A novel mechanism of action of HER2 targeted immunotherapy is explained by inhibition of NRF2 function in ovarian cancer cells

Nuclear erythroid related factor-2 (NRF2) is known to promote cancer therapeutic detoxification and crosstalk with growth promoting pathways. HER2 receptor tyrosine kinase is frequently overexpressed in cancers leading to uncontrolled receptor activation and signaling. A combination of HER2 targeting monoclonal antibodies shows greater anticancer efficacy than the single targeting antibodies, however, its mechanism of action is largely unclear. Here we report novel actions of anti-HER2 drugs, Trastuzumab and Pertuzumab, involving NRF2. HER2 targeting by antibodies inhibited growth in association with persistent generation of reactive oxygen species (ROS), glutathione (GSH) depletion, reduction in NRF2 levels and inhibition of NRF2 function in ovarian cancer cell lines. The combination of antibodies produced more potent effects than single alone; downregulated NRF2 substrates by repressing the Antioxidant Response (AR) pathway with concomitant transcriptional inhibition of NRF2. We showed the antibody combination produced increased methylation at the NRF2 promoter consistent with repression of NRF2 antioxidant function, as HDAC and methylation inhibitors reversed such produced transcriptional effects. These findings demonstrate a novel mechanism and role for NRF2 in mediating the response of cancer cells to the combination of Trastuzumab and Pertuzumab and reinforce the importance of NRF2 in drug resistance and as a key anticancer target.

The dual EGFR/HER2 inhibitor lapatinib synergistically enhances the antitumor activity of the histone deacetylase inhibitor panobinostat in colorectal cancer models

As key molecules that drive progression and chemoresistance in gastrointestinal cancers, epidermal growth factor receptor (EGFR) and HER2 have become efficacious drug targets in this setting. Lapatinib is an EGFR/HER2 kinase inhibitor suppressing signaling through the RAS/RAF/MEK (MAP/ERK kinase)/MAPK (mitogen-activated protein kinase) and PI3K (phosphoinositide 3-kinase)/AKT pathways. Histone deacetylase inhibitors (HDACi) are a novel class of agents that induce cell cycle arrest and apoptosis following the acetylation of histone and nonhistone proteins modulating gene expression and disrupting HSP90 function inducing the degradation of EGFR-pathway client proteins. This study sought to evaluate the therapeutic potential of combining lapatinib with the HDACi panobinostat in colorectal cancer (CRC) cell lines with varying EGFR/HER2 expression and KRAS/BRAF/PIK3CA mutations. Lapatinib and panobinostat exerted concentration-dependent antiproliferative effects in vitro (panobinostat range 7.2-30 nmol/L; lapatinib range 7.6-25.8 μmol/L). Combined lapatinib and panobinostat treatment interacted synergistically to inhibit the proliferation and colony formation in all CRC cell lines tested. Combination treatment resulted in rapid induction of apoptosis that coincided with increased DNA double-strand breaks, caspase-8 activation, and PARP cleavage. This was paralleled by decreased signaling through both the PI3K and MAPK pathways and increased downregulation of transcriptional targets including NF-κB1, IRAK1, and CCND1. Panobinostat treatment induced downregulation of EGFR, HER2, and HER3 mRNA and protein through transcriptional and posttranslational mechanisms. In the LoVo KRAS mutant CRC xenograft model, the combination showed greater antitumor activity than either agent alone, with no apparent increase in toxicity. Our results offer preclinical rationale warranting further clinical investigation combining HDACi with EGFR and HER2-targeted therapies for CRC treatment.

Morphological and molecular characteristics of HER2 amplified urothelial bladder cancer.

Several (pre-) clinical trials are currently investigating the benefit of HER2-targeted therapy in urothelial bladder cancer (UBC). Patients with HER2 amplified UBC could potentially profit from these therapies. However, little is known about histomorphology, HER2 protein expression patterns and occurrence of alterations in the HER2 gene in their tumors. Among 150 metastasizing primary UBC, 13 HER2 amplified tumors were identified. Their histopathological features were compared with 13 matched, non-amplified UBC. HER2 protein expression was determined by immunohistochemistry. The 26 tumors were screened for mutations in exons 19 and 20 of the HER2 gene. UBC with HER2 amplification presented with a broad variety of histological variants (median 2 vs. 1), frequently featured micropapillary tumor components (77 % vs. 8 %) and demonstrated a high amount of tumor associated inflammation. Immunohistochemically, 10 of 13 (77 %) HER2 amplified tumors were strongly HER2 protein positive. Three tumors (23 %) were scored as HER2 negative. One of the HER2 amplified tumors harbored a D769N mutation in exon 19 of the HER2 gene; all other tested tumors were wild type. In conclusion, HER2 amplified UBC feature specific morphological characteristics. They frequently express the HER2 protein diffusely and are, therefore, promising candidates for HER2 targeted therapies. The detection of mutations at the HER2 locus might add new aspects to molecular testing of UBC.

