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.     

Monoclonal antibodies, small molecules, and vaccines in the treatment of breast cancer

The human epidermal growth factor receptor (HER, ErbB) family of receptors is considered an important therapeutic target, and various types of molecularly based small molecules, including monoclonal antibodies, protein tyrosine kinase inhibitors, and therapeutic vaccines, are in development as potential therapies for metastatic breast cancer. Trastuzumab (Herceptin; Genentech, Inc.; South San Francisco, CA), the first approved monoclonal antibody for HER-2 (ErbB-2)-overexpressing metastatic breast cancer, provided the proof of principle that targeting specific receptors results in clinical benefit. Other monoclonal antibodies and the small molecule dual protein tyrosine kinase inhibitors show great promise as treatments for metastatic breast cancer but require evaluation in clinical trials to assess their benefits. Therapeutic vaccines may have a role, particularly in early-stage disease, but they are associated with greater limitations and study design issues that make their evaluation difficult. Optimum combination therapy regimens with a variety of novel approaches that incorporate small molecule targeted therapies need to be developed, and the population most likely to benefit from targeted therapies needs to be identified.     

HER2阳性结直肠癌研究进展

表皮生长因子受体2（HER2）是肿瘤发生、发展过程中的癌基因，在7%的结直肠癌患者中表达，与表皮生长因子受体单克隆抗体的耐药相关。随着CRC治疗困境的出现，以及靶向HER2为乳腺癌、胃癌患者带来生存获益，HER2在CRC中的意义及抗HER2治疗的预后价值被广泛关注，围绕HER2阳性CRC的临床研究亦不断开展。目前，CRC中HER2阳性的诊断标准已逐渐统一，HER2靶向治疗如单克隆抗体、酪氨酸激酶抑制剂、抗体-药物耦联物及HER2相关免疫治疗的单独或联合治疗策略在HER2阳性CRC中显示出较好的疗效，能为患者带来生存获益，本文就此方面的研究进展作一综述。     

A molecular view of anti-ErbB monoclonal antibody therapy

Abnormal activation of the epidermal growth factor receptor (EGFR) and its homolog HER2 (Neu/ErbB2) has been associated with many human cancers, and monoclonal antibodies targeting EGFR and HER2 are effective anticancer therapies. Structural studies of these receptors and antibodies have revealed much about how they function. In this issue of Cancer Cell, Schmiedel et al. report structural and functional studies of the anti-EGFR monoclonal antibody Matuzumab. They show that Matuzumab binds and inhibits EGFR in a manner distinctive from that of other therapeutic anti-EGFR antibodies and suggest that combination therapies with Matuzumab and other antibodies may prove beneficial.     

<Emphasis Type="Italic">ErbB3</Emphasis> is required for ductal morphogenesis in the mouse mammary gland

Introduction  

The receptor ErbB3/HER3 is often over-expressed in human breast cancers, frequently in conjunction with over-expression of the proto-oncogene ERBB2/HER2/NEU. Although the prognostic/predictive value of ErbB3 expression in breast cancer is unclear, ErbB3 is known to contribute to therapeutic resistance. Understanding ErbB3 functions in the normal mammary gland will help to explain its role in cancer etiology and as a modulator of signaling responses to the mammary oncogene ERBB2.     

Oncogenic growth factor receptors: implications for signal transduction therapy

Growth factors of the EGF family and their respective ErbB/HER receptor tyrosine kinases underlie many landmarks of tumor cells, including excessive growth, invasive behavior and attraction of blood vessels. Enhanced expression of ErbB proteins, existence of permanently active receptor mutants and occurrence of autocrine loops are frequently observed in human cancer, and in some cases they associate with poor disease outcome. The four ErbB proteins and their 11 ligands act within a layered signaling network coordinated by ErbB-2/HER2, the most oncogenic family member. Drugs that intercept signals emanating from ErbB-2 and ErbB-1 are already in routine clinical application. Here we review three major strategies to develop new ErbB-targeted therapies. These are monoclonal anti-receptor antibodies, specific tyrosine kinase inhibitors and antagonists of heat shock protein 90. The underlying mechanisms are critically examined, with an emphasis on potential drug combinations, which hold promise for enhanced clinical efficacy.     

