 confers T-DM1 resistance in human epidermal growth factor receptor 2-positive gastric cancer cells.
      ] contribute to therapy resistance.

      Study quality and risk of bias assessment

      From the 73 included studies, 27 (37.0%) were considered high quality, 25 (34.2%) moderate quality, and 21 (28.8%) were of low quality, of which 10 (47.6%) were full text studies and 11 (52.4%) abstracts. The main reason for low study quality was high selection bias or incomplete data reporting (Supplementary Fig. 1). Furthermore, the resistance mechanisms of 30 studies (41.1%) were considered high quality, 28 studies (38.4%) moderate quality, and 20 studies (20.5%) low quality (Supplementary Fig. 2). The main reason for lower quality of the evidence of the proposed resistance mechanism was the absence of further exploration of upstream or downstream targets in the proposed resistance mechanism, a correlation with resistance outcome or restoring compound sensitivity by removal of the mechanism using for instance inhibitors or silencing.

      HER2 receptor changes

      Nineteen studies observed a correlation between HER2 receptor changes and anti-HER2 therapy resistance in HER2-positive GEA. In eight studies, overexpression, loss, or downregulation of the receptor was investigated [
      • Arienti C.
      • Zanoni M.
      • Pignatta S.
      • Del Rio A.
      • Carloni S.
      • Tebaldi M.
      • et al.
      Preclinical evidence of multiple mechanisms underlying trastuzumab resistance in gastric cancer.
      ,
      • Shi J.
      • Li F.
      • Yao X.
      • Mou T.
      • Xu Z.
      • Han Z.
      • et al.
      The HER4-YAP1 axis promotes trastuzumab resistance in HER2-positive gastric cancer by inducing epithelial and mesenchymal transition.
      ,
      • Kashiwada T.
      • Saeki H.
      • Uenosono Y.
      • Makiyama A.
      • Iwatsuki M.
      • Narita Y.
      • et al.
      Multicenter observational study on re-evaluation of HER2 status in patients with HER2-positive advanced or recurrent gastric cancer refractory to trastuzumab.
      ,
      • Shibata T.
      • Kan H.
      • Murakami Y.
      • Ureshino H.
      • Watari K.
      • Kawahara A.
      • et al.
      Y-box binding protein-1 contributes to both HER2/ErbB2 expression and lapatinib sensitivity in human gastric cancer cells.
      ,
      • Yang G.
      • et al.
      Bioinformatics analysis of potential key genes in trastuzumab-resistant gastric cancer.
      ,
      • Kim C.
      • Lee C.-K.
      • Chon H.J.
      • Kim J.H.
      • Park H.S.
      • Heo S.J.
      • et al.
      PTEN loss and level of HER2 amplification is associated with trastuzumab resistance and prognosis in HER2-positive gastric cancer.
      ,
      • Ma L.
      • Zhu W.
      • Wang Q.
      • Yang F.
      • Qian J.
      • Xu T.
      • et al.
      JWA down-regulates HER2 expression via c-Cbl and induces lapatinib resistance in human gastric cancer cells.
      ,
      • Piro G.
      • Carbone C.
      • Cataldo I.
      • Di Nicolantonio F.
      • Giacopuzzi S.
      • Aprile G.
      • et al.
      An FGFR3 autocrine loop sustains acquired resistance to trastuzumab in gastric cancer patients.
      ]. Nine studies described impaired HER2-target binding, either due to mutations in HER2 or coverage of the compound-binding domain [
      • Hong J.
      • Katsha A.
      • Lu P.
      • Shyr Y.u.
      • Belkhiri A.
      • El-Rifai W.
      Regulation of ERBB2 receptor by t-DARPP mediates trastuzumab resistance in human esophageal adenocarcinoma.
      ,
      • Shi M.
      • Yang Z.
      • Hu M.
      • Liu D.
      • Hu Y.
      • Qian L.
      • et al.
      Catecholamine-Induced β2-adrenergic receptor activation mediates desensitization of gastric cancer cells to trastuzumab by upregulating MUC4 expression.
      ,
      • Li G.
      • Zhao L.
      • Li W.
      • Fan K.
      • Qian W.
      • Hou S.
      • et al.
      Feedback activation of STAT3 mediates trastuzumab resistance via upregulation of MUC1 and MUC4 expression.
      ,
      • Deng M.
      • Jing D.-D.
      • Meng X.-J.
      Effect of MUC1 siRNA on drug resistance of gastric cancer cells to trastuzumab.
      ,
      • Zhang C.
      • Chen Z.
      • Chong X.
      • Chen Y.
      • Wang Z.
      • Yu R.
      • et al.
      Clinical implications of plasma ctDNA features and dynamics in gastric cancer treated with HER2-targeted therapies.
      ,
      • Wang D.-S.
      • Liu Z.-X.
      • Lu Y.-X.
      • Bao H.
      • Wu X.
      • Zeng Z.-L.
      • et al.
      Liquid biopsies to track trastuzumab resistance in metastatic HER2-positive gastric cancer.
      ,
      • Yu D.-H.
      • Tang L.
      • Dong H.
      • Dong Z.
      • Zhang L.
      • Fu J.
      • et al.
      Oncogenic HER2 fusions in gastric cancer.
      ,
      • Ding X.
      • et al.
      Systematic molecular profiling of inhibitor response to the clinical missense mutations of ErbB family kinases in human gastric cancer.
      ,
      • Zhang Y.
      • et al.
      Identification of an activating mutation in the extracellular domain of HER2 conferring resistance to pertuzumab.
      ]. Additionally, two studies assessed HER2 receptor glycosylation in gastric cancer cells [
      • Duarte H.O.
      • Rodrigues J.G.
      • Gomes C.
      • Hensbergen P.J.
      • Ederveen A.L.H.
      • de Ru A.H.
      • et al.
      ST6Gal1 targets the ectodomain of ErbB2 in a site-specific manner and regulates gastric cancer cell sensitivity to trastuzumab.
      ,
      • Liu N.
      • et al.
      Increasing HER2 α2,6 sialylation facilitates gastric cancer progression and resistance via the Akt and ERK pathways.
      ].

