Treatment Of ErbB2-positive Breast Cancer Research Articles

Effect of Calcitriol in Inhibiting the Cancer Cell Growth and Promoting Apoptosis in ErbB2-positive Breast Cancer Cells.

Targeted therapies, specifically ErbB family tyrosine kinase inhibitors, have demonstrated potential for improving outcomes in patients with ErbB2-positive breast cancer. Despite their effectiveness, these therapies are associated with limitations, including high costs, side effects, drug resistance, lack of specificity, and toxicity. To overcome these challenges, drug repurposing has emerged as a promising strategy in breast cancer treatment. The aim of this investigation was to assess the influence of calcitriol on breast cancer cell lines expressing ErbB2 and comparing its effects with the conventional treatment, neratinib. We employed an MTT test to determine cell viability and utilized staining techniques to assess cell apoptosis. Flow cytometry was used to evaluate cell cycle arrest, while a scratch wound healing test was performed to examine cancer cell migration ability. Additionally, gene expression studies were conducted for calcitriol and neratinib to support our hypothesis regarding the ErbB2 gene. The repurposing of calcitriol demonstrated enhanced efficacy in suppressing cancer cell growth in ErbB2- positive breast cancer. Proportionally, calcitriol significantly reduced the viability of SK-BR-3 cells, similar to neratinib. Furthermore, calcitriol exhibited significant cytotoxicity against neratinib and substantially reduced breast cancer cell growth. These findings were corroborated by the wound healing assay, cell cycle arrest analysis, and gene expression studies, demonstrating comparable efficacy to the standard treatment, neratinib. The findings from this investigation offer compelling proof that highlights the promising role of calcitriol as an adjuvant drug with antiproliferative and antitumoral effects in the management of ErbB2-positive breast carcinoma patients. Therefore, we recommend further evaluation of calcitriol in clinical settings, particularly for the treatment of ErbB2-positive breast cancer, as it shows promise as a valuable therapeutic option.

Erb-b2 Receptor Tyrosine Kinase 2 (ERBB2) Promotes ATG12-Dependent Autophagy Contributing to Treatment Resistance of Breast Cancer Cells

Simple SummaryExpression of the tyrosine kinase receptor ERBB2 in cancer cells leads to drug resistance. Autophagy, a “self-eating” process inside the cell, is a mechanism for drug resistance in cancer cells. It has been shown that ERBB2 activation leads to increased autophagy in breast cancer cells, but the underlying mechanisms remains unclear. In this study, we demonstrated that ERBB2 promotes autophagy by increasing the protein levels of the autophagy gene ATG12 (autophagy-related 12), contributing to the resistance of breast cancer cells to chemotherapy drugs or ERBB2-targeted antibody treatments. We further showed that ATG12 expression in breast tumors containing ERBB2 correlated with a worse patient survival outcome. Finally, lapatinib is an inhibitor for both EGFR and ERBB2 tyrosine kinases in the EGFR protein family and promotes autophagy in cells containing only EGFR but inhibits autophagy in cells containing only ERBB2. Taken together, this suggests that ERBB2 promotes autophagy through upregulation of ATG12.The epidermal growth factor receptor (EGFR) family member erb-b2 receptor tyrosine kinase 2 (ERBB2) is overexpressed in many types of cancers leading to (radio- and chemotherapy) treatment resistance, whereas the underlying mechanisms are still unclear. Autophagy is known to contribute to cancer treatment resistance. In this study, we demonstrate that ERBB2 increases the expression of different autophagy genes including ATG12 (autophagy-related 12) and promotes ATG12-dependent autophagy. We clarify that lapatinib, a dual inhibitor for EGFR and ERBB2, promoted autophagy in cells expressing only EGFR but inhibited autophagy in cells expressing only ERBB2. Furthermore, breast cancer database analysis of 35 genes in the canonical autophagy pathway shows that the upregulation of ATG12 and MAP1LC3B is associated with a low relapse-free survival probability of patients with ERBB2-positive breast tumors following treatments. Downregulation of ERBB2 or ATG12 increased cell death induced by chemotherapy drugs in ERBB2-positive breast cancer cells, whereas upregulation of ERBB2 or ATG12 decreased the cell death in ERBB2-negative breast cancer cells. Finally, ERBB2 antibody treatment led to reduced expression of ATG12 and autophagy inhibition increasing drug or starvation-induced cell death in ERBB2-positive breast cancer cells. Taken together, this study provides a novel approach for the treatment of ERBB2-positive breast cancer by targeting ATG12-dependent autophagy.

