Isoform Of HER2 Research Articles

P5-07-01: Novel Alternative Splice Variants of HER2 in Invasive Breast Cancer.

Abstract Background: The Human Epidermal Growth Factor Receptor 2 (HER2) is an oncogene expressed in 25–30% of invasive breast cancers. HER2 shares extensive homology with other members of the HER family (HER1, HER3 and HER4), and is constitutively active as an homo- and heterodimer. The HER2 gene encodes an 185kDa transmembrane protein with tyrosine kinase activity. Gene amplification or protein expression of HER2 is a predictor of poor clinical outcome and decreased survival in women with breast cancer, and also indicates a favourable response to Trastuzumab (Herceptin) therapy, or a combinational therapy comprising Herceptin plus chemotherapy. However, resistance to Trastuzumab remains the case in approximately 50% of HER2 amplified/overexpressing tumours. Understanding the molecular mechanisms of Trastuzumab resistance and identifying more effective therapies, is critical in the treatment of patients whose breast cancers express this aggressive disease phenotype. In this study, it is postulated that the abnormal generation of mRNA splice variants may be responsible for the continued tumour growth and progression. Aims: The aim of this study is to increase our understanding of the role of HER-2 splice variants in the development and progression of breast cancer. This will inform the development of more sophisticated and effective therapies that target specific HER-2 isoforms, rather than Herceptin, which targets just the generic wild type HER-2 protein. Materials and Methods: The entire coding region of HER-2 cDNA was PCR amplified using 12 sets of HER-2 specific primers in HER-2 positive cell lines (SKOV-3, SKBR-3, MDA-MB-453 and MDA-MB-361). Results: RT-PCR results showed multiple bands in various regions of the HER2 mRNA. Sequencing of these bands revealed novel alternative splice variants with deletions in exons 13 and 18 of the HER2 gene expressed in addition to the wild type HER2. Bioinformatic analysis of the deletions revealed a cassette exon deletion in exon 13, and a loss of 42 base pairs in the 3’ end of exon 18 compared to the full length HER2. Both the full length HER2 sequence and the sequence containing the deletions were translated using the ExPASy Translate Tool. This revealed an in-frame deletion of 14 amino acids and revealed a novel splice isoform with a deletion in the HER2 protein which encompasses the entire ATP binding pocket. This was determined by analysis using UniProtKB to identify the composition of amino acids for each domain of HER2. Discussion: Our studies have identified novel splice variants in the tyrosine-kinase domain of the HER-2 gene using HER-2 positive cell lines. The loss of an ATP binding site in the HER2 gene may lead to a less active HER2 isoform which may play a significant role in prognosis. Current work is being carried out to study the regulation of these splice variants and to study the role of splice factors ASF/SF2 and SRPK1 in the regulation of HER2 splicing, and to elucidate any significant changes in the HER2 signalling pathways. In addition, the expression of these isoforms are currently being investigated in tissues from formalin fixed, paraffin embedded and frozen breast tumours. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-07-01.

Oncogenes induce senescence with incomplete growth arrest and suppress the DNA damage response in immortalized cells

Activation of the Her2 (ErbB2) oncogene is implicated in the development of breast, ovary and other cancers. Here, we show that expression of NeuT, a mutant-activated rodent isoform of Her2, in immortalized breast epithelial cells, while promoting senescence-associated morphological changes, up-regulation of senescence-associated β-galactosidase activity, and accumulation of the cyclin-dependent kinase inhibitor p21, failed to trigger the major senescence end-point, i.e. permanent growth arrest. Similar senescence-associated phenotype with incomplete growth arrest, which we dubbed senescence with incomplete growth arrest (SWING), could also be triggered by the expression of the Ras oncogene. SWING phenotype was stable, and persisted in tumor xenografts established from NeuT-transduced cells. Furthermore, a significant population of cells in SWING state was found in tumors in the MMTV/NeuT transgenic mouse model. SWING cells showed downregulation of histone H2AX, critical for repair of double-stranded DNA breaks, and impaired activation of Chk1 kinase. Overall, SWING cells were characterized by increased DNA instability and hypersensitivity to genotoxic stresses. We propose that the SWING state could be a stage in the process of cancer development.