Five carboxyl-terminal residues of neuregulin2 are critical for stimulation of signaling by the ErbB4 receptor tyrosine kinase

The neuregulins (NRGs) are members of the epidermal growth factor (EGF) family of peptide growth factors. These hormones are agonists for the ErbB family of receptor tyrosine kinases, a family that includes the epidermal growth factor receptor (EGFR/ErbB1), ErbB2/Neu/HER2, ErbB3/HER3, and ErbB4/HER4. We recently observed that the EGF family hormone NRG2beta is a potent agonist for ErbB4. In contrast, NRG2alpha, a splicing isoform of the same gene that encodes NRG2beta, is a poor ErbB4 agonist. We hypothesized that carboxyl-terminal residues of NRG2beta are critical for stimulation of ErbB4 tyrosine phosphorylation and coupling to downstream signaling events. Here, we demonstrate that the substitution of a lysine residue for Phe45 in NRG2beta results in reduced ligand potency. We also demonstrate that substitution of a phenylalanine for Lys45 in NRG2alpha results in increased ligand potency. Finally, analyses of the gain-of-function NRG2alpha Chg5 mutant demonstrate that Gln43, Met47, Asn49, and Phe50 regulate ligand efficacy. Thus, these data indicate that carboxyl-terminal residues of NRG2beta are critical for activation of ErbB4 signaling. Moreover, these NRG2alpha and NRG2beta mutants reveal new insights into models for ligand-induced ErbB family receptor tyrosine phosphorylation and coupling to downstream signaling events.     

Quantitative assays for the measurement of HER1-HER2 heterodimerization and phosphorylation in cell lines and breast tumors: applications for diagnostics and targeted drug mechanism of action

Introduction  

Ligand-bound and phosphorylated ErbB/HER heterodimers are potent signaling forms of this receptor family, and quantitative measurements of these active receptors may be predictive of patient response to targeted therapies. Using VeraTag™ technology, we developed and characterized quantitative assays measuring epidermal growth factor (EGF)-dependent increases in activated HER receptors in tumor cell line lysates and formalin-fixed, paraffin-embedded (FFPE) tumor sections. We demonstrated the ability of the assays to quantitatively measure changes in activated HER1 and HER2 receptor levels in cell lines following treatment with 2C4, erlotinib, and lapatinib. We utilized these assays to determine the prevalence and distribution of activated HER1, HER2, and HER1-HER2 heterodimers in 43 HER2-positive breast tumors.     

Quantitative multiplexed analysis of ErbB family coexpression for primary breast cancer prognosis in a large retrospective cohort

BACKGROUND:

Assessment of outcome using a single prognostic or predictive marker is the current basis of targeted therapy, but is inherently limited by its simplicity. Multiplexing has provided better classification, but has only been done quantitatively using RNA or DNA. Automated quantitative analysis is a new technology that allows quantitative in situ assessment of protein expression. The authors hypothesized that multiplexed quantitative measurement of ErbB receptor family proteins may allow better prediction of outcome.

METHODS:

The authors quantitatively assessed the expression of 6 proteins in 4 subcellular compartments in 676 patients using breast carcinoma tissue microarrays. Then using Cox proportional hazards modeling and unsupervised hierarchical clustering, they assessed the prognostic value of the expression singly and multiplexed.

RESULTS:

Epidermal growth factor receptor (EGFR), HER‐2, and HER‐3 expression were associated with decreased survival. Multivariate analysis showed high HER‐2 and HER‐3 expression maintained independence as prognostic markers. Hierarchical clustering of expression data defined a small class enriched for HER‐2 expression with 40% 10‐year survival, compared with 55% using conventional methods. Clustering also revealed a similarly poor‐prognostic subgroup coexpressing EGFR and HER‐3 (but low for estrogen receptor, progesterone receptor, and HER‐2) with a 42% 10‐year survival.

CONCLUSIONS:

This work shows that the combined analysis of protein expression improved prognostic classification, and that multiplexed models were superior to any single‐marker–based method for prediction of 10‐year survival. These methods illustrate a protein‐based, multiplexed approach that could more accurately identify patients for targeted therapies. Cancer 2009. © 2009 American Cancer Society.     

