Pan‐HER—An antibody mixture targeting EGFR,HER2 and HER3 abrogates preformed and ligand‐induced EGFR homo‐ and heterodimers

The human epidermal growth factor receptor (HER)‐family is involved in development of many epithelial cancers. Therefore, HER‐family members constitute important targets for anti‐cancer therapeutics such as monoclonal antibodies (mAbs). A limitation to the success of single HER‐targeting mAbs is development of acquired resistance through mechanisms such as alterted receptor dimerization patterns and dependencies. Pan‐HER is a mixture of six mAbs simultaneously targeting epidermal growth factor receptor (EGFR), HER2 and HER3 with two mAbs against each receptor. Pan‐HER has previously demonstrated broader efficacy than targeting single or dual receptor combinations also in resistant settings. In light of this broad efficacy, we decided to investigate the effect of Pan‐HER compared with single HER‐targeting with single and dual mAbs on HER‐family cross‐talk and dimerization focusing on EGFR. The effect of Pan‐HER on cell proliferation and HER‐family receptor degradation was superior to treatment with single mAbs targeting either single receptor, and similar to targeting a single receptor with two non‐overlapping antibodies. Furthermore, changes in EGFR‐dimerization patterns after treatment with Pan‐HER were investigated by in situ proximity ligation assay and co‐immunoprecipitation, demonstrating that Pan‐HER and the EGFR‐targeting mAb mixture efficiently down‐regulate basal EGFR homo‐ and heterodimerization in two tested cell lines, whereas single mAbs had limited effects. Pan‐HER and the EGFR‐targeting mAb mixture also blocked EGF‐binding and thereby ligand‐induced changes in EGFR‐dimerization levels. These results suggest that Pan‐HER reduces the cellular capability to switch HER‐dependency and dimerization pattern in response to treatment and thus hold promise for future clinical development of Pan‐HER in resistant settings.     

Tumor-specific cell surface expression of the -KDEL containing endoplasmic reticular heat shock protein gp96

Heat shock protein (HSP) gp96/grp94 contains a signal peptide at the amino terminus and a -KDEL sequence at the carboxy terminus and is a major component of the lumen of the mammalian endoplasmic reticulum (ER). We show, by a number of immunolocalization methods using light and electron microscopy, that a significant proportion of intact gp96 molecules is also expressed on the cell surface. Surface gp96 molecules truly represent surface expression and do not result from adventitious deposition of gp96 released by dead cells on to the live cells in culture. Cell surface expression of gp96 is enhanced by heat shock and exposure to reducing agents. Gp96 molecules are not released from plasma membranes by repeated salt washes, and gp96 is not an integral membrane protein. Our observations suggest that gp96 and perhaps other HSPs are anchored to the cell surface as part of larger molecular complexes, which also transport them to the cell surface. © 1996 Wiley-Liss, Inc.     

Antitumor activity of pan‐HER inhibitors in HER2‐positive gastric cancer

Molecularly targeted therapy has enabled outstanding advances in cancer treatment. Whereas various anti‐human epidermal growth factor receptor 2 (HER2) drugs have been developed, trastuzumab is still the only anti‐HER2 drug presently available for gastric cancer. In this study, we propose novel treatment options for patients with HER2‐positive gastric cancer. First, we determined the molecular profiles of 12 gastric cancer cell lines, and examined the antitumor effect of the pan‐HER inhibitors afatinib and neratinib in those cell lines. Additionally, we analyzed HER2 alteration in 123 primary gastric cancers resected from Japanese patients to clarify possible candidates with the potential to respond to these drugs. In the drug sensitivity analysis, both afatinib and neratinib produced an antitumor effect in most of the HER2‐amplified cell lines. However, some cells were not sensitive to the drugs. When the molecular profiles of the cells were compared based on the drug sensitivities, we found that cancer cells with lower mRNA expression levels of IGFBP7, a tumor suppressor gene that inhibits the activation of insulin‐like growth factor‐1 receptor (IGF‐1R), were less sensitive to pan‐HER inhibitors. A combination therapy consisting of pan‐HER inhibitors and an IGF‐1R inhibitor, picropodophyllin, showed a notable synergistic effect. Among 123 clinical samples, we found 19 cases of HER2 amplification and three cases of oncogenic mutations. In conclusion, afatinib and neratinib are promising therapeutic options for the treatment of HER2‐amplified gastric cancer. In addition to HER2 amplification, IGFBP7 might be a biomarker of sensitivity to these drugs, and IGF‐1R‐targeting therapy can overcome drug insensitiveness in HER2‐amplified gastric cancer.     