      Altered HER2 receptor expression

      It is previously suggested that the expression levels of membranous HER2 play a role in drug resistance [
      • Creemers A.
      • Ebbing E.A.
      • Hooijer G.K.J.
      • Stap L.
      • Jibodh-Mulder R.A.
      • Gisbertz S.S.
      • et al.
      The dynamics of HER2 status in esophageal adenocarcinoma.
      ]. While Arienti et al. reported increased HER2 expression as secondary resistance mechanism to trastuzumab, the majority of studies showed that downregulation of HER2 reduced sensitivity to lapatinib and trastuzumab in vitro [
      • Arienti C.
      • Zanoni M.
      • Pignatta S.
      • Del Rio A.
      • Carloni S.
      • Tebaldi M.
      • et al.
      Preclinical evidence of multiple mechanisms underlying trastuzumab resistance in gastric cancer.
      ,
      • Shi J.
      • Li F.
      • Yao X.
      • Mou T.
      • Xu Z.
      • Han Z.
      • et al.
      The HER4-YAP1 axis promotes trastuzumab resistance in HER2-positive gastric cancer by inducing epithelial and mesenchymal transition.
      ,
      • Shibata T.
      • Kan H.
      • Murakami Y.
      • Ureshino H.
      • Watari K.
      • Kawahara A.
      • et al.
      Y-box binding protein-1 contributes to both HER2/ErbB2 expression and lapatinib sensitivity in human gastric cancer cells.
      ,
      • Piro G.
      • Carbone C.
      • Cataldo I.
      • Di Nicolantonio F.
      • Giacopuzzi S.
      • Aprile G.
      • et al.
      An FGFR3 autocrine loop sustains acquired resistance to trastuzumab in gastric cancer patients.
      ]. Similarly, treatment resistance correlated with loss of HER2 expression in two studies investigating GEA tumor specimens of patients with progressive disease following trastuzumab-based treatment [
      • Kashiwada T.
      • Saeki H.
      • Uenosono Y.
      • Makiyama A.
      • Iwatsuki M.
      • Narita Y.
      • et al.
      Multicenter observational study on re-evaluation of HER2 status in patients with HER2-positive advanced or recurrent gastric cancer refractory to trastuzumab.
      ,
      • Kim C.
      • Lee C.-K.
      • Chon H.J.
      • Kim J.H.
      • Park H.S.
      • Heo S.J.
      • et al.
      PTEN loss and level of HER2 amplification is associated with trastuzumab resistance and prognosis in HER2-positive gastric cancer.
      ]. More specifically, others showed that gastric cancer cell lines acquired resistance to lapatinib through downregulation of Y-box-binding protein I (YB-1), which in turn downregulated HER2 expression [
      • Shibata T.
      • Kan H.
      • Murakami Y.
      • Ureshino H.
      • Watari K.
      • Kawahara A.
      • et al.
      Y-box binding protein-1 contributes to both HER2/ErbB2 expression and lapatinib sensitivity in human gastric cancer cells.
      ]. Similarly, in gastric cancer cells lapatinib and trastuzumab resistance was obtained by increased degradation of HER2 in the proteasome, potentially caused by JWA-mediated polyubiquitination of HER2 [
      • Yang G.
      • et al.
      Bioinformatics analysis of potential key genes in trastuzumab-resistant gastric cancer.
      ,
      • Ma L.
      • Zhu W.
      • Wang Q.
      • Yang F.
      • Qian J.
      • Xu T.
      • et al.
      JWA down-regulates HER2 expression via c-Cbl and induces lapatinib resistance in human gastric cancer cells.
      ]. Altogether, it is expected that reduced HER2 expression in response to anti-HER2 treatment contributes to resistance.

      Impaired HER2 receptor binding

      Mutations or gene fusion of the HER2 receptor can result in treatment resistance through inability of the drug to bind the receptor. For instance, the SNF270-HER2 fusion leads to aberrant activation of HER2, while the V777L mutation in the kinase domain and the S310F mutation in the extracellular domain prevent binding of lapatinib and pertuzumab to HER2, respectively, contributing to primary resistance [
      • Zhang C.
      • Chen Z.
      • Chong X.
      • Chen Y.
      • Wang Z.
      • Yu R.
      • et al.
      Clinical implications of plasma ctDNA features and dynamics in gastric cancer treated with HER2-targeted therapies.
      ,
      • Wang D.-S.
      • Liu Z.-X.
      • Lu Y.-X.
      • Bao H.
      • Wu X.
      • Zeng Z.-L.
      • et al.
      Liquid biopsies to track trastuzumab resistance in metastatic HER2-positive gastric cancer.
      ,
      • Yu D.-H.
      • Tang L.
      • Dong H.
      • Dong Z.
      • Zhang L.
      • Fu J.
      • et al.
      Oncogenic HER2 fusions in gastric cancer.
      ,
      • Ding X.
      • et al.
      Systematic molecular profiling of inhibitor response to the clinical missense mutations of ErbB family kinases in human gastric cancer.
      ,
      • Zhang Y.
      • et al.
      Identification of an activating mutation in the extracellular domain of HER2 conferring resistance to pertuzumab.
      ]. Furthermore, studies showed that NCI-N87 cells with secondary trastuzumab resistance exert hyperactivation of signal transducer and activator of transcription 3 (STAT3) and subsequent increased expression of membrane-type Mucin 1 and 4 (MUC1, MUC4) [
      • Shi M.
      • Yang Z.
      • Hu M.
      • Liu D.
      • Hu Y.
      • Qian L.
      • et al.
      Catecholamine-Induced β2-adrenergic receptor activation mediates desensitization of gastric cancer cells to trastuzumab by upregulating MUC4 expression.
      ,
      • Li G.
      • Zhao L.
      • Li W.
      • Fan K.
      • Qian W.
      • Hou S.
      • et al.
      Feedback activation of STAT3 mediates trastuzumab resistance via upregulation of MUC1 and MUC4 expression.
      ]. Similar overexpression of MUC1 was found in primary trastuzumab-resistant MKN-45 cells [
      • Deng M.
      • Jing D.-D.
      • Meng X.-J.
      Effect of MUC1 siRNA on drug resistance of gastric cancer cells to trastuzumab.
      ]. These mucins interfere with the recognition and binding of trastuzumab to HER2 and can actively maintain HER2 phosphorylation and activation [
      • Shi M.
      • Yang Z.
      • Hu M.
      • Liu D.
      • Hu Y.
      • Qian L.
      • et al.
      Catecholamine-Induced β2-adrenergic receptor activation mediates desensitization of gastric cancer cells to trastuzumab by upregulating MUC4 expression.
      ,
      • Li G.
      • Zhao L.
      • Li W.
      • Fan K.
      • Qian W.
      • Hou S.
      • et al.
      Feedback activation of STAT3 mediates trastuzumab resistance via upregulation of MUC1 and MUC4 expression.
      ,
      • Deng M.
      • Jing D.-D.
      • Meng X.-J.
      Effect of MUC1 siRNA on drug resistance of gastric cancer cells to trastuzumab.
      ]. Others reported that overexpression of truncated dopamine and cyclic AMP-regulated phosphoprotein of Mr32,000 (t-DARPP-32) prevented trastuzumab binding to HER2 and subsequent receptor dephosphorylation in OE19 and OE33 cells [
      • Hong J.
      • Katsha A.
      • Lu P.
      • Shyr Y.u.
      • Belkhiri A.
      • El-Rifai W.
      Regulation of ERBB2 receptor by t-DARPP mediates trastuzumab resistance in human esophageal adenocarcinoma.
      ]. Thus, impaired HER2 binding through coverage by different proteins contributes to drug resistance.

