Specific targeting of HER2 overexpressing breast cancer cells with doxorubicin-loaded trastuzumab-modified human serum albumin nanoparticles

Specific targeting of tumor cells to achieve higher drug levels in tumor tissue and to overcome cardiotoxic and other secondary effects is the major goal in cancer therapy. With trastuzumab as a humanized monoclonal antibody binding, the HER2 receptor specific targeting is possible. In the present study, target-oriented nanoparticles based on biodegradable human serum albumin (HSA) loaded with cytostatic drug doxorubicin were developed. The surface of the nanoparticles was modified by covalent attachment of trastuzumab. HER2 overexpressing breast cancer cells showed a good cellular binding and uptake of these nanoparticles. The specific transport of the cytostatic drug doxorubicin with this nanoparticulate formulation into the HER2 overexpressing breast cancer cells, their release, and biological activity was demonstrated. The results indicate that these cell-type specific drug-loaded nanoparticles could achieve an improvement in cancer therapy. To our knowledge, this is the first study demonstrating a specific trastuzumab-based targeting of HER2 overexpressing breast cancer cells with doxorubicin-loaded nanoparticles.     

Differential gene expression profiling of human epidermal growth factor receptor 2-overexpressing mammary tumor

Human epidermal growth factor receptor 2 (HER2) is highly expressed in approximately 30% of breast cancer patients, and substantial evidence supports the relationship between HER2 overexpression and poor overall survival. However, the biological function of HER2 signal transduction pathways is not entirely clear. To investigate gene activation within the pathways, we screened differentially expressed genes in HER2-positive mouse mammary tumor using two-directional suppression subtractive hybridization combined with reverse dot-blotting analysis. Forty genes and expressed sequence tags related to transduction, cell proliferation/growth/apoptosis and secreted/extracellular matrix proteins were differentially expressed in HER2-positive mammary tumor tissue. Among these, 19 were already reported to be differentially expressed in mammary tumor, 11 were first identified to be differentially expressed in mammary tumor in this study but were already reported in other tumors, and 10 correlated with other cancers. These genes can facilitate the understanding of the role of HER2 signaling in breast cancer.     

Human Epidermal Growth Factor Receptor 2 (HER2) Impedes MLK3 Kinase Activity to Support Breast Cancer Cell Survival

Human epidermal growth factor receptor 2 (HER2) is amplified in ∼15–20% of human breast cancer and is important for tumor etiology and therapeutic options of breast cancer. Up-regulation of HER2 oncogene initiates cascades of events cumulating to the stimulation of transforming PI3K/AKT signaling, which also plays a dominant role in supporting cell survival and efficacy of HER2-directed therapies. Although investigating the underlying mechanisms by which HER2 promotes cell survival, we noticed a profound reduction in the kinase activity of a pro-apoptotic mixed lineage kinase 3 (MLK3) in HER2-positive (HER2+) but not in HER2-negative (HER2−) breast cancer tissues, whereas both HER2+ and HER2− tumors expressed a comparable level of MLK3 protein. Furthermore, the kinase activity of MLK3 was inversely correlated with HER2+ tumor grades. Moreover, HER2-directed drugs such as trastuzumab and lapatinib as well as depletion of HER2 or HER3 stimulated MLK3 kinase activity in HER2+ breast cancer cell lines. In addition, the noted inhibitory effect of HER2 on MLK3 kinase activity was mediated via its phosphorylation on Ser⁶⁷⁴ by AKT and that pharmacological inhibitors of PI3K/AKT prevented trastuzumab- and lapatinib-induced stimulation of MLK3 activity. Consistent with the pro-apoptotic function of MLK3, stable knockdown of MLK3 in the HER2+ cell line blunted the pro-apoptotic effects of trastuzumab and lapatinib. These findings suggest that HER2 activation inhibits the pro-apoptotic function of MLK3, which plays a mechanistic role in mediating anti-tumor activities of HER2-directed therapies. In brief, MLK3 represents a newly recognized integral component of HER2 biology in HER2+ breast tumors.     