HER3 as biomarker and therapeutic target in pancreatic cancer: new insights in pertuzumab therapy in preclinical models

The anti-HER2 antibody pertuzumab inhibits HER2 dimerization and affects HER2/HER3 dimer formation and signaling. As HER3 and its ligand neuregulin are implicated in pancreatic tumorigenesis, we investigated whether HER3 expression could be a predictive biomarker of pertuzumab efficacy in HER2^low-expressing pancreatic cancer. We correlated in vitro and in vivo HER3 expression and neuregulin dependency with the inhibitory effect of pertuzumab on cell viability and tumor progression. HER3 knockdown in BxPC-3 cells led to resistance to pertuzumab therapy. Pertuzumab treatment of HER3-expressing pancreatic cancer cells increased HER3 at the cell membrane, whereas the anti-HER3 monoclonal antibody 9F7-F11 down-regulated it. Both antibodies blocked HER3 and AKT phosphorylation and inhibited HER2/HER3 heterodimerization but affected differently HER2 and HER3 homodimers. The pertuzumab/9F7-F11 combination enhanced tumor inhibition and the median survival time in mice xenografted with HER3-expressing pancreatic cancer cells. Finally, HER2 and HER3 were co-expressed in 11% and HER3 alone in 27% of the 45 pancreatic ductal adenocarcinomas analyzed by immunohistochemistry. HER3 is essential for pertuzumab efficacy in HER2^low-expressing pancreatic cancer and HER3 expression might be a predictive biomarker of pertuzumab efficacy in such cancers. Further studies in clinical samples are required to confirm these findings and the interest of combining anti-HER2 and anti-HER3 therapeutic antibodies.     

Therapeutic strategies for afatinib‐resistant lung cancer harboring HER2 alterations

Human epidermal growth factor receptor 2 (HER2) plays an important role in the pathogenesis of various cancers. HER2 alterations have been suggested to be a therapeutic target in non‐small‐cell lung cancer (NSCLC), just as in breast and gastric cancers. We previously reported that the pan‐HER inhibitor afatinib could be a useful therapeutic agent as HER2‐targeted therapy for patients with NSCLC harboring HER2 alterations. However, acquired resistance to afatinib was observed in the clinical setting, similar to the case for other HER inhibitors. Thus, elucidation of the mechanisms underlying the development of acquired drug resistance and exploring means to overcome acquired drug resistance are important issues in the treatment of NSCLC. In this study, we experimentally established afatinib‐resistant cell lines from NSCLC cell lines harboring HER2 alterations, and investigated the mechanisms underlying the acquisition of drug resistance. The established cell lines showed several unique afatinib‐resistance mechanisms, including MET amplification, loss of HER2 amplification and gene expression, epithelial‐to‐mesenchymal transition (EMT) and acquisition of cancer stem cell (CSC)‐like features. The afatinib‐resistant cell lines showing MET amplification were sensitive to the combination of afatinib plus crizotinib (a MET inhibitor), both in vitro and in vivo. The resistant cell lines which showed EMT or had acquired CSC‐like features remained sensitive to docetaxel, like the parental cells. These findings may provide clues to countering the resistance to afatinib in NSCLC patients with HER2 alterations.     