Src and ADAM-17-Mediated Shedding of Transforming Growth Factor-alpha Is a Mechanism of Acute Resistance to TRAIL

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo-2L) has emerged as a promising anticancer agent. However, resistance to TRAIL is likely to be a major problem, and sensitization of cancer cells to TRAIL may therefore be an important anticancer strategy. In this study, we examined the effect of the epidermal growth factor receptor (EGFR)tyrosine kinase inhibitor (TKI) gefitinib and a human epidermal receptor 2 (HER2)-TKI (M578440) on the sensitivity of human colorectal cancer (CRC) cell lines to recombinant human TRAIL (rhTRAIL). A synergistic interaction between rhTRAIL and gefitinib and rhTRAIL and M578440 was observed in both rhTRAIL-sensitive and resistant CRC cells. This synergy correlated with an increase in EGFR and HER2 activation after rhTRAIL treatment. Furthermore, treatment of CRC cells with rhTRAIL resulted in activation of the Src family kinases (SFK). Importantly, we found that rhTRAIL treatment induced shedding of transforming growth factor-alpha (TGF-alpha) that was dependent on SFK activity and the protease ADAM-17. Moreover, this shedding of TGF-alpha was critical for rhTRAIL-induced activation of EGFR. In support of this, SFK inhibitors and small interfering RNAs targeting ADAM-17 and TGF-alpha also sensitized CRC cells to rhTRAIL-mediated apoptosis. Taken together, our findings indicate that both rhTRAIL-sensitive and resistant CRC cells respond to rhTRAIL treatment by activating an EGFR/HER2-mediated survival response and that these cells can be sensitized to rhTRAIL using EGFR/HER2-targeted therapies. Furthermore, this acute response to rhTRAIL is regulated by SFK-mediated and ADAM-17-mediated shedding of TGF-alpha, such that targeting SFKs or inhibiting ADAM-17, in combination with rhTRAIL, may enhance the response of CRC tumors to rhTRAIL. [Cancer Res 2008;68(20):8312-21]

Alternative targeted therapy for early HER2 positive breast cancer

Her2 (erbB2) receptor is overexpressed in around twenty percent of early breast cancer and historically conferred a poorer prognosis (1). Targeted anti-Her2 therapy has significantly improved outcomes for these women.

Development of paclitaxel-loaded liposomal systems with anti-her2 antibody for targeted therapy

Purpose: To develop liposome formulations containing monoclonal antibody anti-HER2 (MabHer2), and Paclitaxel (PTX). Methods: Seven different liposomal systems containing PTX, or MabHer2 or a combination of PTX and MabHer2 were made using lipid film hydration technique and sonication. The effects of liposome preparation conditions and extraction methods on antibody structure were investigated by polyacrylamide gel electrophoresis and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The characteristics of the liposomes were determined by a zetasizer, while drug-loading efficiency was evaluated by high-performance liquid chromatography. The cytotoxic effect of the liposome formulations was evaluated on MDA-MB-453 (HER2+) and MCF-7 (HER2-) breast cancer cell lines by MTT assay. Results: The antibody was not significantly affected by the stress conditions and the method of extraction. The particle size of liposomes was < 200 nm while the amount of incorporated PTX was 97.6 % for liposome without cationic agent and 98.2 % for those with cationic agent. Recovery of MabHer2 was 94.38 % after extraction. Combined PTX/MabHer2 liposome was more toxic on HER2 overexpressing positive MDA-MB-453 cell line than PTX-loaded liposomes and MabHer2. MabHer2 and combined PTX/MabHer2 liposomes showed no toxic effects on HER2 overexpressing negative MCF-7 cells relative to cationic PTX-loaded liposomes. Conclusions: This results obtained show that PTX can be encapsulated successfully into liposoma systems and that owing to Her2 specific antibody, these systems can be delivered directly to the target cell.