Epithelial protein-tyrosine phosphatase 1B contributes to the induction of mammary tumors by HER2/Neu but is not essential for tumor maintenance

Protein-tyrosine phosphatase 1B (PTP1B), a well-established metabolic regulator, plays an important role in breast cancer. Using whole-body PTP1B knockout mice, recent studies have shown that PTP1B ablation delays HER2/Neu-induced mammary cancer. Whether PTP1B plays a cell-autonomous or a noncell-autonomous role in HER2/Neu-evoked tumorigenesis and whether it is involved in tumor maintenance was unknown. We generated mice expressing HER2/Neu and lacking PTP1B specifically in the mammary epithelium. We found that mammary-specific deletion of PTP1B delays the onset of HER2/Neu-evoked mammary tumors, establishing a cell autonomous role for PTP1B in such neoplasms. We also deleted PTP1B in established mouse mammary tumors or depleted PTP1B in human breast cancer cell lines grown as xenografts. PTP1B inhibition did not affect tumor growth in either model showing that neither epithelial nor stromal PTP1B is necessary for tumor maintenance. Taken together, our data show that despite the PTP1B contribution to tumor onset, it is not essential for tumor maintenance. This suggests that PTP1B inhibition could be effective in breast tumor prevention.     

The cooperation between hMena overexpression and HER2 signalling in breast cancer

hMena and the epithelial specific isoform hMena(11a) are actin cytoskeleton regulatory proteins belonging to the Ena/VASP family. EGF treatment of breast cancer cell lines upregulates hMena/hMena(11a) expression and phosphorylates hMena(11a), suggesting cross-talk between the ErbB receptor family and hMena/hMena(11a) in breast cancer. The aim of this study was to determine whether the hMena/hMena(11a) overexpression cooperates with HER-2 signalling, thereby affecting the HER2 mitogenic activity in breast cancer. In a cohort of breast cancer tissue samples a significant correlation among hMena, HER2 overexpression, the proliferation index (high Ki67), and phosphorylated MAPK and AKT was found and among the molecular subtypes the highest frequency of hMena overexpressing tumors was found in the HER2 subtype. From a clinical viewpoint, concomitant overexpression of HER2 and hMena identifies a subgroup of breast cancer patients showing the worst prognosis, indicating that hMena overexpression adds prognostic information to HER2 overexpressing tumors. To identify a functional link between HER2 and hMena, we show here that HER2 transfection in MCF7 cells increased hMena/hMena(11a) expression and hMena(11a) phosphorylation. On the other hand, hMena/hMena(11a) knock-down reduced HER3, AKT and p44/42 MAPK phosphorylation and inhibited the EGF and NRG1-dependent HER2 phosphorylation and cell proliferation. Of functional significance, hMena/hMena(11a) knock-down reduced the mitogenic activity of EGF and NRG1. Collectively these data provide new insights into the relevance of hMena and hMena(11a) as downstream effectors of the ErbB receptor family which may represent a novel prognostic indicator in breast cancer progression, helping to stratify patients.     

Targeted Herceptin–dextran iron oxide nanoparticles for noninvasive imaging of HER2/neu receptors using MRI

A novel magnetic resonance imaging (MRI) contrast agent containing Herceptin is reported. The surfaces of superparamagnetic iron oxide nanoparticles were modified with dextran and conjugated with Herceptin (Herceptin–nanoparticles) to improve their dispersion, magnetization, and targeting of the specific receptors on cells. From analytical results, we found that Herceptin–nanoparticles were well dispersed in solutions of various pH range, and had no hysteresis, high saturation magnetization (80 emu/g), and low cytotoxicity to a variety of cells. Notably, the magnetic resonance enhancements for the different breast cancer cell lines (BT-474, SKBR-3, MDA-MB-231, and MCF-7) are proportional to the HER2/neu expression level in vitro. When Herceptin–nanoparticles were administered to mice bearing breast tumor allograft by intravenous injection, the tumor site was detected in T ₂-weighted magnetic resonance images as a 45% enhancement drop, indicating a high level of accumulation of the contrast agent within the tumor sites. Therefore, targeting of cancer cells was observed by in vitro and in vivo MRI studies using Herceptin–nanoparticles contrast agent. In addition, Herceptin–nanoparticles enhancing the magnetic resonance signal intensity were sufficient to detect the cell lines with a low level of HER2/neu expression. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Proteomic characterization of Her2/neu-overexpressing breast cancer cells