      HER2 receptor modifications

      Some studies reported that β-galactoside α2,6-sialyltransferase 1 (ST6Gal1) is overexpressed in gastric cancer specimens and cells, promoting α2,6- and α2,3-sialylation of the HER2 receptor and influencing its regulation [
      • Duarte H.O.
      • Rodrigues J.G.
      • Gomes C.
      • Hensbergen P.J.
      • Ederveen A.L.H.
      • de Ru A.H.
      • et al.
      ST6Gal1 targets the ectodomain of ErbB2 in a site-specific manner and regulates gastric cancer cell sensitivity to trastuzumab.
      ,
      • Liu N.
      • et al.
      Increasing HER2 α2,6 sialylation facilitates gastric cancer progression and resistance via the Akt and ERK pathways.
      ]. In response to trastuzumab, these modifications reduced the half-life and stabilization of the HER2 receptor at the membrane, providing secondary resistance to trastuzumab therapy in vitro.

      Alternative receptor signaling

      In response to HER2 targeting, other receptors can compensate for HER2 loss by maintaining activation of shared downstream pathways. Ten studies demonstrated MET receptor upregulation following inhibition of the HER2 receptor in resistant models [
      • Sanchez-Vega F.
      • Hechtman J.F.
      • Castel P.
      • Ku G.Y.
      • Tuvy Y.
      • Won H.
      • et al.
      EGFR and MET amplifications determine response to HER2 Inhibition in ERBB2-amplified esophagogastric cancer.
      ,
      • Kim J.
      • Fox C.
      • Peng S.
      • Pusung M.
      • Pectasides E.
      • Matthee E.
      • et al.
      Preexisting oncogenic events impact trastuzumab sensitivity in ERBB2-amplified gastroesophageal adenocarcinoma.
      ,
      • Hassan M.S.
      • et al.
      MET activation mediates lapatinib resistance in experimental esophageal adenocarcinoma.
      ,
      • Kang K.H.
      • et al.
      Resistance mechanisms to HER2-targeting treatment in HER2-positive gastric cancer.
      ,
      • Chen C.-T.
      • Kim H.
      • Liska D.
      • Gao S.
      • Christensen J.G.
      • Weiser M.R.
      MET activation mediates resistance to lapatinib inhibition of HER2-amplified gastric cancer cells.
      ,
      • Lee Y.Y.
      • Kim H.-P.
      • Kang M.J.
      • Cho B.-K.
      • Han S.-W.
      • Kim T.-Y.
      • et al.
      Phosphoproteomic analysis identifies activated MET-axis PI3K/AKT and MAPK/ERK in lapatinib-resistant cancer cell line.
      ,
      • Zhang Z.
      • Wang J.
      • Ji D.
      • Wang C.
      • Liu R.
      • Wu Z.
      • et al.
      Functional genetic approach identifies MET, HER3, IGF1R, INSR pathways as determinants of lapatinib unresponsiveness in HER2-positive gastric cancer.
      ,
      • Yoshioka T.
      • et al.
      Acquired resistance mechanisms to afatinib in HER2-amplified gastric cancer cells.
      ,
      • Park J.
      • Choi Y.
      • Ko Y.S.
      • Kim Y.
      • Pyo J.-S.
      • Jang B.G.
      • et al.
      FOXO1 Suppression is a determinant of acquired lapatinib-resistance in HER2-positive gastric cancer cells through MET upregulation.
      ,
      • Kim H.-P.
      • Han S.-W.
      • Song S.-H.
      • Jeong E.-G.
      • Lee M.-Y.
      • Hwang D.
      • et al.
      Testican-1-mediated epithelial-mesenchymal transition signaling confers acquired resistance to lapatinib in HER2-positive gastric cancer.
      ], whilst others described the contributions of other ErbB receptor family members (n = 7) [
      • Shi J.
      • Li F.
      • Yao X.
      • Mou T.
      • Xu Z.
      • Han Z.
      • et al.
      The HER4-YAP1 axis promotes trastuzumab resistance in HER2-positive gastric cancer by inducing epithelial and mesenchymal transition.
      ,
      • Li G.
      • Zhao L.
      • Li W.
      • Fan K.
      • Qian W.
      • Hou S.
      • et al.
      Feedback activation of STAT3 mediates trastuzumab resistance via upregulation of MUC1 and MUC4 expression.
      ,
      • Sanchez-Vega F.
      • Hechtman J.F.
      • Castel P.
      • Ku G.Y.
      • Tuvy Y.
      • Won H.
      • et al.
      EGFR and MET amplifications determine response to HER2 Inhibition in ERBB2-amplified esophagogastric cancer.
      ,
      • Zheng L.
      • Tan W.
      • Zhang J.
      • Yuan D.
      • Yang J.
      • Liu H.
      Combining trastuzumab and cetuximab combats trastuzumab-resistant gastric cancer by effective inhibition of EGFR/ErbB2 heterodimerization and signaling.
      ,
      • Ebbing E.A.
      • Medema J.P.
      • Damhofer H.
      • Meijer S.L.
      • Krishnadath K.K.
      • van Berge Henegouwen M.I.
      • et al.
      ADAM10-mediated release of heregulin confers resistance to trastuzumab by activating HER3.
      ,
      • Sato Y.
      • Yashiro M.
      • Takakura N.
      Heregulin induces resistance to lapatinib-mediated growth inhibition of HER2-amplified cancer cells.
      ,
      • Sampera A.
      • Sánchez-Martín F.J.
      • Arpí O.
      • Visa L.
      • Iglesias M.
      • Menéndez S.
      • et al.
      HER-family ligands promote acquired resistance to trastuzumab in gastric cancer.
      ], fibroblast growth factor receptors (FGFR) (n = 4) [
      • Piro G.
      • Carbone C.
      • Cataldo I.
      • Di Nicolantonio F.
      • Giacopuzzi S.
      • Aprile G.
      • et al.
      An FGFR3 autocrine loop sustains acquired resistance to trastuzumab in gastric cancer patients.
