HER2-targeted Monoclonal Antibody Research Articles

Abstract 1806: TLR2 agonist PSK activates NK cells and enhances the anti-tumor effect of HER2-targeted monoclonal antibody therapy

Abstract Purpose: The therapeutic effect of trastuzumab, the anti-HER2 monoclonal antibody (mAb), is dependent on functional NK cells which mediates antibody-dependent cell-mediated cytotoxicity (ADCC). Novel agents that enhance NK cell function could potentially improve the therapeutic effect of trastuzumab. We recently identified polysaccharide krestin (PSK), a natural product extracted from medicinal mushroom, as a novel TLR2 agonist. The current study was undertaken to evaluate the effect of PSK on human NK cells and the potential of PSK to enhance HER2-targeted mAb therapy. Experimental Design: Human PBMC were stimulated with PSK to evaluate the effect of PSK on NK cell activation and IFN-g secretion. K 562 was used as a target to measure the lytic function of NK cells. Effect of PSK on ADCC was also measured using trastuzumab-coated SKBR3 and MDA-MB-231 breast cancer cells. Finally, in vivo experiment in neu transgenic mice was carried out to determine the potential of PSK to augmented HER2-targeted mAb therapy. Results: PSK upregulated the expression of activation markers, CD25 and CD69, on NK cells, and stimulated IFN-g production by NK cells. The cytolytic potential of NK cells, as shown by CD107a degranulation and lysis of K562, was also augmented by PSK treatment. PSK enhanced trastuzumab-mediated ADCC in lysing both SKBR3 and MDA-MB-231 breast cancer cells. In vivo treatment in neu transgenic mice showed that oral administration of PSK significantly potentiated the anti-tumor effect of anti-rat neu/HER2 mAb therapy. Conclusion: These results demonstrated that natural product PSK activates human NK cells and potentiates trastuzumab-mediated ADCC. Concurrent treatment of PSK and trastuzumab may be a novel way to augment the anti-tumor effect of trastuzumab. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1806. doi:10.1158/1538-7445.AM2011-1806

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

IntroductionLigand-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.MethodsAssays for activated HER1 and HER2 receptors in FFPE and cell lysate formats were developed using VeraTag™ technology, which requires the proximity of an antibody pair for light-dependent release of a fluorescently labeled tag, followed by capillary electrophoresis-based quantitation.ResultsLigand-dependent and independent HER1-HER2 heterodimer levels measured by lysate and FFPE VeraTag™ assays trended with HER1 and HER2 expression levels in tumor cell lines, which was confirmed by co-immunoprecipitation. The formation of EGF-dependent HER1-HER2 heterodimers were inhibited by the HER2-targeted monoclonal antibody 2C4 and stabilized by the HER1 tyrosine kinase inhibitor (TKI) erlotinib. EGF-dependent HER1 and HER2 phosphorylation was inhibited by lapatinib and erlotinib. Further, we observed that dominant receptor signaling patterns may switch between HER1-HER1 and HER1-HER2, depending on drug mechanism of action and relative levels of HER receptors. In FFPE breast tumors that expressed both HER1 and HER2, HER1-HER2 heterodimers were detected in 25 to 50% of tumors, depending on detection method. The levels of activated phospho-HER1-HER2 heterodimers correlated with HER1 or HER2 levels in an analysis of 43 HER2-positive breast tumors.ConclusionsVeraTag™ lysate assays can be used as a tool for understanding the mechanism of action of targeted HER-family inhibitors in the preclinical setting, while VeraTag™ FFPE assays of activated HER receptors combined with total HER2 measurements (HERmark®) in tumor samples may provide a more accurate prediction of clinical response to both HER1 and HER2 targeted therapies.