The receptor tyrosine kinase HER2 is an oncogene amplified in invasive breast cancer and its overexpression in mammary epithelial cell lines is a strong determinant of a tumorigenic phenotype. Accordingly, HER2-overexpressing mammary tumors are commonly indicative of a poor prognosis in patients. Several quantitative proteomic studies have employed two-dimensional gel electrophoresis in combination with MS/MS, which provides only limited information about the molecular mechanisms underlying HER2/neu signaling. In the present study, we used a SILAC-based approach to compare the proteomic profile of normal breast epithelial cells with that of Her2/neu-overexpressing mammary epithelial cells, isolated from primary mammary tumors arising in mouse mammary tumor virus-Her2/neu transgenic mice. We identified 23 proteins with relevant annotated functions in breast cancer, showing a substantial differential expression. This included overexpression of creatine kinase, retinol-binding protein 1, thymosin 4 and tumor protein D52, which correlated with the tumorigenic phenotype of Her2-overexpressing cells. The differential expression pattern of two genes, gelsolin and retinol binding protein 1, was further validated in normal and tumor tissues. Finally, an in silico analysis of published cancer microarray data sets revealed a 23-gene signature, which can be used to predict the probability of metastasis-free survival in breast cancer patients.     

A gene-protein assay for human epidermal growth factor receptor 2 (HER2): brightfield tricolor visualization of HER2 protein, the HER2 gene, and chromosome 17 centromere (CEN17) in formalin-fixed, paraffin-embedded breast cancer tissue sections

ABSTRACT: BACKGROUND: The eligibility of breast cancer patients for human epidermal growth factor receptor 2 (HER2)-directed therapies is determined by the HER2 gene amplification and/or HER2 protein overexpression status of the breast tumor as determined by in situ hybridization (ISH) or immunohistochemistry (IHC), respectively. Our objective was to combine the US Food and Drug Administration (FDA)-approved HER2 & chromosome 17 centromere (CEN17) brightfield ISH (BISH) and HER2 IHC assays into a single automated HER2 gene-protein assay allowing simultaneous detection of all three targets in a single tissue section. METHODS: The HER2 gene-protein assay was optimized using formalin-fixed, paraffin-embedded (FFPE) samples of the xenograft tumors MCF7 [HER2 negative (non-amplified gene, protein negative)] and Calu-3 [HER2 positive (amplified gene, protein positive)]. HER2 IHC was performed using a rabbit monoclonal anti-HER2 antibody (clone 4B5) and a conventional 3,3' -diaminobenzidine IHC detection. The HER2 & CEN17 BISH signals were visualized using horseradish peroxidase-based silver and alkaline phosphatase-based red detection systems, respectively with a cocktail of 2,4-dinitrophenyl-labeled HER2 and digoxigeninlabeled CEN17 probes. The performance of the gene-protein assay on tissue microarray slides containing 189 randomly selected FFPE clinical breast cancer tissue cores was compared to that of the separate HER2 IHC and HER2 & CEN17 BISH assays. RESULTS: HER2 protein detection was optimal when the HER2 IHC protocol was used before (rather than after) the BISH protocol. The sequential use of HER2 IHC and HER2 & CEN17 BISH detection steps on FFPE xenograft tumor sections appropriately co-localized the HER2 protein, HER2 gene, and CEN17 signals after mitigating the silver background staining by using a naphthol phosphate-containing hybridization buffer for the hybridization step. The HER2 protein and HER2 gene status obtained using the multiplex HER2 gene-protein assay demonstrated high concordance with those obtained using the separate HER2 IHC and HER2 & CEN17 BISH assays, respectively. CONCLUSIONS: We have developed a protocol that allows simultaneous visualization of the HER2 IHC and HER2 & CEN17 BISH targets. This automated protocol facilitated the determination of HER2 protein and HER2 gene status in randomly selected breast cancer samples, particularly in cases that were equivocal or exhibited tumor heterogeneity. The HER2 gene-protein assay produced results virtually equivalent to those of the single FDA-approved HER2 IHC and HER2 & CEN17 BISH assays. Virtual slides The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2041964038705297.     