      ,
      • Gambardella V.
      • Sampera A.
      • Castillo J.
      • Sánchez-Martín F.
      • Gimeno-Valiente F.
      • Tarazona N.
      • et al.
      42 - SRC-S6 axis as a potential mechanism of resistance to anti HER2 treatment in gastric cancer (GC) cell lines.
      ,
      • Guo J.
      • Zhong X.
      • Tan Q.
      • Yang S.
      • Liao J.
      • Zhuge J.
      • et al.
      miR-301a-3p induced by endoplasmic reticulum stress mediates the occurrence and transmission of trastuzumab resistance in HER2-positive gastric cancer.
      ,
      • Yu Y.
      • Yu X.
      • Liu H.
      • Song Q.
      • Yang Y.
      miR-494 inhibits cancer-initiating cell phenotypes and reverses resistance to lapatinib by downregulating FGFR2 in HER2-positive gastric cancer.
      ], or other receptors (n = 6) [
      • Shi M.
      • Yang Z.
      • Hu M.
      • Liu D.
      • Hu Y.
      • Qian L.
      • et al.
      Catecholamine-Induced β2-adrenergic receptor activation mediates desensitization of gastric cancer cells to trastuzumab by upregulating MUC4 expression.
      ,
      • Zhang Z.
      • Wang J.
      • Ji D.
      • Wang C.
      • Liu R.
      • Wu Z.
      • et al.
      Functional genetic approach identifies MET, HER3, IGF1R, INSR pathways as determinants of lapatinib unresponsiveness in HER2-positive gastric cancer.
      ,
      • Guo J.
      • Zhong X.
      • Tan Q.
      • Yang S.
      • Liao J.
      • Zhuge J.
      • et al.
      miR-301a-3p induced by endoplasmic reticulum stress mediates the occurrence and transmission of trastuzumab resistance in HER2-positive gastric cancer.
      ,
      • Zuo Q.
      • et al.
      Development of trastuzumab-resistant human gastric carcinoma cell lines and mechanisms of drug resistance.
      ,
      • Su B.
      • Huang T.
      • Jin Y.
      • Yin H.
      • Qiu H.
      • Yuan X.
      Apatinib exhibits synergistic effect with pyrotinib and reverses acquired pyrotinib resistance in HER2-positive gastric cancer via stem cell factor/c-kit signaling and its downstream pathways.
      ,
      • Chen Z.
      • Liu Z.
      • Zhang M.
      • Huang W.
      • Li Z.
      • Wang S.
      • et al.
      EPHA2 blockade reverses acquired resistance to afatinib induced by EPHA2-mediated MAPK pathway activation in gastric cancer cells and avatar mice.
      ].

      ErbB receptor upregulation

      As shown by a phase II study in trastuzumab-resistant HER2-positive GEA patients, upregulation of the ErbB receptor family member epidermal growth factor receptor (EGFR) was associated with afatinib resistance [
      • Sanchez-Vega F.
      • Hechtman J.F.
      • Castel P.
      • Ku G.Y.
      • Tuvy Y.
      • Won H.
      • et al.
      EGFR and MET amplifications determine response to HER2 Inhibition in ERBB2-amplified esophagogastric cancer.
      ]. Furthermore, overexpression of EGFR results in increased heterodimerization with HER2, maintaining downstream signaling in a trastuzumab-resistant in vitro model [
      • Zheng L.
      • Tan W.
      • Zhang J.
      • Yuan D.
      • Yang J.
      • Liu H.
      Combining trastuzumab and cetuximab combats trastuzumab-resistant gastric cancer by effective inhibition of EGFR/ErbB2 heterodimerization and signaling.
      ]. The overexpression of EGFR could be explained by feedback activation via STAT3 [
      • Li G.
      • Zhao L.
      • Li W.
      • Fan K.
      • Qian W.
      • Hou S.
      • et al.
      Feedback activation of STAT3 mediates trastuzumab resistance via upregulation of MUC1 and MUC4 expression.
      ]. Moreover, HER3 and HER4 were upregulated in acquired trastuzumab and lapatinib resistant GEA cells [
      • Shi J.
      • Li F.
      • Yao X.
      • Mou T.
      • Xu Z.
      • Han Z.
      • et al.
      The HER4-YAP1 axis promotes trastuzumab resistance in HER2-positive gastric cancer by inducing epithelial and mesenchymal transition.
      ,
      • Ebbing E.A.
      • Medema J.P.
      • Damhofer H.
      • Meijer S.L.
      • Krishnadath K.K.
      • van Berge Henegouwen M.I.
      • et al.
      ADAM10-mediated release of heregulin confers resistance to trastuzumab by activating HER3.
      ,
      • Sato Y.
      • Yashiro M.
      • Takakura N.
      Heregulin induces resistance to lapatinib-mediated growth inhibition of HER2-amplified cancer cells.
      ,
      • Sampera A.
      • Sánchez-Martín F.J.
      • Arpí O.
      • Visa L.
      • Iglesias M.
      • Menéndez S.
      • et al.
      HER-family ligands promote acquired resistance to trastuzumab in gastric cancer.
      ]. More specifically, HER3 upregulation was accompanied by increased levels of its ligand Heregulin, which was released from the membrane by matrix metalloprotease ADAM10. The activation of HER3 subsequently led to increased HER2/HER3 heterodimer formation that conferred secondary resistance [
      • Ebbing E.A.
      • Medema J.P.
      • Damhofer H.
      • Meijer S.L.
      • Krishnadath K.K.
      • van Berge Henegouwen M.I.
      • et al.
      ADAM10-mediated release of heregulin confers resistance to trastuzumab by activating HER3.
      ,
      • Sato Y.
      • Yashiro M.
      • Takakura N.
      Heregulin induces resistance to lapatinib-mediated growth inhibition of HER2-amplified cancer cells.
      ].