Targeted therapies in non-small cell lung cancer

The majority of non-small cell lung cancer (NSCLC) patients present with advanced disease and with a 5 year survival rate of <15% for these patients, treatment outcomes are considered extremely disappointing. Standard chemotherapy regimens provide some improvement to ~40% of patients. However, intrinsic and acquired chemoresistance are a significant problem and hinder sustained long term benefits of such treatments. Advances in proteomic and genomic profiling have increased our understanding of the aberrant molecular mechanisms that are driving an individual's tumour. The increased sensitivity of these technologies has enabled molecular profiling at the stage of initial biopsy thus paving the way for a more personalised approach to the treatment of cancer patients. Improvements in diagnostics together with a wave of new targeted small molecule inhibitors and monoclonal antibodies have revolutionised the treatment of cancer. To date there are essentially three targeted agents approved for clinical use in NSCLC. The tyrosine kinase inhibitor (TKI) erlotinib, which targets the epidermal growth factor receptor (EGFR) TK domain, has proven to be an effective treatment strategy in patients who harbour activating mutations in the EGFR TK domain. Bevacizumab a monoclonal antibody targeting the vascular endothelial growth factor (VEGF) can improve survival, response rates, and progression-free survival when used in combination with chemotherapy. Crizotinib, a small-molecule drug, inhibits the tyrosine kinase activity of the echinoderm microtubule-associated protein-like 4 anaplastic lymphoma kinase (EML4-ALK) fusion protein, resulting in decreased tumour cell growth, migration, and invasiveness in patients with locally advanced or metastatic NSCLC. The clinical relevance of several other targeted agents are under investigation in distinct molecular subsets of patients with key "driver" mutations including: KRAS, HER2, BRAF, MET, PIK3CA, AKT1,MAP2K1, ROS1 and RET. Often several pathways are activated simultaneously and crosstalk between pathways allows tumour cells to escape the inhibition of a single targeted agent. This chapter will explore the clinical development of currently available targeted therapies for NSCLC as well as those in clinical trials and will examine the synergy between cytotoxic therapies.

HER2 positive gastric and gastroesophageal adenocarcinoma; An Irish tertiary center experience

Background: Trastuzumab has been approved for patients with human epidermal growth factor receptor 2 (HER2) over expression and gene amplification metastatic gastric cancer. Here we present the prevalence of HER2 positive gastric cancer in an Irish population, the use of Trastuzumab in first line and beyond progression. Methods: The study was conducted in St James's Hospital, Dublin. A retrospective analysis of the date of patients with HER2 positive gastric cancer over a period of 3 years was carried out. Her2 positive was defined as immunohistochemistry (IHC) score of +3, of IHC score of +2 and increased gene copy number by fluorescence in situ hybridization (FISH). Overall survival was calculated from the day of initiation of treatment with Trastuzumab until death. Results: During the study period 140 patients with gastric and gastro-esophageal junction adenocarcinoma were treated. Out of those, 30 (21.4%) had HER2 positive disease. Among HER2 positive disease patients 18 (12.8%) were treated with first line Trastuzumab containing regimen with a median overall survival of 13 months. Nine (50%) developed progressive disease while on Trastuzumab and of those, 4 (22.2%) patients continued on Trastuzumab beyond progression, two (11.1%) of whom achieved stable disease and a prolonged survival. Conclusion: HER2 positivity rate in an Irish population with advanced gastric and gastro-esophageal junction adenocarcinoma is 21.4%. Treatment with Trastuzumab in the first line in combination with chemotherapy is a reasonable approach. Continuation of Trastuzumab beyond progression is a feasible strategy that requires further exploration.

A study of HER2 gene amplification and protein expression in gastric cancer

Background Gastric cancer is a leading cause of cancer-related mortality, and current treatment outcomes for advanced disease remain poor. HER2 has been identified as a potential candidate for targeted therapy in gastric cancers displaying HER2 gene amplification and protein overexpression.

Overcoming Resistance to Targeted Therapies in Cancer

The recent discovery of oncogenic drivers and subsequent development of novel targeted strategies has significantly added to the therapeutic armamentarium of anti-cancer therapies. Targeting BCR-ABL in chronic myeloid leukemia (CML) or HER2 in breast cancer has led to practice-changing clinical benefits, while promising therapeutic responses have been achieved by precision medicine approaches in EGFR mutant lung cancer, colorectal cancer and BRAF mutant melanoma. However, although initial therapeutic responses to targeted therapies can be substantial, many patients will develop disease progression within 6-12 months. An increasing application of powerful omics-based approaches and improving preclinical models have enabled the rapid identification of secondary resistance mechanisms. Herein, we discuss how this knowledge has translated into rational, novel treatment strategies for relapsed patients in genomically selected cancer populations.