Cholesterol content in cell membrane maintains surface levels of ErbB2 and confers a therapeutic vulnerability in ErbB2-positive breast cancer

BackgroundErbB2 overexpression identifies a subset of breast cancer as ErbB2-positive and is frequently associated with poor clinical outcomes. As a membrane-embedded receptor tyrosine kinase, cell surface levels of ErbB2 are regulated dynamically by membrane physical properties. The present study aims to investigate the influence of membrane cholesterol contents on ErbB2 status and cellular responses to its tyrosine kinase inhibitors.MethodsThe cholesterol abundance was examined in ErbB2-positive breast cancer cells using filipin staining. Cellular ErbB2 localizations were investigated by immunofluorescence with altered membrane cholesterol contents. The inhibitory effects of the cholesterol-lowering drug lovastatin were assessed using cell proliferation, apoptosis, immunoblotting and immunofluorescence assays. The synergistic effects of lovastatin with the ErbB2 inhibitor lapatinib were evaluated using an ErbB2-positive breast cancer xenograft mouse model.ResultsMembrane cholesterol contents positively correlated with cell surface distribution of ErbB2 through increasing the rigidity and decreasing the fluidity of cell membranes. Reduction in cholesterol abundance assisted the internalization and degradation of ErbB2. The cholesterol-lowering drug lovastatin significantly potentiated the inhibitory effects of ErbB2 kinase inhibitors, accompanied with enhanced ErbB2 endocytosis. Lovastatin also synergized with lapatinib to strongly suppress the in vivo growth of ErbB2-positive breast cancer xenografts.ConclusionThe cell surface distribution of ErbB2 was closely regulated by membrane physical properties governed by cholesterol contents. The cholesterol-lowering medications can hence be exploited for potential combinatorial therapies with ErbB2 kinase inhibitors in the clinical treatment of ErbB2-positive breast cancer.

ERBB2 overexpression suppresses stress-induced autophagy and renders ERBB2-induced mammary tumorigenesis independent of monoallelic Becn1 loss

Defective autophagy has been implicated in mammary tumorigenesis, as the gene encoding the essential autophagy regulator BECN1 is deleted in human breast cancers and Becn1+/− mice develop mammary hyperplasias. In agreement with a recent study, which reports concurrent allelic BECN1 loss and ERBB2 amplification in a small number of human breast tumors, we found that low BECN1 mRNA correlates with ERBB2-overexpression in breast cancers, suggesting that BECN1 loss and ERBB2 overexpression may functionally interact in mammary tumorigenesis. We now report that ERBB2 overexpression suppressed autophagic response to stress in mouse mammary and human breast cancer cells. ERBB2-overexpressing Becn1+/+ and Becn1+/− immortalized mouse mammary epithelial cells (iMMECs) formed mammary tumors in nude mice with similar kinetics, and monoallelic Becn1 loss did not alter ERBB2- and PyMT-driven mammary tumorigenesis. In human breast cancer databases, ERBB2-expressing tumors exhibit a low autophagy gene signature, independent of BECN1 mRNA expression, and have similar gene expression profiles with non-ERBB2-expressing breast tumors with low BECN1 levels. We also found that ERBB2-expressing BT474 breast cancer cells, despite being partially autophagy-deficient under stress, can be sensitized to the anti-ERBB2 antibody trastuzumab (tzb) by further pharmacological or genetic autophagy inhibition. Our results indicate that ERBB2-driven mammary tumorigenesis is associated with functional autophagy suppression and ERBB2-positive breast cancers are partially autophagy-deficient even in a wild-type BECN1 background. Furthermore and extending earlier findings using tzb-resistant cells, exogenously imposed autophagy inhibition increases the anticancer effect of trastuzumab on tzb-sensitive ERBB2-expressing breast tumor cells, indicating that pharmacological autophagy suppression has a wider role in the treatment of ERBB2-positive breast cancer.