      MET receptor upregulation

      As reported by multiple studies, MET was upregulated and phosphorylated in cells treated with lapatinib, afatinib, or trastuzumab, or in cells demonstrating secondary resistance to these anti-HER2 agents. Thus, MET upregulation contributes to drug resistance by maintaining downstream signaling and stimulating cell cycle progression in vitro [
      • Hassan M.S.
      • et al.
      MET activation mediates lapatinib resistance in experimental esophageal adenocarcinoma.
      ,
      • Kang K.H.
      • et al.
      Resistance mechanisms to HER2-targeting treatment in HER2-positive gastric cancer.
      ,
      • Chen C.-T.
      • Kim H.
      • Liska D.
      • Gao S.
      • Christensen J.G.
      • Weiser M.R.
      MET activation mediates resistance to lapatinib inhibition of HER2-amplified gastric cancer cells.
      ,
      • Lee Y.Y.
      • Kim H.-P.
      • Kang M.J.
      • Cho B.-K.
      • Han S.-W.
      • Kim T.-Y.
      • et al.
      Phosphoproteomic analysis identifies activated MET-axis PI3K/AKT and MAPK/ERK in lapatinib-resistant cancer cell line.
      ,
      • Zhang Z.
      • Wang J.
      • Ji D.
      • Wang C.
      • Liu R.
      • Wu Z.
      • et al.
      Functional genetic approach identifies MET, HER3, IGF1R, INSR pathways as determinants of lapatinib unresponsiveness in HER2-positive gastric cancer.
      ,
      • Yoshioka T.
      • et al.
      Acquired resistance mechanisms to afatinib in HER2-amplified gastric cancer cells.
      ,
      • Park J.
      • Choi Y.
      • Ko Y.S.
      • Kim Y.
      • Pyo J.-S.
      • Jang B.G.
      • et al.
      FOXO1 Suppression is a determinant of acquired lapatinib-resistance in HER2-positive gastric cancer cells through MET upregulation.
      ,
      • Kim H.-P.
      • Han S.-W.
      • Song S.-H.
      • Jeong E.-G.
      • Lee M.-Y.
      • Hwang D.
      • et al.
      Testican-1-mediated epithelial-mesenchymal transition signaling confers acquired resistance to lapatinib in HER2-positive gastric cancer.
      ]. These results are supported by sequencing results of HER2-positive GEA patients specimens, showing upregulation and amplification of MET in untreated and trastuzumab-treated specimens [
      • Sanchez-Vega F.
      • Hechtman J.F.
      • Castel P.
      • Ku G.Y.
      • Tuvy Y.
      • Won H.
      • et al.
      EGFR and MET amplifications determine response to HER2 Inhibition in ERBB2-amplified esophagogastric cancer.
      ,
      • Kim J.
      • Fox C.
      • Peng S.
      • Pusung M.
      • Pectasides E.
      • Matthee E.
      • et al.
      Preexisting oncogenic events impact trastuzumab sensitivity in ERBB2-amplified gastroesophageal adenocarcinoma.
      ].