YES1 activation induces acquired resistance to neratinib in HER2‐amplified breast and lung cancers

Molecular‐targeted therapies directed against human epidermal growth factor receptor 2 (HER2) are evolving for various cancers. Neratinib is an irreversible pan‐HER tyrosine kinase inhibitor and has been approved by the FDA as an effective drug for HER2‐positive breast cancer. However, acquired resistance of various cancers to molecular‐targeted drugs is an issue of clinical concern, and emergence of resistance to neratinib is also considered inevitable. In this study, we established various types of neratinib‐resistant cell lines from HER2‐amplified breast and lung cancer cell lines using several drug exposure conditions. We analyzed the mechanisms of emergence of the resistance in these cell lines and explored effective strategies to overcome the resistance. Our results revealed that amplification of YES1, which is a member of the SRC family, was amplified in two neratinib‐resistant breast cancer cell lines and one lung cancer cell line. Knockdown of YES1 by siRNA and pharmacological inhibition of YES1 by dasatinib restored the sensitivity of the YES1‐amplified cell lines to neratinib in vitro. Combined treatment with dasatinib and neratinib inhibited tumor growth in vivo. This combination also induced downregulation of signaling molecules such as HER2, AKT and MAPK. Our current results indicate that YES1 plays an important role in the emergence of resistance to HER2‐targeted drugs, and that dasatinib enables such acquired resistance to neratinib to be overcome.     

CLEOPATRA: a phase III evaluation of pertuzumab and trastuzumab for HER2-positive metastatic breast cancer

IntroductionPatients with human epidermal growth factor receptor 2 (HER2)-positive breast cancer have a poor prognosis. Despite proven efficacy with the standard of care, trastuzumab, most patients with HER2-positive metastatic breast cancer (MBC) will eventually progress, requiring further intervention to prolong life. Pertuzumab, a humanized monoclonal antibody, is the first HER2 dimerization inhibitor. It binds to the dimerization site on the HER2 domain and prevents ligand-driven pairing of HER2 with other HER receptors, thus inhibiting tumor cell growth and survival. Combining trastuzumab and pertuzumab therefore provides a more comprehensive blockade of the HER signaling pathways, resulting in greater antitumor efficacy.Study DesignCLEOPATRA (CLinical Evaluation Of Pertuzumab And TRAstuzumab) is an international, randomized, double-blind, placebo-controlled phase III trial that aims to recruit approximately 800 patients from around 250 centers worldwide. Adults with HER2-positive adenocarcinoma of the breast with locally recurrent or metastatic disease will be randomized (1:1) to receive docetaxel, trastuzumab, and pertuzumab or docetaxel, trastuzumab, and placebo. The primary endpoint of the study is progression-free survival (PFS), based on tumor assessments conducted by an independent review facility. Secondary endpoints include PFS evaluated by investigator assessment, overall survival, objective response rate, duration of response, time to symptom progression, and safety. Data will also be collected to identify biomarkers that may correlate with clinical outcomes.ConclusionThe CLEOPATRA study will provide important information about the efficacy and safety of adding pertuzumab to one current standard of care in patients with HER2-positive MBC.     

A novel splice variant of HER2 with increased transformation activity

The HER2 proto-oncogene (also known as neu or c-erbB-2) belongs to the epidermal growth factor receptor family. HER2 is frequently amplified in human carcinomas. Gene amplification or overexpression of HER2 has been correlated with poor prognosis in several human cancers. Point mutation in the rat HER2 homolog, neu, is involved in the formation of rat neuroblastomas. However, no similar mutation in HER2 has been found in human cancers. Here we report the identification of a novel alternative splicing form of HER2 (ΔHER2) in human cell lines. An exon 16 amino acids long in the extracellular domain was deleted in ΔHER2. Deletion mutations in the corresponding region were shown previously to be involved in the formation of mammary carcinomas in transgenic mice. In the focus-formation assay, ΔHER2 showed much stronger transformation activity than did wild-type HER2. This result suggests that the deleted 16–amino acid exon may play a regulatory role in HER2 transformation activity. Mol. Carcinog. 23:62–68, 1998. © 1998 Wiley-Liss, Inc.     

Trastuzumab and lapatinib modulation of HER2 tyrosine/threonine phosphorylation and cell signaling