Comparison of preclinical cardiotoxic effects of different ErbB2 inhibitors

Two novel human antitumor immunoconjugates, made up of a human anti-ErbB2 scFv, Erbicin, fused with either a human RNase or the Fc region of a human IgG1, are selectively cytotoxic for ErbB2-positive cancer cells in vitro and in vivo. The Erbicin-derived immunoagents (EDIA) target an epitope different from that of trastuzumab, the only humanized antibody currently prescribed for treatment of ErbB2-positive breast cancer (BC). As Trastuzumab has shown cardiotoxic effects, in this study, we evaluated if any side effects were exerted also by EDIA, used as single agents or in combination with anthracyclines. Furthermore, we compared the in vitro and in vivo cardiotoxic effects of EDIA with those of the other available anti-ErbB2 drugs: Trastuzumab, 2C4 (Pertuzumab), and Lapatinib. In this article, we show that EDIA, in contrast with Trastuzumab, 2C4, and Lapatinib, have no toxic effects on human fetal cardiomyocytes in vitro, and do not induce additive toxicity when combined with doxorubicin. Furthermore, EDIA do not impair cardiac function in vivo in mice, as evaluated by Color Doppler echocardiography, whereas Trastuzumab significantly reduces radial strain (RS) at day 2 and fractional shortening (FS) at day 7 of treatment in a fashion similar to doxorubicin. Also 2C4 and Lapatinib significantly reduce RS after only 2 days of treatment, even though they showed cardiotoxic effects less pronounced than those of Trastuzumab. These results strongly indicate that RS could become a reliable marker to detect early cardiac dysfunction and that EDIA could fulfill the therapeutic need of patients ineligible to Trastuzumab treatment because of cardiac dysfunction.

A Single-Dose, Crossover, Placebo- and Moxifloxacin-Controlled Study to Assess the Effects of Neratinib (HKI-272) on Cardiac Repolarization in Healthy Adult Subjects

Neratinib is an orally administered, small-molecule, irreversible pan-ErbB inhibitor in development for the treatment of ErbB2-positive breast cancer. This study assessed the effects of therapeutic and supratherapeutic neratinib concentrations on cardiac repolarization, in accordance with current regulatory guidance. This was a two-part study in healthy subjects. In part 1, subjects were randomized to receive placebo, 400 mg moxifloxacin, or 240 mg neratinib (therapeutic dose) following a high-fat meal. In part 2, after a washout period, subjects received placebo plus 400 mg ketoconazole or 240 mg neratinib plus ketoconazole (supratherapeutic dose). ANOVA was used to compare the baseline-adjusted QTc interval for neratinib with that of placebo (reference), and for neratinib plus ketoconazole with that of placebo plus ketoconazole (reference). Pharmacokinetic/pharmacodynamic analyses and categorical summaries of interval data were done. Assay sensitivity was evaluated by the effect of moxifloxacin on QTc compared with placebo. Sixty healthy subjects were enrolled in this study. The upper bounds of the 90% confidence interval for baseline-adjusted QTcN (population-specific corrected QT) were </=10 milliseconds greater than the corresponding reference at all postdose time points under conditions of both therapeutic and supratherapeutic plasma concentrations of neratinib. Pharmacokinetic/pharmacodynamic analysis revealed no relationship between neratinib concentrations and QTc interval. No subjects had QTcI, QTcF, or QTcN intervals >450 milliseconds or change from baseline >30 milliseconds. Moxifloxacin produced a significant increase in QTcN compared with placebo (P < 0.05). Therapeutic and supratherapeutic plasma concentrations of neratinib do not prolong the QTc interval in healthy subjects.

Cardiotoxic effects, or lack thereof, of anti-ErbB2 immunoagents.

e11054 Background: Herceptin (H), an anti-ErbB2 humanized antibody prescribed for treatment of ErbB2-positive breast cancer, has proved to be an essential tool in immunotherapy. However, large-scale clinical studies with H have shown that it engenders cardiomyopathy, particularly in patients treated either concurrently or previously with anthracyclines. Two novel human antitumor immunoconjugates were engineered in our laboratory by fusion of a human anti-ErbB2 scFv, Erbicin, with either a human RNase or the Fc region of a human IgG1, and thus called Erb- hRNase and Erb-hcAb (human anti-ErbB2-compact antibody), respectively. Both immunoagents are selectively cytotoxic for ErbB2-positive cancer cells in vitro and vivo. The Erbicin-derived immunoagents (EDIA) target on breast cancer cells an ErbB2 epitope different from that of H. We report that EDIA did not show in vitro cardiotoxic effects on rat and human cardiomyocytes, whereas H was strongly toxic. Methods: Methods for measuring cardiac function in M-mode with shortening fraction (FA) and in B-mode with ejection fraction are not very sensitive in detecting early myocardial damage. In this study we evaluated myocardial strain by speckle tracking technique (ST) by Color Doppler echocardiography (Visual sonic Vevo 2100) to identify early left ventricular dysfunction in mice treated with EDIA, doxorubicin (D), H, their associations and in a sham group. We measured ST and FA by assessing M-mode short axis projection in C57BL/6 mice at time 0, 2 and 6 days of daily administration. Results: EDIA did not impair cardiac function in vivo in a mouse model whereas H significantly reduced radial strain at 3 days of treatment and fractional shortening (% FS) at 6 days of treatment compared to the sham group in a fashion similar to D. Similarly, cardiac fibrosis, an index of collagen accumulation following cardiac muscle deterioration, was significantly attenuated in mice treated with either Erb-hcAb or Erb-hRNase as compared to those treated with H or D. Conclusions: These results suggest that the Erb-hcAb and the immuno RNase are immunoagents which can fulfil the therapeutic need of patients ineligible to H treatment due to cardiac dysfunction. No significant financial relationships to disclose.