Fhit Expression Protects Against HER2-Driven Breast tumor Development: Unraveling the Molecular Interconnections

The tumor suppressor gene FHIT is inactivated by genetic and epigenetic changes, i.e., loss of heterozygosity or promoter hypermethylation, in common human cancers. We recently showed that Fhit protein levels can be regulated by Fhit proteasome degradation mediated by EGF-dependent activation of EGFR family members, including HER2, whose overexpression is linked to poor prognosis in breast cancer. Analysis of a series of 384 human primary breast carcinomas revealed low/absent Fhit protein levels more frequently in HER2-overexpressing tumors. To test for a possible complementation of the FHIT and HER2 genes, tumor incidence was assessed in mice carrying one inactivated Fhit allele (Fhit+/-) crossed with FVB/N mice carrying the rat HER2/neu proto-oncogene driven by the mouse mammary tumor virus promoter. All Fhit heterozygous mice developed mammary tumors, whereas when both Fhit alleles (Fhit+/+) were present, tumor incidence was reduced in 27% of the mice, which remained tumor-free at 20 months. These findings suggest a protective role for FHIT in HER2-driven mammary tumors. Together, these data argue for the cooperation between Fhit and HER2 in breast carcinogenesis.     

Concomitant Detection of HER2 Protein and Gene Alterations by Immunohistochemistry (IHC) and Silver Enhanced In Situ Hybridization (SISH) Identifies HER2 Positive Breast Cancer with and without Gene Amplification

Introduction

HER2 status assessment became a mandatory test assay in breast cancer, giving prognostic and predictive information including eligibility for adjuvant anti-HER2 therapy. Precise and reliable assessment of HER2 status is therefore of utmost importance. In this study we analyzed breast cancer samples by a novel technology for concomitant detection of the HER2 protein and gene copy number.

Methods

Tissue microarrays containing 589 invasive breast cancer samples were analyzed with a double immunohistochemistry (IHC) and silver labeled in situ hybridization (SISH) assay simultaneously detecting HER2 protein and gene copy number in the same tumor cells. This bright-field assay was analyzed using scores according to the modified ASCO guidelines and the results were correlated with patient prognosis.

Results

Overall concordance rate between protein expression and the presence of gene amplification was 98%. Fifty-seven of 60 tumors (95%) with IHC score 3+, 6 of 10 tumors with IHC score 2+ (60%) and only 3 of 519 tumors (0.6%) with IHC score 0/1+ were amplified by SISH. Patients with gene amplification despite IHC score 0/1+ had a tendency for worse overall survival (p = 0.088, reaching nearly statistical significance) compared to IHC score 0/1+ without amplification. In contrast, there was no difference in overall survival in IHC score 3+/2+ tumors with and without gene amplification.

Conclusions

The novel double IHC and SISH assay for HER2 is efficient in the identification of breast cancer with discordant HER2 protein and HER2 gene status, especially for the prognostically relevant groups of HER2 protein negative tumors with HER2 amplification and HER2 protein positive tumors without HER2 amplification. Breast cancer without HER2 amplification among IHC score 2+/3+ tumors (10% in our cohort) suggests that other mechanisms than gene amplification contribute to protein overexpression in these cells.     

Egr-1 is required for neu/HER2-induced mammary tumors

Egr-1 is known to function mainly as a tumor suppressor through direct regulation of multiple tumor suppressor genes. To determine the role of Egr-1 in breast tumors in vivo, we used mouse models of breast cancer induced by HER2/neu. We compared neu-overexpressing Egr-1 knockout mice (neu/Egr-1 KO) to neu-overexpressing Egr-1 wild type or heterozygote mice (neu/Egr-1 WT or neu/Egr-1 het) with regard to onset of tumor appearance and number of tumors per mouse. In addition, to examine the role of Egr-1 in vitro, we established neu/Egr-1 WT and KO tumor cell lines derived from breast tumors developed in each mouse. Egr-1 deletion delayed tumor development in vivo and decreased the rate of cell growth in vitro. These results suggest that Egr-1 plays an oncogenic role in HER2/neu-driven mammary tumorigenesis.     