Mechanisms of resistance to trastuzumab: an updated review

Human epidermal growth factor receptor 2 (HER2) is over-expressed in 20%–25% of invasive breast cancer and is associated with an aggressive tumor phenotype and reduced survival rate. As a clinically widely applied HER2-targeted monoclonal antibody, trastuzumab (Herceptin®), combined with chemotherapy significantly increases the no tumor survival time of the patient. However, the majority of the cancers that initially respond to Herceptin begin to counter against the treatment within 1 year. This review describes several important well-known trastuzumab resistance mechanisms as well as the updated advancement in this field. The sufficiently investigated mechanisms are over-activation of PI3K/AKT pathway, activation of PI3K/AKT via alternative pathway, steric hindrance of receptor-antibody interaction, increase of serum HER2 extracellular domain, and abnormal expression of epidermal growth factor receptor (EGFR) family and their ligands. And the newly investigated mechanisms involve Darpp-32 and t-Darpp, autophagy of tumor cells, HSP27, HSP90, c-MYC, ADAM proteases, EphA2, Rac1, MUC1*, HER2Δ16, and mesenchymal CD44(pos)CD24(neg/low) phenotype.

Growth factor receptor 1 and 2 (HER1 and HER2) gene and protein assessment and evaluation of lapatinib plasma concentrations (PCs) in breast cancer patients

Trastuzumab is a monoclonal antibody targeting HER2. Lapatinib, a tyrosine kinase inhibitor, targets HER2 and HER1 (also EGFR), but the HER1 status is not assessed in clinical practice. Drug plasma concentrations (PCs) may influence the treatment outcome. This study focuses on HER1 status, lapatinib PCs, and lapatinib treatment response. Retrospective tumor and blood samples from 28 lapatinib plus capecitabine treated patients (Pts) were used. HER1 gene, chromosome 7 (Ch7), and protein status were assessed by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) in archival tumors. Lapatinib PCs were evaluated by ultra performance liquid chromatography–tandem mass spectrometry. Blood samples were taken in 16 to 30 hours after lapatinib administration. The data were evaluated by standard statistical methods. Pts were treated with lapatinib plus capecitabine for advanced disease. Most tumors were invasive ductal carcinomas 82% (23/28); 43% (12/28) displayed hormonal receptor positivity. Lapatinib treatment lasted from 1.2 to 15.6 months. Twenty Pts ended lapatinib due to progression, 6 owing to toxicity. Ch7 polysomy was found in 30% (6/20) of Pts. HER1 amplification was not observed. HER1 membrane expression was found in 22% (5/23). Median of lapatinib PCs was 5.09 g/ml, with small interindividual variations. The exception was 1 patient (11.25 g/ml) with hepatotoxicity. HER1 IHC membrane positivity correlated negatively with disease-free survival (DFS) (p < 0.014) and cytoplasmic positivity correlated negatively with DFS (p < 0.017) and overall survival (OS) (p < 0.035). Longer trastuzumab to lapatinib intervals correlated with longer OS (p < 0.008). Intercalation of 5-FU based regimens between trastuzumab and lapatinib influenced negatively survival on lapatinib plus capacitabine (p < 0.02). Intercalation of chemotherapy based on 5-FU before lapatinib plus capecitabine regimen reduces the treatment effect and suggests preselection of resistant cells before lapatinib plus capecitabine administration. Lapatinib PCs were higher in one patient who developed liver toxicity. Thus, measurement of PCs may be a way of identifying hepatotoxicity high risk patients and titering their lapatinib doses to avoid side effects. Supported by internal grant of the Faculty of Medicine and Dentistry, Palacky University, no. 91110301 and MSM 6198959216.

Trastuzumab‐based neoadjuvant therapy in patients with HER2‐positive breast cancer