Breast cancer ErbB2 status in Asian women: A review of local literature

e22164 Background: Breast cancer incidence is increasing throughout Asia. However, the characteristics of Asian breast cancer are not always well understood due to the limited availability of local data and their inadequate inclusion in global literature databases. Effective breast cancer treatment in this region requires a better understanding of Asian breast cancer biology, including ErbB2 status. Methods: We performed a literature search in seven Asian countries on breast cancer studies in which tumour ErbB2 overexpression was assessed. The keywords erbB2 OR HER2 OR ErbB-2 OR HER-2 AND breast cancer AND (country) were used to search PubMed, international and local conference abstracts and local-language journals from the year 2000 onwards. Where available, we selected up to five representative studies from each country on the basis of population size, multi-institution patient populations, institution reputation and journal impact factor. We excluded studies containing only mRNA, serum, or cell line data or those conducted on biased populations or in western countries on Asian populations. Results: ErbB2 data from 26 Asian breast cancer studies are summarized in the table . The mean or median age of the study populations ranged from 46–56 years, with one exception. In most studies that evaluated tumour ErbB2 and hormone status, ErbB2 over-expression correlated negatively with ER positivity. Conclusions: The larger studies in particular confirm that the proportion of ErbB2-positive breast cancer in Asia is generally similar to the 20–30% reported for western women. Definitions of ErbB2 positivity using IHC vary between institutions and FISH is not routinely performed in several Asian countries. This may contribute to the fact that reliable, representative data on breast tumour ErbB2 status is scarce in Malaysia, the Philippines, India and Thailand. The increased availability of accurate ErbB2 testing and data would aid improved treatment of ErbB2-positive breast cancer in these countries. [Table: see text] [Table: see text]

IκB kinase α kinase activity is required for self-renewal of ErbB2/Her2-transformed mammary tumor-initiating cells

NF-kappaB is constitutively active in many solid tumors, including breast cancer. However, the role of NF-kappaB in breast carcinogenesis is unknown. Ikkalpha(AA/AA) "knockin" mice in which activation of IkappaB kinase alpha (IKKalpha) is prevented by replacement of activation loop serines with alanines exhibit delayed mammary gland growth during pregnancy, because IKKalpha activity is required for cyclin D1 induction and proliferation of lobuloalveolar epithelial cells. Given the role of cyclin D1 in breast and mammary cancer, we examined involvement of IKKalpha in mammary carcinogenesis induced by oncogenes or a chemical carcinogen, 7,12-dimethylbenz[a]anthracene (DMBA). The Ikkalpha(AA) mutation retarded tumor development in response to either 7,12-dimethylbenzaanthracene or the MMTV-c-neu (ErbB2/Her2) transgene but had no effect on MMTV-v-Ha-ras-induced cancer, although both oncogenes rely on cyclin D1. Strikingly, primary Ikkalpha(AA/AA)/MMTV-c-neu carcinoma cells exhibited diminished self-renewal capacity, resulting in the inability to establish secondary tumors. Ikkalpha(AA/AA)/MMTV-c-neu carcinoma cells underwent premature senescence when cultured under conditions used for propagation of mammary gland stem cells. Thus, IKKalpha is not only a regulator of mammary epithelial proliferation, but is also an important contributor to ErbB2-induced oncogenesis, providing signals that maintain mammary tumor-initiating cells. IKKalpha may represent a novel and specific target for treatment of ErbB2-positive breast cancer.