Antibody-drug conjugates in breast cancer: Marching from HER2-overexpression into HER2-low

The recent decade has witnessed a vigorous prosper of antibody-drug conjugates (ADCs) in solid tumors including breast cancer. Integrating the specificity of monoclonal antibodies and potency of cytotoxic drugs, ADCs are capable of delivering cytotoxic agents directly to tumor cells and surrounding accomplices with heterogeneous antigen expression by exerting the distinctive bystander effect. Up till now, three ADCs (T-DM1, T-DXd and SG) have attained the official approval and stepped into clinical practices in breast cancer, with numerous promising products in the pipeline. As an unprecedented breast cancer subgroup identified following solidified drug benefit, the cognitive and therapeutic paradigm of HER2-low population which was previously thought lacking definite targets and efficacious regimens has been thoroughly rewritten by ADCs, and several encouraging achievements are expected in ongoing trials. Herein, we discuss the contrived knowledge, latest advancements and future perspectives of ADCs in HER2-overexpressing and HER2-low breast cancer.     

pH-Responsive Artemisinin Derivatives and Lipid Nanoparticle Formulations Inhibit Growth of Breast Cancer Cells In Vitro and Induce Down-Regulation of HER Family Members

Artemisinin (ART) dimers show potent anti-proliferative activities against breast cancer cells. To facilitate their clinical development, novel pH-responsive artemisinin dimers were synthesized for liposomal nanoparticle formulations. A new ART dimer was designed to become increasingly water-soluble as pH declines. The new artemisinin dimer piperazine derivatives (ADPs) remained tightly associated with liposomal nanoparticles (NPs) at neutral pH but were efficiently released at acidic pH''s that are known to exist within solid tumors and organelles such as endosomes and lysosomes. ADPs incorporated into nanoparticles down regulated the anti-apoptotic protein, survivin, and cyclin D1 when incubated at low concentrations with breast cancer cell lines. We demonstrate for the first time, for any ART derivative, that ADP NPs can down regulate the oncogenic protein HER2, and its counterpart, HER3 in a HER2+ cell line. We also show that the wild type epidermal growth factor receptor (EGFR or HER1) declines in a triple negative breast cancer (TNBC) cell line in response to ADP NPs. The declines in these proteins are achieved at concentrations of NP109 at or below 1 µM. Furthermore, the new artemisinin derivatives showed improved cell-proliferation inhibition effects compared to known dimer derivatives.     

Abstracts for a conference on trace elements in diet,nutrition, and health: essentiality and toxicity

Overexpression of HER2/neu is associated with drug resistance and poor outcome in breast cancer. Solamargine (SM), a glycoalkaloid purified from the herb Solanum incanum, exhibits HER2/neu gene modulation of HER2/neu high-expressing human breast cancer cell line ZR-75-1. SM downregulation of HER2/neu gene expression was determined by RT-PCR and Southern hybridization. Additionally, the membrane-bound HER2/neu receptor in highly HER2/neu-expressing breast cancer cells was determined by radioimmunoassay, immunocytochemistry, fluorescent immunocytochemistry, and flow cytometry. SM significantly decreased the number of HER2/neu receptors on the cell membrane. Methotrexate (MTX), 5-florouracil (5-Fu), and cisplatin (CDDP) are commonly used for breast carcinoma treatment in clinics; however, patients with HER2/neu overexpression exhibit resistance to these anticancer drugs. Notably, combination of MTX, 5-Fu, and CDDP with SM individually increased the susceptibility of breast cancer cells to these chemotherapeutic agents. Experimental results indicated that downregulation of HER2/neu by SM might be an effective strategy for enhancing drug susceptibility of breast cancer cells expressing high levels of HER2/neu.     