      FGFR and other receptor upregulation

      In acquired lapatinib and trastuzumab resistant in vitro or in vivo models, FGFR1, FGFR2, and FGFR3 were upregulated and contributed to secondary drug resistance [
      • Piro G.
      • Carbone C.
      • Cataldo I.
      • Di Nicolantonio F.
      • Giacopuzzi S.
      • Aprile G.
      • et al.
      An FGFR3 autocrine loop sustains acquired resistance to trastuzumab in gastric cancer patients.
      ,
      • Gambardella V.
      • Sampera A.
      • Castillo J.
      • Sánchez-Martín F.
      • Gimeno-Valiente F.
      • Tarazona N.
      • et al.
      42 - SRC-S6 axis as a potential mechanism of resistance to anti HER2 treatment in gastric cancer (GC) cell lines.
      ,
      • Guo J.
      • Zhong X.
      • Tan Q.
      • Yang S.
      • Liao J.
      • Zhuge J.
      • et al.
      miR-301a-3p induced by endoplasmic reticulum stress mediates the occurrence and transmission of trastuzumab resistance in HER2-positive gastric cancer.
      ,
      • Yu Y.
      • Yu X.
      • Liu H.
      • Song Q.
      • Yang Y.
      miR-494 inhibits cancer-initiating cell phenotypes and reverses resistance to lapatinib by downregulating FGFR2 in HER2-positive gastric cancer.
      ]. Some studies reported that the expression of FGFR1 and FGFR2 was controlled by the expression of microRNAs miR-301a-3p and miR-494. While miR-301a-3p promoted resistance by stimulating FGFR1 expression, miR-494 reversed resistance by downregulating FGFR2 [
      • Guo J.
      • Zhong X.
      • Tan Q.
      • Yang S.
      • Liao J.
      • Zhuge J.
      • et al.
      miR-301a-3p induced by endoplasmic reticulum stress mediates the occurrence and transmission of trastuzumab resistance in HER2-positive gastric cancer.
      ,
      • Yu Y.
      • Yu X.
      • Liu H.
      • Song Q.
      • Yang Y.
      miR-494 inhibits cancer-initiating cell phenotypes and reverses resistance to lapatinib by downregulating FGFR2 in HER2-positive gastric cancer.
      ]. Additionally, the insulin-like growth factor 1 receptor (IGF1R) was found to contribute to primary anti-HER2 therapy resistance, while the c-kit receptor, the β2-adrenergic receptor (β2-AR), and erythropoietin-producing hepatocellular receptor A2 (EPHA2) were described to contribute to secondary resistance [
      • Shi M.
      • Yang Z.
      • Hu M.
      • Liu D.
      • Hu Y.
      • Qian L.
      • et al.
      Catecholamine-Induced β2-adrenergic receptor activation mediates desensitization of gastric cancer cells to trastuzumab by upregulating MUC4 expression.
      ,
      • Zhang Z.
      • Wang J.
      • Ji D.
      • Wang C.
      • Liu R.
      • Wu Z.
      • et al.
      Functional genetic approach identifies MET, HER3, IGF1R, INSR pathways as determinants of lapatinib unresponsiveness in HER2-positive gastric cancer.
      ,
      • Guo J.
      • Zhong X.
      • Tan Q.
      • Yang S.
      • Liao J.
      • Zhuge J.
      • et al.
      miR-301a-3p induced by endoplasmic reticulum stress mediates the occurrence and transmission of trastuzumab resistance in HER2-positive gastric cancer.
      ,
      • Zuo Q.
      • et al.
      Development of trastuzumab-resistant human gastric carcinoma cell lines and mechanisms of drug resistance.
      ,
      • Su B.
      • Huang T.
      • Jin Y.
      • Yin H.
      • Qiu H.
      • Yuan X.
      Apatinib exhibits synergistic effect with pyrotinib and reverses acquired pyrotinib resistance in HER2-positive gastric cancer via stem cell factor/c-kit signaling and its downstream pathways.
      ,
      • Chen Z.
      • Liu Z.
      • Zhang M.
      • Huang W.
      • Li Z.
      • Wang S.
      • et al.
      EPHA2 blockade reverses acquired resistance to afatinib induced by EPHA2-mediated MAPK pathway activation in gastric cancer cells and avatar mice.
      ]. Chen et al. identified the latter receptor as a new dimerization partner of HER2 that contributed to secondary afatinib resistance [
      • Chen Z.
      • Liu Z.
      • Zhang M.
      • Huang W.
      • Li Z.
      • Wang S.
      • et al.
      EPHA2 blockade reverses acquired resistance to afatinib induced by EPHA2-mediated MAPK pathway activation in gastric cancer cells and avatar mice.
      ].