Le CpG et le poly(I:C) agissent en synergie avec le trastuzumab contre le cancer du sein HER2+

Chez la souris, la thérapie anti-HER2 est dépendante de la présence de cellules T CD8+IFN-γ+ et des réponses IFN de type I. Ces IFN sont induits par les TLRs suite à la reconnaissance de signaux de danger, appelés PAMPs et DAMPs. Les TLR-3 et TLR-9 sont tous deux de bons inducteurs d’IFN de type I et sont également capable d’agir en synergie afin d’augmenter les niveaux d’IFN-γ, de TNF-α et d’IL-12. Notre hypothèse fut que la stimulation de ces deux TLRs mènerait à l’amélioration de l’activité anti-tumorale du trastuzumab via le recrutement et l’activation des cellules immunitaires. Nos buts furent de confirmer le potentiel thérapeutique de la combinaison de l’anticorps anti-HER2, de l’agoniste de TLR-3, le poly(I:C), et de l’agoniste de TLR-9, le CpG ODN. Des études in vivo et in vitro nous ont permis de découvrir une synergie entre ces agents qui résulte en une cytotoxicité ciblée plus efficace. De plus, cette thérapie s’avéra efficace chez des modèles CD8-dépendants et CD8-indépendents. Les souris purent rejeter leur tumeur et demeurer sains plusieurs semaines après l’arrêt des injections. Ces souris étaient également protégées lors d’un challenge, soulignant ainsi la présence d’une immunité mémoire. Nous avons aussi découvert que l’administration combine de trastuzumab des deux agonistes de TLRs mène à des réponses systémiques. Des études de déplétion confirmèrent que les cellules T CD8+ sont cruciales pour la protection à long terme des animaux, mais que les pDC sont moins impliquées que ce que l’on pourrait croire. Leur absence n’a que modestement affecté les effets de notre thérapie. À l’opposé, les cellules NK sont d’importants médiateurs des effets thérapeutiques. Des expériences d’ADCC ont révélé que le CpG ODN et poly(I:C) ont tous deux la capacité d’améliorer les fonctions des cellules NK, mais que la stimulation simultanée des TLR-3 et TLR-9 permet de maximiser les effets bénéfiques du trastuzumab. De la même manière, l’addition de CpG ODN et de poly(I:C) aux anticorps anti-HER2 a permis d’augmenter les réponses pro-inflammatoires, plus spécifiquement l’IFN-γ, le TNF-α, l’IP-10 et l’IL-12.

Chromogenic in situ hybridization compared with other approaches to evaluate HER2/neu status in breast carcinomas

Human epidermal growth factor receptor 2 (HER2) has been evaluated in breast cancer patients to identify those most likely to benefit from herceptin-targeted therapy. HER2 amplification, detected in 20-30% of invasive breast tumors, is associated with reduced survival and metastasis. The most frequently used technique for evaluating HER2 protein status as a routine procedure is immunohistochemistry (IHC). HER2 copy number alterations have also been evaluated by fluorescence in situ hybridization (FISH) in moderate immunoexpression (IHC 2+) cases. An alternative procedure to evaluate gene amplification is chromogenic in situ hybridization (CISH), which has some advantages over FISH, including the correlation between HER2 status and morphological features. Other methodologies have also been used, such as silver-enhanced in situ hybridization (SISH) and quantitative real-time RT-PCR, to determine the number of HER2 gene copies and expression, respectively. Here we will present a short and comprehensive review of the current advances concerning HER2 evaluation in human breast cancer.