Cell surface transmembrane signaling receptors EGFR, HER3, and HER4 are activated by ligand-binding-mediated dimerization and phosphorylation. In contrast, HER2 amplification promotes signaling by increasing homo/heterodimerization and ligand binding. Trastuzumab or lapatinib therapy of HER2 amplicon-positive breast cancer cells induces growth inhibition and intracellular growth pathway signaling modulation. The mechanism(s) by which trastuzumab, an IgG1 humanized antibody, induces modification of cell signaling upon binding to an extracellular determinant on a ligand-less ??receptor?? membrane protein remains unexplained. Using immune detection methodology comprised of antibodies detecting three distinct domains of HER and five tyrosine/threonine phosphorylation sites, the effects of trastuzumab and lapatinib were defined during steady state growth inhibition. Here, we show that lapatinib markedly reduces HER2 tyrosine phosphorylation, while in contrast, no change in tyrosine phosphate levels is detected during trastuzumab-mediated cell growth inhibition. As trastuzumab treatment does not change either the steady state HER2 protein levels or HER2 mRNA, these findings argue against an antibody-dependent alteration in internalization kinetics. We further show a sequenced relationship between lapatinib-induced blockage of phosphorylation (6?C8?h) and induction of delayed cell death (5?C6?days), while trastuzumab-treated cells showed no evidence of cell death up to 9?days. Taken together, these results demonstrate that inhibition of HER2 phosphorylation by lapatinib is sufficient to induce apoptosis while trastuzumab binding to the extracellular HER2 domain may function by sterically modulating the detection of phosphate moieties by cytoplasmic signal transducers. This investigation also detected a 20?kD protein, which is down-regulated by lapatinib, further demonstrating the complexity of this signal transduction system.     

Modulation of breast cancer progression and differentiation by thegp30/neregulin

Summary In the last decade we have come to understand that the growth of cancer cells in general and of breast cancer in particular depends, in many cases, upon growth factors that will bind to and activate their receptors. One of these growth factor receptors is theerbB-2 protein which plays an important role in the prognosis of breast cancer and is overexpressed in nearly 30% of human breast cancer patients. While evidence accumulates to support the relationship betweenerbB-2 overexpression and poor overall survival in breast cancer, understanding of the biological consequence(s) oferbB-2 overexpression remains elusive. Our recent discovery of thegp30 has allowed us to identify a number of related but distinct biological endpoints which appear responsive to signal transduction through theerbB-2 receptor. These endpoints of growth, invasiveness, and differentiation have clear implications for the emergence, maintenance and/or control of malignancy, and represent established endpoints in the assessment of malignant progression in breast cancer. We have shown thatgp30 induces a biphasic growth effect on cells witherbB-2 over-expression. We have recently determined the protein sequence ofgp30 and cloned its full length cDNA sequence. We have also cloned two additional forms to the ligand, that are believed to be different isoforms. We are currently expressing the different forms in order to determine their biological effects. To elucidate the cellular mechanisms underlying cell growth inhibition bygp30, we tested the effect of this ligand on cell growth and differentiation of the human breast cancer cells which overexpresserbB-2 and cells which express low levels of this protooncogene. High concentrations of ligand induced differentiation of cells overexpressingerbB-2, as measured by inhibition of cell growth, and increased synthesis of milk components, and modulation of E-cadherin and up-regulation of c-jun and c-fos. These findings indicate that ligand-induced growth inhibition in cells overexpressingerbB-2 is associated with an apparent induction of differentiation. The availability ofgp30 derived synthetic peptides and its full cDNAs provides tools necessary to acquire a better understanding of the mechanism of action of the this ligands and theerbB-2 receptor in breast cancer.     

Acquired resistance mechanisms to afatinib in HER2‐amplified gastric cancer cells

Cancer treatment, especially that for breast and lung cancer, has entered a new era and continues to evolve, with the development of genome analysis technology and the advent of molecular targeted drugs including tyrosine kinase inhibitors. Nevertheless, acquired drug resistance to molecular targeted drugs is unavoidable, creating a clinically challenging problem. We recently reported the antitumor effect of a pan‐HER inhibitor, afatinib, against human epidermal growth factor receptor 2 (HER2)‐amplified gastric cancer cells. The purpose of the present study was to identify the mechanisms of acquired afatinib resistance and to investigate the treatment strategies for HER2‐amplified gastric cancer cells. Two afatinib‐resistant gastric cancer cell lines were established from 2 HER2‐amplified cell lines, N87 and SNU216. Subsequently, we investigated the molecular profiles of resistant cells. The activation of the HER2 pathway was downregulated in N87‐derived resistant cells, whereas it was upregulated in SNU216‐derived resistant cells. In the N87‐derived cell line, both MET and AXL were activated, and combination treatment with afatinib and cabozantinib, a multikinase inhibitor that inhibits MET and AXL, suppressed the cell growth of cells with acquired resistance both in vitro and in vivo. In the SNU216‐derived cell line, YES1, which is a member of the Src family, was remarkably activated, and dasatinib, a Src inhibitor, exerted a strong antitumor effect in these cells. In conclusion, we identified MET and AXL activation in addition to YES1 activation as novel mechanisms of afatinib resistance in HER2‐driven gastric cancer. Our results also indicated that treatment strategies targeting individual mechanisms of resistance are key to overcoming such resistance.     