Overexpression, or gene amplification, of the human epidermal growth factor receptor 2 (HER2) is evident in 20% to 25% of breast cancers. The biologic agent trastuzumab is an HER2-targeted monoclonal antibody that inhibits the proliferation of tumor cells and induces tumor cell death through multiple mechanisms of action. Currently, trastuzumab is approved for use in the adjuvant and metastatic settings. Trials combining trastuzumab with neoadjuvant chemotherapy suggest that patients with HER2-positive breast cancer also may benefit from preoperative trastuzumab. For this article, the author reviewed efficacy and safety data from key studies of patients who received neoadjuvant trastuzumab-based therapy. Studies were identified from literature searches of publication and congress databases. The results of 3 large phase 3 trials (the M. D. Anderson Cancer Center neoadjuvant trastuzumab trial, the Neoadjuvant Herceptin [NOAH] trial, and the German Breast Group/Gynecologic Oncology Study Group "GeparQuattro" trial) demonstrated that, compared with chemotherapy alone, neoadjuvant trastuzumab plus chemotherapy significantly increased pathologic complete response rates to as high as 65%. Improvements in disease-free, overall, and event-free survival also were reported in the NOAH trial. In addition to demonstrated efficacy, a low incidence of cardiac dysfunction suggests that neoadjuvant trastuzumab is both effective and well tolerated. Similar results have been reported in a range of phase 2 studies using different trastuzumab-based regimens. These encouraging data led the National Comprehensive Cancer Network to recommend treating patients who have operable, locally advanced, HER2-positive breast cancer with neoadjuvant paclitaxel plus trastuzumab followed by 5-fluorouracil, epirubicin, and cyclophosphamide plus trastuzumab.

Phase II Trial of Pertuzumab and Trastuzumab in Patients With Human Epidermal Growth Factor Receptor 2–Positive Metastatic Breast Cancer That Progressed During Prior Trastuzumab Therapy

PURPOSE; Pertuzumab, a human epidermal growth factor receptor 2 (HER2) -targeted monoclonal antibody, potently inhibits HER2 dimerization and HER-mediated signaling pathways. Pertuzumab and the approved HER2-targeted monoclonal antibody trastuzumab have complementary mechanisms of action and result in enhanced antitumor activity when combined. This phase II trial assessed the efficacy and safety profile of the combination in patients with HER2-positive breast cancer whose disease had progressed during prior trastuzumab-based therapy. This was a multicenter, open-label, single-arm, Simon two-stage study. Patients with advanced HER2-positive breast cancer in whom disease progression had occurred during prior trastuzumab-based therapy received trastuzumab weekly (4 mg/kg loading dose, then 2 mg/kg every week) or every 3 weeks (8 mg/kg loading dose, then 6 mg/kg every 3 weeks) and pertuzumab every 3 weeks (840 mg loading dose, then 420 mg every 3 weeks). Treatment continued until disease progression or excessive toxicity. All 66 patients were assessable for efficacy and safety. The objective response rate was 24.2%, and the clinical benefit rate was 50%. Five patients (7.6%) experienced a complete response, 11 patients (16.7%) experienced a partial response, and 17 patients (25.8%) experienced stable disease of > or = 6 months. Median progression-free survival was 5.5 months. Overall, the combination of pertuzumab and trastuzumab was well tolerated, and adverse events were mild to moderate. Cardiac dysfunction was minimal, and no patients withdrew as a result of cardiac-related adverse events. The combination of pertuzumab and trastuzumab is active and well tolerated in patients with metastatic HER2-positive breast cancer who had experienced progression during prior trastuzumab therapy.