Association between HSP90 and Her2 in gastric and gastroesophageal carcinomas

BACKGROUND Her2 expression and amplification occurs in a significant subset of gastro-esophageal carcinomas. Her2 is a client protein of molecular chaperones, e.g. heat shock protein (HSP) 90, rendering targeted therapies against Her2/HSP90 an interesting approach. This study aimed to investigate the role and relationship of Her2 and HSP90 in gastric and gastro-esophageal adenocarcinomas. MATERIAL AND METHODS Immunohistochemical determination of HSP90 and Her2 expression was performed on 347 primary resected tumors. Her2 amplification was additionally determined by fluorescence in situ hybridization for all cases. Expression and amplification results were correlated with pathologic parameters (UICC pTNM category, tumor grading) and survival. RESULTS Elevated Her2 copy numbers were observed in 87 tumors, 21 of them showing amplification. 174 tumors showed Her2 immunoreactivity/expression. HSP 90 immunoreactivity was found in 125 tumors. There was no difference between gastric carcinomas and carcinomas of the gastroesophageal junction regarding Her2 or HSP90. Both high HSP90 and Her2 expression/amplification were associated with earlier tumor stages (p<0.01), absence of lymph node metastases (p<0.02) and Laurens intestinal type (p<0.001). HSP90 correlated with Her2 expression and amplification (p<0.001 each). Expressions of HSP90 and Her2, but not Her2 amplification were associated with better prognosis (p=0.02; p=0.004; p=0.802). Moreover, Her2 expression was an independent prognostic factor for overall survival in the subgroup of gastric carcinoma patients (p=0.014) besides pT category, pN category and distant metastases. CONCLUSION Her2 expression and gene amplification occurred in a significant subset of cases. Our results suggest a favorable prognostic impact of Her2 expression. This warrants further investigations regarding the significance of Her2 non-amplified tumors showing Her2 immunoreactivity and the definition of Her2 status in gastric cancers. Moreover, the correlation of Her2 expression with the expression of Her2 chaperoning HSP90 may indicate a synergistic regulation. Targeting HSP90 with or without Her2 may offer additional therapeutic options for gastric carcinoma treatment.

Application of cell line based genomic predictors to predict response to targeted therapies in breast cancer

Cancer cell lines can be treated with a drug and the molecular comparison of responders and non-responders may yield potential predictors that could be tested in the clinic. It is a bioinformatics challenge to apply the cell line-derived multivariable response predictors to patients who respond to therapy. Using the gene expression data from 23 breast cancer cell lines, I developed three predictors of dasatinib sensitivity by selecting differentially expressed genes and applying different classification algorithms. The performance of these predictors on independent cell lines with known dasatinib response was tested. The predictor based on weighted voting method has the best overall performance. It correctly predicted dasatinib sensitivity in 11 out of 12 (92%) breast and 17 out of 23 (74%) lung cancer cell lines. These predictors were then applied to the gene expression data from 133 breast cancer patients in an attempt to predict how the patients might respond to dasatinib therapy. Two predictors identified 13 patients in common to be dasatinib sensitive. Sixty two percent of these cases are triple negative (ER-negative, HER2-negative and PR-negative) and 76% are double negative. The result is consistent with the findings from other studies, which identified a target population for dasatinib treatment to be triple negative or basal breast cancer subtype. In conclusion, we think that the cell line-derived dasatinib classifiers can be applied to the human patients. ^

Targeting the actin cytoskeleton in HER2+ metastatic cancer cells using a macrolide toxin

Breast and ovarian cancers are among the leading causes of cancer related deaths in women worldwide. In a subset of these cancers, dysregulation of the human epidermal growth factor receptor 2 (HER2) leads to overexpression of the receptor on the cell surface. Previous studies have found that these HER2+ cancers show high rates of progression to metastatic disease. Metastasis is driven by cytoskeletal rearrangements that produce filamentous actin (F-actin) based structures that penetrate and degrade extracellular matrix to facilitate tumour invasion. Advancements in targeted therapy have made F-actin an attractive target for the development of new cancer therapies. In this thesis, we tested the actin-depolymerizing macrolide toxin, Mycalolide B (MycB), as a potential warhead for a novel antibody drug conjugate (ADC) to target highly metastatic HER2+ breast and ovarian cancers. We found that MycB treatment of HER2+ breast (SKBR3, MDA-MB-453) and ovarian (SKOV3) cancer cells led to loss of viability (IC50 values ≤ 64 nM). Sub-lethal doses of MycB treatment caused potent suppression of leading edge protrusions, migration and invasion potential of HER2+ cancer cells (IC50 ≤ 32 nM). In contrast, other F-actin based processes such as receptor endocytosis were less sensitive to MycB treatment. MycB treatment skewed the size of endocytic vesicles, which may reflect defects in F-actin based vesicle motility or maturation. Given that HER2+ cancers have been effectively targeted by Trastuzumab and Trastuzumab-based ADCs, we tested the effects of a combination of Trastuzumab and MycB on cell migration and invasion. We found that MycB/ Trastuzumab combination treatments inhibited motility of SKOV3 cells to a greater degree than either treatment alone. Altogether, our results provide proof-of-principle that actin toxins such as MycB can be used as a novel class of warheads for ADCs to target and combat highly metastatic cancers.