Assessment of EGFR/HER2 dimerization by FRET-FLIM utilizing Alexa-conjugated secondary antibodies in relation to targeted therapies in cancers

The expression level of the HER family is unreliable as a predictive marker for targeted therapies in cancer. Thus, there is a need to develop other biomarkers, which can be used to accurately select responsive patients for targeted therapies. The HER dimerization status may be more important than HER receptor expression per se in determining sensitivity or resistance to a given therapeutic agent. The aim of the study is to develop a FRET assay using dye conjugated secondary antibodies to assess HER receptor dimerization. Using primary antibodies from different species in conjunction with Alexa488 and Alexa546 conjugated secondary antibodies, we validated our EGFR/HER2 dimerization assay in three cell lines, EGFR positive A431 cells as well as HER2 positive breast cell lines BT474 and SKBR3 cells. Finally, we applied our assay to assess EGFR/HER2 dimerization in paraffin embedded cell pellets. Our results show promise for the assay to be applied to tumor samples in order to assess the prognostic significance and predictive value of HER receptor dimerization in various cancers.     

Plasma membrane gp96 enhances invasion and metastatic potential of liver cancer via regulation of uPAR

Targeted therapy is currently under intensive investigation due to the resistance of liver cancer to cytotoxic chemotherapies. Dissecting the molecular events that drive the progression of liver cancer and defining specific targets are urgently needed to develop efficient tailored therapies. Cell membrane gp96 (mgp96) has been implicated in tumor growth and malignancy. Here, we explored the functional and clinical relevance of mgp96 in liver cancer. We found that elevated mgp96 abundance was associated with tumor metastasis and recurrence in patients with primary liver tumors. Decreased KDELR1 levels in hepatoma cells contribute to cell membrane translocation of the normally ER‐resident gp96. Urokinase‐type plasminogen activator receptor (uPAR) was identified as a mgp96 client protein, and mgp96 stabilized uPAR protein. Our clinical results proved that elevated mgp96 abundance is positively correlated with uPAR expression levels in liver tumors. We further provided evidence that targeting mgp96 with siRNA or a specific mAb that blocked the mgp96‐uPAR interaction led to inhibited cell growth, survival, and invasion in vitro, as well as the suppression of liver tumor growth and metastasis in vivo. mgp96 promotes liver cancer progression through increasing the protein stability and signaling of uPAR, and may be a new promising target for suppressing uPAR‐mediated tumor growth and metastasis in liver cancer.     

Pancreatic endocrine tumours: mutational and immunohistochemical survey of protein kinases reveals alterations in targetable kinases in cancer cell lines and rare primaries

BackgroundKinases represent potential therapeutic targets in pancreatic endocrine tumours (PETs).Patients and methodsThirty-five kinase genes were sequenced in 36 primary PETs and three PET cell lines: (i) 4 receptor tyrosine kinases (RTK), epithelial growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tyrosine-protein kinase KIT (KIT), platelet-derived growth factor receptor alpha (PDGFRalpha); (ii) 6 belonging to the Akt/mTOR pathway; and (iii) 25 frequently mutated in cancers. The immunohistochemical expression of the four RTKs and the copy number of EGFR and HER2 were assessed in 140 PETs.ResultsSomatic mutations were found in KIT in one and ATM in two primary neoplasms. Among 140 PETs, EGFR was immunopositive in 18 (13%), HER2 in 3 (2%), KIT in 16 (11%), and PDGFRalpha in 135 (96%). HER2 amplification was found in 2/130 (1.5%) PETs. KIT membrane immunostaining was significantly associated with tumour aggressiveness and shorter patient survival. PET cell lines QGP1, CM and BON harboured mutations in FGFR3, FLT1/VEGFR1 and PIK3CA, respectively.ConclusionsOnly rare PET cases, harbouring either HER2 amplification or KIT mutation, might benefit from targeted drugs. KIT membrane expression deserves further attention as a prognostic marker. ATM mutation is involved in a proportion of PET. The finding of specific mutations in PET cell lines renders these models useful for preclinical studies involving pathway-specific therapies.     