Progression and treatment of HER2-positive breast cancer

Approximately 20-30% of breast cancer tumors overexpress or amplify human epidermal growth factor receptor 2 (HER2). The role of this receptor in the progression of HER2+ breast cancer and resistance to certain anticancer monotherapies was investigated. The results of several pre-clinical and clinical trials, with the aim of determining the most safe and effective course of treatment for HER2+ breast cancer, were also thoroughly examined. A thorough search of databases including Pubmed, Springer, and The American Society of Clinical Oncology was performed, and pertinent studies were identified. The most relevant studies were preclinical, phase II, and III clinical trials identifying the function of the HER2 receptor in HER2+ breast cancer progression, as well as studies assessing the efficacy of monotherapy and combination therapy in the treatment of this aggressive form of cancer. The HER2 receptor belongs to a family of receptors that consists of four cell-surface receptors (HER1-4) that share strong homology with the epidermal growth factor receptor (EGFR). All HER receptors interact with specific types of ligands to induce receptor activation, except for HER2, for which no known ligand has yet been identified. HER2 is activated by forming dimers with other HER receptors, and this results in a stronger and more prolonged signal transduction event. When expressed at normal levels, HER2 regulates cell growth, differentiation, and survival. However, under pathological conditions of HER2 overexpression, numerous HER2 heterodimers are formed resulting in aggressive tumor growth. Therefore, the prognosis associated with HER2-positive breast cancer is usually poor. A specific cohort of patients with breast cancer whose tumors test both hormone receptor (estrogen receptor [ER] and progesterone receptor [PR]) and HER2 positive have been found to be resistant to targeted hormone therapy. Studies investigating the etiology of this resistance have found that the cell membrane estrogen receptor communicates with HER2 in promoting the release of ER coactivators that cause the endocrine drug and selective estrogen receptor modulator, tamoxifen, to act as an agonist rather than an antagonist of the hormone estrogen. Thus, research has directed its inquiry toward the development of therapies specifically targeting HER2. The development of trastuzumab, a recombinant monoclonal antibody against HER2, initially proved to be a well-tolerated first line of treatment. However, in the long-term patients, trastuzumab was shown to develop resistance to this monotherapy. Therefore, research on HER2 positive breast cancer has focused on the study of different anti-HER2 combination therapies over the past decade. While the development and approval of the HER2-targeted recombinant monoclonal antibody trastuzumab (Herceptin) has been efficacious in slowing HER2 cancer progression, combining this and other anti-HER2 therapy with either chemotherapy or endocrine therapy has proven more effective in improving overall and progression free survival.

Strongly Enhanced Antitumor Activity of Trastuzumab and Pertuzumab Combination Treatment on HER2-Positive Human Xenograft Tumor Models

The human epidermal growth factor receptor (HER) family plays an important role in cell survival and proliferation, and is implicated in oncogenesis. Overexpression of HER2 is associated with aggressive disease and poor prognosis. Trastuzumab is a humanized monoclonal antibody targeting HER2 and has proven survival benefit for women with HER2-positive early and metastatic breast cancer. Pertuzumab, another monoclonal antibody, is a HER2 dimerization inhibitor that binds to a different epitope on HER2 than trastuzumab and inhibits HER2 dimer formation with other HER family members such as HER3 and HER1. We investigated the antitumor activity of these agents alone and in combination in HER2-positive breast and non-small cell lung cancer xenografts. Our data show that the combination of trastuzumab and pertuzumab has a strongly enhanced antitumor effect and induces tumor regression in both xenograft models, something that cannot be achieved by either monotherapy. The enhanced efficacy of the combination was also observed after tumor progression during trastuzumab monotherapy. Near-IR fluorescence imaging experiments confirm that pertuzumab binding to tumors is not impaired by trastuzumab pretreatment. Furthermore, we show by in vitro assay that both trastuzumab and pertuzumab potently activate antibody-dependent cellular cytotoxicity. However, our data suggest that the strongly enhanced antitumor activity is mainly due to the differing but complementary mechanisms of action of trastuzumab and pertuzumab, namely inhibition of HER2 dimerization and prevention of p95HER2 formation.

Breast Cancer: Understanding Sensitivity and Resistance to Chemotherapy and Targeted Therapies to Aid in Personalised Medicine

Breast cancer is the second leading cause of cancer deaths. This disease is estimated to be diagnosed in over one million people worldwide and to cause more than 400,000 deaths each year. This is a significant health problem in terms of both morbidity and mortality. Chemotherapy forms part of a successful treatment regime in many cases; however, as few as half of the patients treated may benefit from this, as a result of intrinsic or acquired multiple drug resistance (MDR). A range of mechanisms of MDR has been identified using in vitro cell culture models; many, if not all, of which may contribute to breast cancer resistance in the clinical setting. This phenomenon is complicated by the heterogenous nature of breast cancer and the likely multi-factorial nature of clinical resistance. It has been very well established that a "one treatment fits all" approach is not relevant and significant advances have been made through identifying and appropriately treating sub-groups of patients; particularly with newer rationally-targeted therapies, such as the HER2-targeted monoclonal antibody, Trastuzumab, and the dual HER2 and EGFR tyrosine kinase inhibitor, Lapatinab. Furthermore, large defined collaborative studies, using standardised global profiling approaches to study mRNA, microRNAs and proteins, followed by functional genomics studies, by ourselves and others, are underway in order to definitively establish the degree of complexity contributing to drug resistance. The overall vision is to identify the optimum therapeutic regime for individual patients -possibly involving novel targeted therapies, drug resistance modulators, and chemotherapy- to overcome breast cancer.