Adoptive transfer of autologous, HER2-specific, cytotoxic T lymphocytes for the treatment of HER2-overexpressing breast cancer

The human epidermal growth factor receptor 2 (HER2) has been targeted as a breast cancer-associated antigen by immunotherapeutical approaches based on HER2-directed monoclonal antibodies and cancer vaccines. We describe the adoptive transfer of autologous HER2-specific T-lymphocyte clones to a patient with metastatic HER2-overexpressing breast cancer. The HLA/multimer-based monitoring of the transferred T lymphocytes revealed that the T cells rapidly disappeared from the peripheral blood. The imaging studies indicated that the T cells accumulated in the bone marrow (BM) and migrated to the liver, but were unable to penetrate into the solid metastases. The disseminated tumor cells in the BM disappeared after the completion of adoptive T-cell therapy. This study suggests the therapeutic potential for HER2-specific T cells for eliminating disseminated HER2-positive tumor cells and proposes the combination of T cell-based therapies with strategies targeting the tumor stroma to improve T-cell infiltration into solid tumors.     

Profiling the HER3/PI3K pathway in breast tumors using proximity-directed assays identifies correlations between protein complexes and phosphoproteins

Background

The identification of patients for targeted antineoplastic therapies requires accurate measurement of therapeutic targets and associated signaling complexes. HER3 signaling through heterodimerization is an important growth-promoting mechanism in several tumor types and may be a principal resistance mechanism by which EGFR and HER2 expressing tumors elude targeted therapies. Current methods that can study these interactions are inadequate for formalin-fixed, paraffin-embedded (FFPE) tumor samples.

Methodology and Principal Findings

Herein, we describe a panel of proximity-directed assays capable of measuring protein-interactions and phosphorylation in FFPE samples in the HER3/PI3K/Akt pathway and examine the capability of these assays to inform on the functional state of the pathway. We used FFPE breast cancer cell line and tumor models for this study. In breast cancer cell lines we observe both ligand-dependent and independent activation of the pathway and strong correlations between measured activation of key analytes. When selected cell lines are treated with HER2 inhibitors, we not only observe the expected molecular effects based on mechanism of action knowledge, but also novel effects of HER2 inhibition on key targets in the HER receptor pathway. Significantly, in a xenograft model of delayed tumor fixation, HER3 phosphorylation is unstable, while alternate measures of pathway activation, such as formation of the HER3PI3K complex is preserved. Measurements in breast tumor samples showed correlations between HER3 phosphorylation and receptor interactions, obviating the need to use phosphorylation as a surrogate for HER3 activation.

Significance

This assay system is capable of quantitatively measuring therapeutically relevant responses and enables molecular profiling of receptor networks in both preclinical and tumor models.     

Reverse-phase protein microarray highlights HER2 signaling activation in immunohistochemistry/FISH/HER2-negative breast cancers

Evaluation of: Wulfkuhle JD, Berg D, Wolff C et al. Molecular analysis of HER2 signaling in human breast cancer by functional protein pathway activation mapping. Clin. Cancer Res. 18(23), 6426–6435 (2012).

Exhaustive characterization and mapping of pivotal molecules and downstream effectors deregulated in breast cancer is of fundamental clinical value to define the most effective therapy. Wulfkuhle et al. applied reverse-phase protein microarray, a highly sensitive immunoassay able to perform quantitative and multiplexed analysis of total and/or modified cellular proteins, to assess protein levels and activation/phosphorylation status of the HER family (EGFR, HER2, HER3) and downstream signaling molecules in HER2⁺ and HER2^- breast cancers. The research was performed using laser capture microdissected tumor epithelial cells from frozen samples and formalin-fixed paraffin embedded specimens, which were also analyzed by immunohistochemistry (IHC) and FISH. This study identified a subgroup of IHC/FISH/HER2^- patients with HER2 activation/phosphorylation levels comparable with those obtained from IHC/FISH/HER2⁺ tumors. HER2 signaling activation was independent from total HER2 expression and involved HER3 and EGFR activation. These findings indicate that molecular characterization by reverse-phase protein microarray of HER2 and its partners/effectors in the signaling cascade enables the identification of a subgroup of IHC/FISH/HER2^- patients showing HER2 signaling activation. These patients, currently excluded from targeted therapy administration, could potentially benefit from this and it could improve prognosis and survival.