β1 integrin mediates an alternative survival pathway in breast cancer cells resistant to lapatinib

Introduction  

The overexpression of human epidermal growth factor receptor (HER)-2 in 20% of human breast cancers and its association with aggressive growth has led to widespread use of HER2-targeted therapies, such as trastuzumab (T) and lapatinib (L). Despite the success of these drugs, their efficacy is limited in patients whose tumors demonstrate de novo or acquired resistance to treatment. The β1 integrin resides on the membrane of the breast cancer cell, activating several elements of breast tumor progression including proliferation and survival.     

[fam-] trastuzumab deruxtecan,antitumor activity is dependent on HER2 expression level rather than on HER2 amplification

Therapies targeted to human epidermal growth factor receptor 2 (HER2) have proven effective against tumors positive for HER2 amplification, but there is an unmet clinical need for the treatment of tumors that express HER2 protein in the absence of HER2 amplification. [fam-] trastuzumab deruxtecan (DS-8201a) is a novel antibody–drug conjugate composed of the anti-HER2 antibody and the topoisomerase I inhibitor, an exatecan derivative. It has shown efficacy against tumors that express HER2 and is currently under evaluation in clinical trials. We here show that the antitumor activity of [fam-] trastuzumab deruxtecan is dependent on the expression level of HER2 protein in colorectal cancer (CRC) cell lines negative for HER2 amplification. We established isogenic CRC cell lines that express various levels of HER2 protein in the absence of HER2 amplification, and we found that cells that express HER2 at a high level were sensitive to [fam-] trastuzumab deruxtecan but not to conventional HER2-targeted therapies. Furthermore, [fam-] trastuzumab deruxtecan manifested a bystander killing effect both in vitro and in vivo, with cells essentially negative for HER2 expression also being killed in the presence of HER2-expressing cells, an effect that has the potential to overcome heterogeneity of HER2 expression in CRC tumors. Our results thus suggest that [fam-] trastuzumab deruxtecan warrants further study as a potential treatment for CRC tumors that express HER2 protein in the absence of HER2 amplification.     

Differential action of small molecule HER kinase inhibitors on receptor heterodimerization: therapeutic implications

Deregulation of ErbB/HER receptor tyrosine kinases has been linked to several types of cancer. The mechanism of activation of these receptors includes establishment of receptor dimers. Here, we have analyzed the action of different small molecule HER tyrosine kinase inhibitors (TKIs) on HER receptor dimerization. Breast cancer cell lines were treated with distinct TKIs and the formation of HER2‐HER3 dimers was analyzed by coimmunoprecipitation and western blot or by Förster resonance energy transfer assays. Antibody‐dependent cellular cytotoxicity was analyzed by measuring the release of lactate dehydrogenase and cell viability. Lapatinib and neratinib interfered with ligand‐induced dimerization of HER receptors; while pelitinib, gefitinib, canertinib or erlotinib did not. Moreover, lapatinib and neratinib were able to disrupt previously formed receptor dimers. Structural analyses allowed the elucidation of the mechanism by which some TKIs prevent the formation of HER receptor dimers, while others do not. Experiments aimed at defining the functional importance of dimerization indicated that TKIs that impeded dimerization prevented down‐regulation of HER2 receptors, and favored the action of trastuzumab. We postulate that TKIs that prevent dimerization and down‐regulation of HER2 may augment the antitumoral action of trastuzumab, and this mechanism of action should be considered in the treatment of HER2 positive tumors which combine TKIs with antireceptor antibodies.     