An overview of HER-targeted therapy with lapatinib in breast cancer

Breast cancer is a global public health burden with more than one million new diagnoses worldwide each year. As a significant proportion of women with early-stage breast cancer experience a relapse and metastatic breast cancer is generally incurable, therapeutic innovations are ongoing. One notable innovation in recent decades has been the identification of a subset of breast cancers that overexpress the transmembrane glycoprotein human epidermal growth factor receptor 2 (HER2) and the consequent development of HER2-targeted therapy. Given the significant benefits demonstrated with the HER2-targeted monoclonal antibody, trastuzumab, in the adjuvant and metastatic settings, investigators have endeavored to develop novel mechanisms for disrupting HER2-mediated signaling. Lapatinib, an orally available HER1- and HER2-targeted tyrosine kinase inhibitor, represents one such notable innovation. Lapatinib is currently being evaluated in both the adjuvant and metastatic settings and was recently approved by the United States Food and Drug Administration in combination with capecitabine, for the treatment of women with HER2-positive, pretreated, metastatic breast cancer. However, the ideal strategy for incorporating novel HER2-targeted agents, including lapatinib, into existing management paradigms is uncertain.

A Review of Trastuzumab-Based Therapy in Patients with HER2-positive Metastatic Breast Cancer

The ERBB2 or HER2 receptor is overexpressed in 25% of breast cancers and is associated with poor prognosis. Trastuzumab, a monoclonal antibody targeting HER2 has been demonstrated to improve survival when combined with chemotherapy for the treatment of HER2 overexpressing metastatic breast cancer (MBC). Further studies have endeavoured to clarify the optimum chemotherapy regimen in combination with trastuzumab for MBC and its use together with novel biological agents. This review summarises these data together with preclinical studies exploring the mechanism of trastuzumab action and causes of drug resistance. The frequent incidence of brain metastases in patients on trastuzumab is highlighted, and data on the continuation of trastuzumab following CNS and non-CNS progression reviewed.

HER2-Positive Early Breast Cancer and Trastuzumab: A Surgeon’s Perspective

Although the treatment of patients with early-stage breast cancer is provided by a multidisciplinary team, surgeons must ensure they are well informed about all aspects of patient care. For example, understanding the importance of human epidermal growth factor receptor 2 (HER2) gene amplification and/or protein overexpression and the effect on patient prognosis can guide therapeutic decision making. In addition, surgeons should also be knowledgeable about the wide variety of available postsurgical treatments, from traditional chemotherapy and radiotherapy to novel agents such as the HER2-targeted monoclonal antibody trastuzumab. Trastuzumab was recently approved for adjuvant treatment of invasive, HER2-positive, node-positive breast cancer. Its approval was based on the combined results of two large-scale trials, which demonstrated that adding trastuzumab to chemotherapy significantly improves disease-free and overall survival. Knowledge about the indications, schedules, and safety profiles of therapies such as trastuzumab will enable surgeons to optimize the timing of surgery in relation to these therapies, and to make informed decisions about the team member to whom a particular patient should ideally be referred for postsurgical care. In the future, results from large-scale trials evaluating the clinical utility of neoadjuvant trastuzumab will become available. Early results from ongoing phase III trials of the addition of trastuzumab to presurgical chemotherapy suggest that high response rates, including pathologic complete responses, are achievable. If trastuzumab is approved for use in neoadjuvant regimens, the need for surgeons to be well informed about the appropriate use of this particular agent will become even more important.