Resistance to HER2-directed antibodies and tyrosine kinase inhibitors: mechanisms and clinical implications

The antibody trastuzumab and the tyrosine kinase inhibitor lapatinib are approved by the FDA for the treatment of HER2-overexpressing breast cancer. These anti-HER2 drugs are changing the natural history of HER2-overexpressing breast cancer. However, therapeutic resistance to trastuzumab or lapatinib, as either single-agents or in combination with chemotherapy in the metastatic setting, typically occurs within months of starting therapy. Several mechanisms of trastuzumab-resistance have been reported that include signaling from other HER receptors, signaling from receptor tyrosine kinases (RTKs) outside of the HER (ErbB) family, increased phosphatidylinositol 3-kinase signaling, and the presence of truncated forms of HER2. Mechanisms of resistance to lapatinib also point to increased phosphatidylinositol 3-kinase signaling as well as derepression/activation of compensatory survival pathways. In this review, we discuss how these models and mechanisms enhance our understanding of the clinical resistance to HER2-directed therapies.     

ErbB tyrosine kinase receptor inhibitors in breast cancer treatment

The ErbB tyrosine kinase receptor family comprises 4 receptors (HER1 [EGFR/ErbB1], HER2 [ErbB2], HER3 [ErbB3] and HER4 [ErbB4]), and at least 10 ligands. Pre-clinical and clincial studies have indicated that members of this family have important roles in breast cancer development and progression, and which leads to the concept of targeting ErbB receptors as a promising treatment strategy. Trastuzumab, an anti-HER2 antibody, provided the first definitive evidence for the clinical efficacy of targeting an ErbB receptor in patients with breast cancer. Over the past 5 years, much interest has been generated as a result of the development of orally-administered ErbB tyrosine kinase receptor inhibitors. Pre-clinical and early clinical studies with these agents are reviewed here.     

HER2/neu as a predictive factor in breast cancer

One of the current trends in breast cancer research is to identify markers that can predict response to specific anticancer therapies; intense laboratory research and therapeutic trials are exploiting this strategy. The combination of cytotoxic drugs directed at the tumor population with the highest probability of being sensitive to them with molecules targeted at intracellular signaling and cell cycle control pathways, which may be deregulated as part of the malignant process, represents our best hope for improved survival in both early and advanced disease. The transmembrane tyrosine kinase receptor, HER2/neu, has been the subject of much investigation with respect to its prognostic value, predictive value, and as a target of antibody-mediated therapy. Retrospective evidence strongly suggests that HER2 overexpression is associated with decreased disease-free and overall survival in node-positive, and possibly also node-negative, breast cancer. Prospective trials have demonstrated that antibodies to HER2 can produce tumor responses in women with advanced disease that overexpresses this molecule. Moreover, the combination of such antibodies with cytotoxic drugs has been one of the few recent strategies to improve survival duration in metastatic breast cancer. The evidence supporting the role of HER2 as a factor predictive of response to hormone therapy and cytotoxic drugs is more ambiguous and requires prospective assessment. The available literature is reviewed herein, with a focus on the predictive value of HER2, potential mechanisms of resistance and sensitivity to various drugs, and future research directions involving this important molecule.     

Developments in the Management of Metastatic HER2-Positive Breast Cancer: A Review

Approximately 20% of breast cancers overexpress human epidermal growth factor receptor 2 (HER2), providing an actionable target for many different therapies. In the metastatic setting, prognosis has improved greatly with the use of anti-HER2 drugs such as trastuzumab, pertuzumab, and trastuzumab-emtansine. In the third line setting and beyond, several emerging treatments have shown benefits, including novel small molecule targeted agents and antibody-drug conjugates. Systemic treatment of brain metastases in HER2-positive patients and the role of endocrine-based treatment for patients with hormone receptor (HR) positive disease remain areas of research interest. This article will review the current approach to systemic management of metastatic HER2-positive breast cancer in Canada, and present novel treatments that may be available in the near future.     