      Activation of downstream signaling

      Several studies (n = 23) investigated alterations in downstream signaling cascades that correlate with HER2-targeted therapy resistance. Whereas often activation of downstream signaling results from alternative receptor signaling on the cell surface, others described downstream activation through direct activation by overexpression of several genes. In ten studies, the MAPK and PI3K/AKT pathways were reported to contribute to resistance [
      • Ma L.
      • Zhu W.
      • Wang Q.
      • Yang F.
      • Qian J.
      • Xu T.
      • et al.
      JWA down-regulates HER2 expression via c-Cbl and induces lapatinib resistance in human gastric cancer cells.
      ,
      • Shi M.
      • Yang Z.
      • Hu M.
      • Liu D.
      • Hu Y.
      • Qian L.
      • et al.
      Catecholamine-Induced β2-adrenergic receptor activation mediates desensitization of gastric cancer cells to trastuzumab by upregulating MUC4 expression.
      ,
      • Wang D.-S.
      • Liu Z.-X.
      • Lu Y.-X.
      • Bao H.
      • Wu X.
      • Zeng Z.-L.
      • et al.
      Liquid biopsies to track trastuzumab resistance in metastatic HER2-positive gastric cancer.
      ,
      • Kim J.
      • Fox C.
      • Peng S.
      • Pusung M.
      • Pectasides E.
      • Matthee E.
      • et al.
      Preexisting oncogenic events impact trastuzumab sensitivity in ERBB2-amplified gastroesophageal adenocarcinoma.
      ,
      • Liu W.
      • Chang J.
      • Liu M.
      • Yuan J.
      • Zhang J.
      • Qin J.
      • et al.
      Quantitative proteomics profiling reveals activation of mTOR pathway in trastuzumab resistance.
      ,
      • Shi W.
      • Zhang G.
      • Ma Z.
      • Li L.
      • Liu M.
      • Qin L.
      • et al.
      Hyperactivation of HER2-SHCBP1-PLK1 axis promotes tumor cell mitosis and impairs trastuzumab sensitivity to gastric cancer.
      ,
      • Gambardella V.
      • Gimeno-Valiente F.
      • Tarazona N.
      • Ciarpaglini C.M.
      • Roda D.
      • Fleitas T.
      • et al.
      NRF2 through RPS6 activation is related to anti-HER2 drug resistance in HER2-amplified gastric cancer.
      ,
      • Tang L.
      • Long Z.
      • Zhao N.a.
      • Feng G.
      • Guo X.
      • Yu M.
      NES1/KLK10 promotes trastuzumab resistance via activation of PI3K/AKT signaling pathway in gastric cancer.
      ,
      • Liu J.
      • Pan C.
      • Guo L.
      • Wu M.
      • Guo J.
      • Peng S.
      • et al.
      A new mechanism of trastuzumab resistance in gastric cancer: MACC1 promotes the Warburg effect via activation of the PI3K/AKT signaling pathway.
      ,
      • Yang Z.
      • Guo L.
      • Liu D.
      • Sun L.
      • Chen H.
      • Deng Q.
      • et al.
      Acquisition of resistance to trastuzumab in gastric cancer cells is associated with activation of IL-6/STAT3/Jagged-1/Notch positive feedback loop.
      ]. Furthermore, six studies assessed the loss of phosphatase and tensin homolog (PTEN) expression [
      • Kim C.
      • Lee C.-K.
      • Chon H.J.
      • Kim J.H.
      • Park H.S.
      • Heo S.J.
      • et al.
      PTEN loss and level of HER2 amplification is associated with trastuzumab resistance and prognosis in HER2-positive gastric cancer.
      ,
      • Zuo Q.
      • et al.
      Development of trastuzumab-resistant human gastric carcinoma cell lines and mechanisms of drug resistance.
      ,
      • Ning G.
      • Zhu Q.
      • Kang W.
      • Lee H.
      • Maher L.
      • Suh Y.-S.
      • et al.
      A novel treatment strategy for lapatinib resistance in a subset of HER2-amplified gastric cancer.
      ,
      • Eto K.
      • Iwatsuki M.
      • Watanabe M.
      • Ida S.
      • Ishimoto T.
      • Iwagami S.
      • et al.
      The microRNA-21/PTEN pathway regulates the sensitivity of HER2-positive gastric cancer cells to trastuzumab.
      ,
      • Kim H.S.
      • Zhang X.
      • Park K.H.
      • Park J.S.
      • Kim K.H.
      • Chung H.C.
      • et al.
      PI3K pathway as a major determinant of resistance to HER2-targeted therapy in advanced gastric cancer.
      ,
      • Yokoyama D.
      • Hisamori S.
      • Deguchi Y.
      • Nishigori T.
      • Okabe H.
      • Kanaya S.
      • et al.
      PTEN is a predictive biomarker of trastuzumab resistance and prognostic factor in HER2-overexpressing gastroesophageal adenocarcinoma.
      ,
      • Deguchi Y.
      • Okabe H.
      • Oshima N.
      • Hisamori S.
      • Minamiguchi S.
      • Muto M.
      • et al.
      PTEN loss is associated with a poor response to trastuzumab in HER2-overexpressing gastroesophageal adenocarcinoma.
      ], six studies investigated Src kinase signaling [
      • Yoshioka T.
      • et al.
      Acquired resistance mechanisms to afatinib in HER2-amplified gastric cancer cells.
      ,
      • Sampera A.
      • Sánchez-Martín F.J.
      • Arpí O.
      • Visa L.
      • Iglesias M.
      • Menéndez S.
      • et al.
      HER-family ligands promote acquired resistance to trastuzumab in gastric cancer.
      ,
      • Gambardella V.
      • Sampera A.
      • Castillo J.
      • Sánchez-Martín F.
      • Gimeno-Valiente F.
      • Tarazona N.
      • et al.
      42 - SRC-S6 axis as a potential mechanism of resistance to anti HER2 treatment in gastric cancer (GC) cell lines.
      ,
      • Sampera A.
      • Gelabert-Baldrich M.
      • Sánchez-Martín F.J.
      • Dalmases A.
      • Arpi O.
      • Iglesias M.
      • et al.
      Identification of molecular mechanisms of acquired resistance to trastuzumab in gastric cancer.
      ,
      • Jin M.H.
      • Nam A.-R.
      • Park J.E.
      • Bang J.-H.
      • Bang Y.-J.
      • Oh D.-Y.
      Resistance mechanism against trastuzumab in HER2-positive cancer cells and its negation by Src inhibition.
      ,
      • Hong Y.S.
      • Kim J.
      • Pectasides E.
      • Fox C.
      • Hong S.-W.
      • Ma Q.
      • et al.
      Src mutation induces acquired lapatinib resistance in ERBB2-amplified human gastroesophageal adenocarcinoma models.
      ], and one investigated both [
      • Ning G.
      • Zhu Q.
      • Kang W.
      • Lee H.
      • Maher L.
      • Suh Y.-S.
      • et al.
      A novel treatment strategy for lapatinib resistance in a subset of HER2-amplified gastric cancer.
      ].