Bleomycin-induced pulmonary fibrosis is attenuated by a monoclonal antibody targeting HER2

The importance of HER2/HER3 signaling in decreasing the effects of lung injury was recently demonstrated. Transgenic mice unable to signal through HER2/HER3 had significantly less bleomycin-induced pulmonary fibrosis and showed a survival benefit. Based on these data, we hypothesized that pharmacological blockade of HER2/HER3 in vivo in wild-type mice would have the same beneficial effects. We tested this hypothesis in a bleomycin lung injury model using 2C4, a monoclonal antibody directed against HER2 that blocks HER2/HER3 signaling. The administration of 2C4 before injury decreased the effects of bleomycin at days 15 and 21 after injury. HER2/HER3 blockade resulted in less collagen deposition (362.8 +/- 37.9 compared with 610.5 +/- 27.1 microg/mg; P = 0.03) and less lung morphological changes (injury score of 1.99 +/- 1.55 vs. 3.90 +/- 0.76; P < 0.04). In addition, HER2/HER3 blockade resulted in a significant survival advantage with 50% vs. 25% survival at 30 days (P = 0.04). These results confirm that HER2 signaling can be pharmacologically targeted to reduce lung fibrosis and remodeling after injury.

NK-cell and T-cell functions in patients with breast cancer: effects of surgery and adjuvant chemo- and radiotherapy

Breast cancer is globally the most common malignancy in women. Her2-targeted monoclonal antibodies are established treatment modalities, and vaccines are in late-stage clinical testing in patients with breast cancer and known to promote tumour-killing through mechanisms like antibody-dependent cellular cytotoxicity. It is therefore increasingly important to study immunological consequences of conventional treatment strategies. In this study, functional tests and four-colour flow cytometry were used to detect natural killer (NK)-cell functions and receptors as well as T-cell signal transduction molecules and intracellular cytokines in preoperative breast cancer patients, and patients who had received adjuvant radiotherapy or adjuvant combined chemo-radiotherapy as well as in age-matched healthy controls. The absolute number of NK cells, the density of NK receptors as well as in vitro quantitation of functional NK cytotoxicity were significantly higher in preoperative patients than the post-treatments group and controls. A similar pattern was seen with regard to T-cell signalling molecules, and preoperative patients produced significantly higher amounts of cytokines in NK and T cells compared to other groups. The results indicate that functions of NK and T cells are well preserved before surgery but decrease following adjuvant therapy, which may speak in favour of early rather than late use of immunotherapeutic agents such as trastuzumab that may depend on intact immune effector functions.

Selecting Endocrine Therapy for Breast Cancer: What Role Does HER-2/neu Status Play?

Most women with breast cancer that overexpresses either estrogen (ER+) or progesterone receptors (PR+) will be treated with an endocrine agent. Although ER and PR are the only validated predictive markers of response to hormonal therapy, many women with hormone receptor-positive (HR+) tumors will fail to respond to endocrine treatment or develop hormone resistant disease. Another tumor marker, HER-2/neu, is an oncogene whose amplification or protein overexpression predicts response to a monoclonal antibody targeting HER-2/neu, as well as to anthracycline-based chemotherapy. Preclinical and some clinical data suggest that HER-2/neu positivity may also predict for relative resistance to endocrine therapy. Interpretation of clinical data addressing this issue is confounded by largely retrospective study designs and considerable heterogeneity in patient populations and methods of marker detection and interpretation. At this writing, HER-2/neu expression should not influence the decision to use endocrine therapy.

Phase I trial of the trifunctional anti-HER2 x anti-CD3 antibody ertumaxomab in metastatic breast cancer

2530 Background: Ertumaxomab is an intact bispecific monoclonal antibody targeting HER2 and CD3. The heavy chain is composed of murine IgG2a and rat IgG2b subclasses with strong binding to activato...