Antibody targeting of HER2/HER3 signaling overcomes heregulin‐induced resistance to PI3K inhibition in prostate cancer

Dysregulated expression and/or mutations of the various components of the phosphoinositide 3‐kinase (PI3K)/Akt pathway occur with high frequency in prostate cancer and are associated with the development and progression of castration resistant tumors. However, small molecule kinase inhibitors that target this signaling pathway have limited efficacy in inhibiting tumor growth, primarily due to compensatory survival signals through receptor tyrosine kinases (RTKs). Although members of the epidermal growth factor receptor (EGFR), or HER, family of RTKs are strongly implicated in the development and progression of prostate cancer, targeting individual members of this family such as EGFR or HER2 has resulted in limited success in clinical trials. Multiple studies indicate a critical role for HER3 in the development of resistance against both HER‐targeted therapies and PI3K/Akt pathway inhibitors. In this study, we found that the growth inhibitory effect of GDC‐0941, a class I PI3K inhibitor, is markedly reduced in the presence of heregulin. Interestingly, this effect is more pronounced in cells lacking phosphatase and tensin homolog function. Heregulin‐mediated resistance to GDC‐0941 is associated with reactivation of Akt downstream of HER3 phosphorylation. Importantly, combined blockade of HER2 and HER3 signaling by an anti‐HER2/HER3 bispecific antibody or a mixture of anti‐HER2 and anti‐HER3 antibodies restores sensitivity to GDC‐0941 in heregulin‐treated androgen‐dependent and ‐independent prostate cancer cells. These studies indicate that the combination of PI3K inhibitors with HER2/HER3 targeting antibodies may constitute a promising therapeutic strategy for prostate cancer.     

表皮生长因子受体家族特点及与肿瘤关系的研究进展

人类表皮生长因子受体(human epidermal growth factor receptor,HER)家族属于酪氨酸激酶Ⅰ亚族的跨膜蛋白受体家族，包括4个成员，分别是HER1(EGFR/ErbB1)、HER2(ErbB2)、HER3(ErbB3)和HER4(ErbB4)，由erb基因编码，在细胞生长、增殖及凋亡等活动中起到重要的调节作用。同时，作为原癌基因家族，HER家族在许多人类肿瘤中异常激活及过度表达，是这些肿瘤发生和发展的关键因素，与多种肿瘤的临床病理特征及肿瘤患者的不良预后密切相关。HER家族一直是肿瘤领域基础实验研究和临床诊治研究的热点之一，以其为靶点的综合抗肿瘤治疗方案获得了良好的临床疗效。本文通过查阅对有关HER家族及其与肿瘤关系的相关文献，总结人表皮生长因子受体家族特点及其在肿瘤发生发展、生物靶向诊治方面的最新研究进展。相信随着HER家族临床研究成果的不断丰富及分子生物学技术的快速发展可为肿瘤临床诊治提供新的思路和帮助。     

Neuregulins and cancer.

The neuregulins represent the largest subclass of polypeptide factors of the epidermal growth factor family of ligands. These molecules are synthesized as membrane-bound, biologically active growth factors that act by binding to the HER/ErbB receptor tyrosine kinases. Preclinical data have indicated that increased expression and function of neuregulins may provoke cancer. Furthermore, neuregulin expression has been detected in several neoplasias, and their presence may correlate with response to treatments that target the HER receptors such as trastuzumab. In addition, the neuregulins have also been implicated in resistance to anti-HER therapies. Therefore, targeting of the neuregulins may be helpful in neoplastic diseases in which these polypeptide factors contribute to tumor generation and/or maintenance.     

Assessment of HER2 status in breast cancer: why, when and how?

Human epidermal growth factor receptor 2 (HER2) is overexpressed, usually as a result of HER2 proto-oncogene amplification, in 20-30% of breast cancers. A HER2-positive status is generally associated with more aggressive disease and a worse prognosis. Furthermore, a positive HER2 status may predict the likelihood of resistance to some conventional therapies, as well as probably being predictive of sensitivity to anthracycline dose intensification. In addition to this prognostic/predictive value, HER2 is a target for specific therapy, with anti-HER2 monoclonal antibody therapy available in the USA. This article reviews the different assays used to determine HER2 status, discussing their relative advantages/disadvantages and the need for their standardisation before integration alongside other pathological indices into the clinical management of breast cancer.     