      PI3K/AKT and MAPK pathway activation

      The PI3K/AKT and MAPK signaling pathways are frequently activated in malignancies by receptors such as HER2, and play important roles in cell metabolism, proliferation, differentiation, migration, and survival [
      • Vara J.Á.F.
      • Casado E.
      • de Castro J.
      • Cejas P.
      • Belda-Iniesta C.
      • González-Barón M.
      PI3K/Akt signalling pathway and cancer.
      ,
      • Dhillon A.S.
      • Hagan S.
      • Rath O.
      • Kolch W.
      MAP kinase signalling pathways in cancer.
      ]. Some studies reported that primary and secondary trastuzumab resistance was initiated by increased activation of PI3K/AKT and MAPK signaling pathways, caused by overexpression of the scaffold protein SHC1, nuclear factor erythroid 2-related factor 2 (NRF2), and normal epithelial cell-specific-1 protein (NES1) [
      • Liu W.
      • Chang J.
      • Liu M.
      • Yuan J.
      • Zhang J.
      • Qin J.
      • et al.
      Quantitative proteomics profiling reveals activation of mTOR pathway in trastuzumab resistance.
      ,
      • Shi W.
      • Zhang G.
      • Ma Z.
      • Li L.
      • Liu M.
      • Qin L.
      • et al.
      Hyperactivation of HER2-SHCBP1-PLK1 axis promotes tumor cell mitosis and impairs trastuzumab sensitivity to gastric cancer.
      ,
      • Gambardella V.
      • Gimeno-Valiente F.
      • Tarazona N.
      • Ciarpaglini C.M.
      • Roda D.
      • Fleitas T.
      • et al.
      NRF2 through RPS6 activation is related to anti-HER2 drug resistance in HER2-amplified gastric cancer.
      ,
      • Tang L.
      • Long Z.
      • Zhao N.a.
      • Feng G.
      • Guo X.
      • Yu M.
      NES1/KLK10 promotes trastuzumab resistance via activation of PI3K/AKT signaling pathway in gastric cancer.
      ]. In addition, others showed that tumor suppressor JWA and metastasis associated with the colon cancer 1 (MACC1) were both overexpressed in primary and secondary trastuzumab-resistant models, respectively, and directly activated the PI3K/AKT and MAPK pathways [
      • Ma L.
      • Zhu W.
      • Wang Q.
      • Yang F.
      • Qian J.
      • Xu T.
      • et al.
      JWA down-regulates HER2 expression via c-Cbl and induces lapatinib resistance in human gastric cancer cells.
      ,
      • Liu J.
      • Pan C.
      • Guo L.
      • Wu M.
      • Guo J.
      • Peng S.
      • et al.
      A new mechanism of trastuzumab resistance in gastric cancer: MACC1 promotes the Warburg effect via activation of the PI3K/AKT signaling pathway.
      ]. Analysis of HER2-positive GEA tissues revealed that activating mutations in PI3K induced primary resistance to lapatinib and trastuzumab and were correlated with worse progression free survival (PFS) [
      • Wang D.-S.
      • Liu Z.-X.
      • Lu Y.-X.
      • Bao H.
      • Wu X.
      • Zeng Z.-L.
      • et al.
      Liquid biopsies to track trastuzumab resistance in metastatic HER2-positive gastric cancer.
      ,
      • Kim J.
      • Fox C.
      • Peng S.
      • Pusung M.
      • Pectasides E.
      • Matthee E.
      • et al.
      Preexisting oncogenic events impact trastuzumab sensitivity in ERBB2-amplified gastroesophageal adenocarcinoma.
      ]. Yang et al. reported that NCI-N87 and MKN-45 cells with secondary trastuzumab resistance were dependent on IL-6/STAT3/Jagged-1/Notch-mediated survival signaling instead of PI3K/AKT signaling [
      • Yang Z.
      • Guo L.
      • Liu D.
      • Sun L.
      • Chen H.
      • Deng Q.
      • et al.
      Acquisition of resistance to trastuzumab in gastric cancer cells is associated with activation of IL-6/STAT3/Jagged-1/Notch positive feedback loop.
      ].

      Loss of PTEN expression

      Inactivation of the tumor suppressor PTEN due to genetic alterations or transcriptional modifications leads to major changes in signaling and contributes to cancer treatment efficacy [
      • Keniry M.
      • Parsons R.
      The role of PTEN signaling perturbations in cancer and in targeted therapy.
      ]. Mutations or downregulation of PTEN resulted in increased PI3K/AKT and MAPK signaling and secondary resistance to trastuzumab and lapatinib in vitro [
      • Zuo Q.
      • et al.
      Development of trastuzumab-resistant human gastric carcinoma cell lines and mechanisms of drug resistance.
      ,
      • Ning G.
      • Zhu Q.
      • Kang W.
      • Lee H.
      • Maher L.
      • Suh Y.-S.
      • et al.
      A novel treatment strategy for lapatinib resistance in a subset of HER2-amplified gastric cancer.
      ,
      • Eto K.
      • Iwatsuki M.
      • Watanabe M.
      • Ida S.
      • Ishimoto T.
      • Iwagami S.
      • et al.
      The microRNA-21/PTEN pathway regulates the sensitivity of HER2-positive gastric cancer cells to trastuzumab.
      ]. Likewise, PTEN loss is common in HER2-positive GEA patients (34–67%), and associated with shorter PFS and OS and acquired trastuzumab resistance [
      • Kim C.
      • Lee C.-K.
      • Chon H.J.
      • Kim J.H.
      • Park H.S.
      • Heo S.J.
      • et al.
      PTEN loss and level of HER2 amplification is associated with trastuzumab resistance and prognosis in HER2-positive gastric cancer.
      ,
      • Kim H.S.
      • Zhang X.
      • Park K.H.
      • Park J.S.
      • Kim K.H.
      • Chung H.C.
      • et al.
      PI3K pathway as a major determinant of resistance to HER2-targeted therapy in advanced gastric cancer.
      ,
      • Yokoyama D.
      • Hisamori S.
      • Deguchi Y.
      • Nishigori T.
      • Okabe H.
      • Kanaya S.
      • et al.
      PTEN is a predictive biomarker of trastuzumab resistance and prognostic factor in HER2-overexpressing gastroesophageal adenocarcinoma.
      ,
      • Deguchi Y.
      • Okabe H.
      • Oshima N.
      • Hisamori S.
      • Minamiguchi S.
      • Muto M.
      • et al.
      PTEN loss is associated with a poor response to trastuzumab in HER2-overexpressing gastroesophageal adenocarcinoma.
      ].

      Src kinase activation

      Src tyrosine kinases interact with other receptors to activate downstream signaling cascades such as MAPK, PI3K/AKT, and STAT3 pathways [
      • Wheeler D.L.
      • Iida M.
      • Dunn E.F.
      The role of Src in solid tumors.
      ]. Multiple studies showed that NCI-N87 cells with secondary trastuzumab, lapatinib, or afatinib resistance have increased phosphorylation of Src or YES1, another member of the Src kinase family. Whereas the majority of studies demonstrated individual Src or YES1 upregulation and activation, others described this mechanism as a response to upregulation of HER-receptors on the cell membrane. Increased phosphorylation led to increased activation of downstream pathways [
      • Yoshioka T.
      • et al.
      Acquired resistance mechanisms to afatinib in HER2-amplified gastric cancer cells.
      ,
      • Sampera A.
      • Sánchez-Martín F.J.
      • Arpí O.
      • Visa L.
      • Iglesias M.
      • Menéndez S.
      • et al.
      HER-family ligands promote acquired resistance to trastuzumab in gastric cancer.
      ,
      • Gambardella V.
      • Sampera A.
      • Castillo J.
      • Sánchez-Martín F.
      • Gimeno-Valiente F.
      • Tarazona N.
      • et al.
      42 - SRC-S6 axis as a potential mechanism of resistance to anti HER2 treatment in gastric cancer (GC) cell lines.