Antisense clusterin oligodeoxynucleotides increase the response of HER‐2 gene amplified breast cancer cells to Trastuzumab

Clusterin (CLU) is a heterodimeric secreted glycoprotein implicated in several physiological and pathological processes including cancer. Although recent data showed that overexpression of CLU is closely associated with disease progression in patients with breast tumor, the functional role of CLU expression in this tumor hystotype remains to be determined. The objectives in this study were to evaluate CLU expression levels after treatment with Trastuzumab, a HER2-targeted monoclonal antibody used in the clinical management of advanced breast cancer patients, and to test the usefulness of combined treatment with OGX-011, the second generation 2'-methoxyethyl gapmer oligonucleotides targeting the CLU gene, and Trastuzumab in this tumor hystotype. By using the HER-2 gene amplified-BT474 human breast cancer cells, we found Trastuzumab decreased HER-2 expression and inhibited cell proliferation without affecting apoptosis. Interestingly, Trastuzumab treatment up-regulated CLU protein expression in a dose-dependent fashion. We therefore hypothesized that the treatment with OGX-011, by blocking Trastuzumab-induced CLU expression, might potentiate the growth-inhibitory effect of Trastuzumab alone. Although OGX-011 had no effect on the behavior of the BT474 cells when used alone, it significantly enhanced the sensitivity of cells to Trastuzumab. A significant increase in the percentage of apoptotic cells, analyzed in terms of annexin V positivity and cleavage of poly(ADP-ribose) polymerase, was observed after combined treatment with OGX-011 plus Trastuzumab but not with either agent alone. Altogether our findings suggest that combined targeting of HER-2 and CLU may represent a novel, rational approach to breast cancer therapy.

Exquisite antitumour response to trastuzumab in a patient with no evidence of Ras–Raf–MAPK and PI3K–Akt pathways activation

We report on the case of a patient with a diagnosis of a HER2-overexpressing metastatic breast cancer which was refractory to a combination of a Raf kinase inhibitor and docetaxel, but highly sensitive to trastuzumab, a HER2-targeted monoclonal antibody. Interestingly, there was no evidence of Ras–Raf–MAPK or PI3K–Akt pathways activation.

Overexpression of Her-2/NEU in epithelial ovarian carcinoma induces vascular endothelial growth factor C by activating NF-kappa B: implications for malignant ascites formation and tumor lymphangiogenesis.

Vascular endothelial growth factor C (VEGF-C) is an important growth factor that governs lymphatic spread and the development of intraperitoneal tumors associated with epithelial ovarian cancer; however, its regulation is not yet understood. Overexpression of Her-2/NEU is related to poor survival in advanced epithelial ovarian carcinoma patients. Accordingly, this study attempted to analyze the association between the Her-2/NEU oncogene and VEGF-C in ovarian carcinoma and to elucidate the molecular mechanism of VEGF-C induction by Her-2/NEU. Immunohistochemistry was used to determine the expression of Her-2/NEU and VEGF-C in tissues from 41 patients with epithelial ovarian carcinoma. Several Her-2/NEU-stably-transfected Caov-3 ovarian carcinoma cells were used to evaluate the effect of Her-2/NEU on VEGF-C, the possible regulation mechanism, and the biological function of VEGF-C. Our experimental results identified a significant association between the Her-2/NEU oncogene and VEGF-C expression in both epithelial ovarian cancer patients (p < 0.05; Fisher's exact test) and in vitro cell lines. The overexpression of Her-2/NEU in Caov-3 ovarian cancer cells resulted in induction of a considerable amount of VEGF-C mRNA and protein; this process was dose-dependently inhibited by herceptin. The generation of VEGF-C significantly increased endothelial permeability. Pharmacological and genetic inhibition assays revealed that the cytoplasmic signaling molecule, p38 MAPK, and the transcriptional factor, NF-kappa B, are critically involved in the transcriptional activation of the VEGF-C gene by Her-2/NEU. In conclusion, this work clearly establishes that the Her-2/NEU oncogene is essential for the regulation of VEGF-C in ovarian carcinoma. It may be possible to use the monoclonal antibody targeting Her-2/NEU receptor to limit the formation of malignant ascites and lymphatic spread in ovarian carcinoma.