650 POSTER A Systems approach to identifying patient responders to EGFR-targeted therapy of colorectal cancer

The human epidermal growth factor receptor (EGFR) family consists of four members that belong to the ErbB lineage of proteins (ErbB1–4). These receptors consist of a glycosylated extracellular domain, a single hydrophobic transmembrane segment, and an intracellular portion with a juxtamembrane segment, a protein kinase domain, and a carboxyterminal tail. Seven ligands bind to EGFR including epidermal growth factor and transforming growth factor α, none bind to ErbB2, two bind to ErbB3, and seven ligands bind to ErbB4. The ErbB proteins function as homo and heterodimers. The heterodimer consisting of ErbB2, which lacks a ligand, and ErbB3, which is kinase impaired, is surprisingly the most robust signaling complex of the ErbB family. Growth factor binding to EGFR induces a large conformational change in the extracellular domain, which leads to the exposure of a dimerization arm in domain II of the extracellular segment. Two ligand-EGFR complexes unite to form a back-to-back dimer in which the ligands are on opposite sides of the aggregate. Following ligand binding, EGFR intracellular kinase domains form an asymmetric homodimer that resembles the heterodimer formed by cyclin and cyclin-dependent kinase. The carboxyterminal lobe of the activator kinase of the dimer interacts with the amino-terminal lobe of the receiver kinase thereby leading to its allosteric stimulation. Downstream ErbB signaling modules include the phosphatidylinositol 3-kinase/Akt (PKB) pathway, the Ras/Raf/MEK/ERK1/2 pathway, and the phospholipase C (PLCγ) pathway. Several malignancies are associated with the mutation or increased expression of members of the ErbB family including lung, breast, stomach, colorectal, head and neck, and pancreatic carcinomas and glioblastoma (a brain tumor). Gefitinib, erlotinib, and afatinib are orally effective protein-kinase targeted quinazoline derivatives that are used in the treatment of ERBB1-mutant lung cancer. Lapatinib is an orally effective quinazoline derivative used in the treatment of ErbB2-overexpressing breast cancer. Trastuzumab, pertuzumab, and ado-trastuzumab emtansine, which are given intravenously, are monoclonal antibodies that target the extracellular domain and are used for the treatment of ErbB2-positive breast cancer; ado-trastuzumab emtansine is an antibody-drug conjugate that delivers a cytotoxic drug to cells overexpressing ErbB2. Cetuximab and panitumumab are monoclonal antibodies that target ErbB1 and are used in the treatment of colorectal cancer. Cancers treated with these targeted drugs eventually become resistant to them. The role of combinations of targeted drugs or targeted drugs with cytotoxic therapies is being explored in an effort to prevent or delay drug resistance in the treatment of these malignancies.

423 POSTER A markov model to evaluate the cost effectiveness of five bisphosphonate therapies in the prevention of bone complications in breast cancer patients with bone metastases: a German outpatient perspective

The human epidermal growth factor receptor (EGFR) family consists of four members that belong to the ErbB lineage of proteins (ErbB1–4). These receptors consist of a glycosylated extracellular domain, a single hydrophobic transmembrane segment, and an intracellular portion with a juxtamembrane segment, a protein kinase domain, and a carboxyterminal tail. Seven ligands bind to EGFR including epidermal growth factor and transforming growth factor α, none bind to ErbB2, two bind to ErbB3, and seven ligands bind to ErbB4. The ErbB proteins function as homo and heterodimers. The heterodimer consisting of ErbB2, which lacks a ligand, and ErbB3, which is kinase impaired, is surprisingly the most robust signaling complex of the ErbB family. Growth factor binding to EGFR induces a large conformational change in the extracellular domain, which leads to the exposure of a dimerization arm in domain II of the extracellular segment. Two ligand-EGFR complexes unite to form a back-to-back dimer in which the ligands are on opposite sides of the aggregate. Following ligand binding, EGFR intracellular kinase domains form an asymmetric homodimer that resembles the heterodimer formed by cyclin and cyclin-dependent kinase. The carboxyterminal lobe of the activator kinase of the dimer interacts with the amino-terminal lobe of the receiver kinase thereby leading to its allosteric stimulation. Downstream ErbB signaling modules include the phosphatidylinositol 3-kinase/Akt (PKB) pathway, the Ras/Raf/MEK/ERK1/2 pathway, and the phospholipase C (PLCγ) pathway. Several malignancies are associated with the mutation or increased expression of members of the ErbB family including lung, breast, stomach, colorectal, head and neck, and pancreatic carcinomas and glioblastoma (a brain tumor). Gefitinib, erlotinib, and afatinib are orally effective protein-kinase targeted quinazoline derivatives that are used in the treatment of ERBB1-mutant lung cancer. Lapatinib is an orally effective quinazoline derivative used in the treatment of ErbB2-overexpressing breast cancer. Trastuzumab, pertuzumab, and ado-trastuzumab emtansine, which are given intravenously, are monoclonal antibodies that target the extracellular domain and are used for the treatment of ErbB2-positive breast cancer; ado-trastuzumab emtansine is an antibody-drug conjugate that delivers a cytotoxic drug to cells overexpressing ErbB2. Cetuximab and panitumumab are monoclonal antibodies that target ErbB1 and are used in the treatment of colorectal cancer. Cancers treated with these targeted drugs eventually become resistant to them. The role of combinations of targeted drugs or targeted drugs with cytotoxic therapies is being explored in an effort to prevent or delay drug resistance in the treatment of these malignancies.