HER family kinase domain mutations promote tumor progression and can predict response to treatment in human breast cancer

Resistance to HER2‐targeted therapies remains a major obstacle in the treatment of HER2‐overexpressing breast cancer. Understanding the molecular pathways that contribute to the development of drug resistance is needed to improve the clinical utility of novel agents, and to predict the success of targeted personalized therapy based on tumor‐specific mutations. Little is known about the clinical significance of HER family mutations in breast cancer. Because mutations within HER1/EGFR are predictive of response to tyrosine kinase inhibitors (TKI) in lung cancer, we investigated whether mutations in HER family kinase domains are predictive of response to targeted therapy in HER2‐overexpressing breast cancer. We sequenced the HER family kinase domains from 76 HER2‐overexpressing invasive carcinomas and identified 12 missense variants. Patients whose tumors carried any of these mutations did not respond to HER2 directed therapy in the metastatic setting. We developed mutant cell lines and used structural analyses to determine whether changes in protein conformation could explain the lack of response to therapy. We also functionally studied all HER2 mutants and showed that they conferred an aggressive phenotype and altered effects of the TKI lapatinib. Our data demonstrate that mutations in the finely tuned HER kinase domains play a critical function in breast cancer progression and may serve as prognostic and predictive markers.     

HER3 is a determinant for poor prognosis in melanoma

PURPOSE: The epidermal growth factor receptor family member HER3 is overexpressed in diverse human cancers and has been associated with poor prognosis in breast, lung, and ovarian cancer. However, the relevance of HER3 with regard to its prognostic significance and function in primary melanoma and metastases remains largely elusive. EXPERIMENTAL DESIGN: HER3 protein expression was analyzed immunohistochemically using tissue microarrays of 130 primary melanoma and 87 metastases relative to established clinical variables. The possibility of an influence of HER3 on melanoma cell proliferation, migration, invasion, and chemotherapy-induced apoptosis was studied in human melanoma cell lines. RESULTS: We show that HER3 is frequently expressed in malignant melanoma and metastases at elevated levels. High HER3 expression may serve as a prognostic marker because it correlates with cell proliferation, tumor progression, and reduced patient survival. Suppression of HER3 expression by RNA interference reduces melanoma cell proliferation, migration, and invasion in vitro. In addition, down-regulation of HER3 synergistically enhances dacarbazine-induced apoptosis. Moreover, monoclonal antibodies specific for the extracellular portion of HER3 efficiently block heregulin-induced proliferation, migration, and invasion of melanoma cell lines. CONCLUSION: Our results provide novel insights into the role of HER3 in melanoma and point out new possibilities for therapeutic intervention.     

Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhances targeting selectivity and induces a therapeutic effect in vitro

Inappropriate signalling through the EGFR and ErbB2/HER2 members of the epidermal growth factor family of receptor tyrosine kinases is well recognised as being causally linked to a variety of cancers. Consequently, monoclonal antibodies specific for these receptors have become increasingly important components of effective treatment strategies for cancer. Increasing evidence suggests that ErbB3 plays a critical role in cancer progression and resistance to therapy. We hypothesised that co-targeting the preferred ErbB2/ErbB3 heterodimer with a bispecific single-chain Fv (bs-scFv) antibody would promote increased targeting selectivity over antibodies specific for a single tumour-associated antigen (TAA). In addition, we hypothesised that targeting this important heterodimer could induce a therapeutic effect. Here, we describe the construction and evaluation of the A5-linker-ML3.9 bs-scFv (ALM), an anti-ErbB3/ErbB2 bs-scFv. The A5-linker-ML3.9 bs-scFv exhibits selective targeting of tumour cells in vitro and in vivo that co-express the two target antigens over tumour cells that express only one target antigen or normal cells that express low levels of both antigens. The A5-linker-ML3.9 bs-scFv also exhibits significantly greater in vivo targeting of ErbB2'+'/ErbB3'+' tumours than derivative molecules that contain only one functional arm targeting ErbB2 or ErbB3. Binding of ALM to ErbB2'+'/ErbB3'+' cells mediates inhibition of tumour cell growth in vitro by effectively targeting the therapeutic anti-ErbB3 A5 scFv. This suggests both that ALM could provide the basis for an effective therapeutic agent and that engineered antibodies selected to co-target critical functional pairs of TAAs can enhance the targeting specificity and efficacy of antibody-based cancer therapeutics.