In vitro and in vivo evidence that a combination of lapatinib plus S‐1 is a promising treatment for pancreatic cancer

Lapatinib is a small molecule inhibitor of both HER2 and the epidermal growth factor receptor (EGFR). We investigated the effect of treatment with lapatinib alone or in combination with a fluoropyrimidine derivative S‐1 against pancreatic cancer. The HER2/EGFR expression in each of the four pancreatic cancer cell lines MiaPaca‐2, PANC‐1, Capan‐1 and Capan‐2 was measured by flow cytometry. The anti‐tumor effects of lapatinib (30 mg/kg) and/or S‐1 (10 mg/kg) were evaluated using female BALB/c nude mice xenografts generated using these four cell lines. Synergy between lapatinib and S‐1 was examined by median effect analysis in vitro. Resected pancreatic cancer tissues from 137 patients were immunohistochemically stained with anti‐human HER2 and EGFR antibodies. The administration of lapatinib as a single agent substantially suppressed tumor growth in vivo of all pancreatic cancer cell lines examined. A strong correlation was observed between HER2 expression and the anti‐tumor effect of lapatinib in vivo. Lapatinib synergized with S‐1 to inhibit the tumor growth of MiaPaca‐2 and PANC‐1 xenografts. When used as a single agent in vitro, lapatinib barely inhibit the cell growth of any cell line. However, lapatinib synergized with the anti‐tumor activity of the S‐1 components 5‐fluorouracil and 5‐chloro‐2,4‐dihydrogenase against all cell lines. Immunohistochemical staining demonstrated that 70% of the pancreatic cancers overexpressed HER2 and/or EGFR. Both lapatinib monotherapy and combined treatment with S‐1 may be promising treatments for patients with pancreatic cancers; the majority these cancers express lapatinib target molecules. (Cancer Sci 2009; 00: 000–000)     

Estrogen-dependent,tamoxifen-resistant tumorigenic growth of MCF-7 cells transfected with HER2/neu

Summary Since the poor prognosis associated with HER2 amplified breast cancers might be explained by a mechanistic association between p185^HER2 overexpression and therapeutic resistance, we assessed the chemo-endocrine sensitivity of estrogen receptor (ER) containing MCF-7 breast cancer cells transfected with full-length HER2 cDNA. Of the 36 isolated MCF/HER2 subclones, 7 were found to overexpress p185^HER2 surface receptor at levels 3 to 45-fold greater than parental or control transfected cells (MCF/neo). The overexpressing transfectants possessed increased inositol-1,4,5-trisphosphate-3'-kinase activity comparable to enzyme activity in the endogenously HER2 amplified breast cancer cell lines SK-Br-3 and BT-474. The anti-p185^HER2 monoclonal antibody and receptor-specific partial agonist, muMAb4D5 (4D5), known to inhibit growth of SK-Br-3 and BT-474 cells, produced no significant growth inhibitory effect on any of the transfectants including the 45-fold overexpressing MCF/HER2–18 cells which were studied in greater detail. MCF/HER2–18 cells contained at least partially functioning exogenous receptor since 4D5 (3µg/ml) specifically stimulated phosphorylation of p185^HER2 and its co-precipitating ptyr56 substrate within 5 min, and this was followed at 1 h by a transient induction ofc-myc but notc-fos mRNA. ER content and thein vitro sensitivity of MCF/HER2–18 cells to 5-fluorouracil and adriamycin were identical to those of control transfectants and parental cells. However, these highly overexpressing transfectants had acquired low level (2 to 4-fold) resistance to cisplatin and were no longer sensitive to the antiestrogen tamoxifen (TAM). To compare the hormone-dependent tumorigenicity of the HER2 transfectants, MCF/HER2–18 and control cells (MCF, MCF/neo-3) were implanted into ovariectomized athymic nude mice. No tumors were produced in the absence of estradiol (E₂) administration. In E₂ supplemented mice, MCF/HER2–18 tumors grew most rapidly. When E₂ treatment was stopped and daily TAM injections were initiated, MCF-7 and MCF/neo-3 tumor growth ceased immediately, while MCF/HER2–18 tumors continued to show an accelerated growth rate lasting weeks. This pattern of hormone-dependent, TAM-resistant growth exhibited by the MCF/HER2–18 tumors in nude mice supports the possibility that p185^HER2 overexpression in human breast cancers may be linked to therapeutic resistance.