Downregulation of miR-342 is associated with tamoxifen resistant breast tumors

BackgroundTumor resistance to the selective estrogen receptor modulator tamoxifen remains a serious clinical problem especially in patients with tumors that also overexpress HER2. We have recently demonstrated that the clinically important isoform of HER2, HERΔ16, promotes therapeutically refractory breast cancer including resistance to endocrine therapy. Likewise additional breast tumor cell models of tamoxifen resistance have been developed that do not involve HER2 overexpression. However, a unifying molecular mechanism of tamoxifen resistance has remained elusive.ResultsHere we analyzed multiple cell models of tamoxifen resistance derived from MCF-7 cells to examine the influence of microRNAs (miRNAs) on tamoxifen resistance. We compared miRNA expression profiles of tamoxifen sensitive MCF-7 cells and tamoxifen resistant MCF-7/HER2Δ16 cells. We observed significant and dramatic downregulation of miR-342 in the MCF-7/HER2Δ16 cell line as well as the HER2 negative but tamoxifen resistant MCF-7 variants TAMR1 and LCC2. Restoring miR-342 expression in the MCF-7/HER2Δ16 and TAMR1 cell lines sensitized these cells to tamoxifen-induced apoptosis with a dramatic reduction in cell growth. Expression of miR-342 was also reduced in a panel of tamoxifen refractory human breast tumors, underscoring the potential clinical importance of miR-342 downregulation. Towards the goal of identifying direct and indirect targets of miR-342 we restored miR-342 expression in MCF-7/HER2Δ16 cells and analyzed changes in global gene expression by microarray. The impact of miR-342 on gene expression in MCF-7/HER2Δ16 cells was not limited to miR-342 in silica predicted targets. Ingenuity Pathways Analysis of the dataset revealed a significant influence of miR-342 on multiple tumor cell cycle regulators.ConclusionsOur findings suggest that miR-342 regulates tamoxifen response in breast tumor cell lines and our clinical data indicates a trend towards reduced miR-342 expression and tamoxifen resistance. In addition, our results suggest that miR-342 regulates expression of genes involved in tamoxifen mediated tumor cell apoptosis and cell cycle progression. Restoring miR-342 expression may represent a novel therapeutic approach to sensitizing and suppressing the growth of tamoxifen refractory breast tumors.

Oncogenic HER2Δ16 suppresses miR-15a/16 and deregulates BCL-2 to promote endocrine resistance of breast tumors

Tamoxifen is the most commonly prescribed therapy for patients with estrogen receptor (ER)α-positive breast tumors. Tumor resistance to tamoxifen remains a serious clinical problem especially in patients with tumors that also overexpress human epidermal growth factor receptor 2 (HER2). Current preclinical models of HER2 overexpression fail to recapitulate the clinical spectrum of endocrine resistance associated with HER2/ER-positive tumors. Here, we show that ectopic expression of a clinically important oncogenic isoform of HER2, HER2Δ16, which is expressed in >30% of ER-positive breast tumors, promotes tamoxifen resistance and estrogen independence of MCF-7 xenografts. MCF-7/HER2Δ16 cells evade tamoxifen through upregulation of BCL-2, whereas mediated suppression of BCL-2 expression or treatment of MCF-7/HER2Δ16 cells with the BCL-2 family pharmacological inhibitor ABT-737 restores tamoxifen sensitivity. Tamoxifen-resistant MCF-7/HER2Δ16 cells upregulate BCL-2 protein levels in response to suppressed ERα signaling mediated by estrogen withdrawal, tamoxifen treatment or fulvestrant treatment. In addition, HER2Δ16 expression results in suppression of BCL-2-targeting microRNAs miR-15a and miR-16. Reintroduction of miR-15a/16 reduced tamoxifen-induced BCL-2 expression and sensitized MCF-7/HER2Δ16 to tamoxifen. Conversely, inhibition of miR-15a/16 in tamoxifen-sensitive cells activated BCL-2 expression and promoted tamoxifen resistance. Our results suggest that HER2Δ16 expression promotes endocrine-resistant HER2/ERα-positive breast tumors and in contrast to wild-type HER2, preclinical models of HER2Δ16 overexpression recapitulate multiple phenotypes of endocrine-resistant human breast tumors. The mechanism of HER2Δ16 therapeutic evasion, involving tamoxifen-induced upregulation of BCL-2 and suppression of miR-15a/16, provides a template for unique therapeutic interventions combining tamoxifen with modulation of microRNAs and/or ABT-737-mediated BCL-2 inhibition and apoptosis.

An oncogenic isoform of HER2 associated with locally disseminated breast cancer and trastuzumab resistance

The HER2-targeted therapy trastuzumab is widely used for the treatment of patients with metastatic breast tumors overexpressing HER2. However, an objective response is observed in only 12% to 24% of patients treated with trastuzumab as a single agent and initial responders regress in <6 months (1-3). The reason for the clinical failure of trastuzumab in this setting remains unclear. Here we show that local lymph node-positive disease progression in 89% of breast cancer patients with HER2-positive tumors involves the HER2 oncogenic variant HER2Delta16. We further show that ectopic expression of HER2Delta16, but not wild-type HER2, promotes receptor dimerization, cell invasion, and trastuzumab resistance of NIH3T3 and MCF-7 tumor cell lines. The potentiated metastatic and oncogenic properties of HER2Delta16 were mediated through direct coupling of HER2Delta16 to Src kinase. Cotargeting of HER2Delta16 and Src kinase with the single-agent tyrosine kinase inhibitor dasatinib resulted in Src inactivation, destabilization of HER2Delta16, and suppressed tumorigenicity. Activated Src kinase was also observed in 44% of HER2Delta16-expressing breast carcinomas underscoring the potential clinical implications of coupled HER2Delta16 and Src signaling. Our results suggest that HER2Delta16 expression is an important genetic event driving trastuzumab-refractory breast cancer. We propose that successful targeted therapeutics for intervention of aggressive HER2-positive breast cancers will require a strategy to suppress HER2Delta16 oncogenic signaling. One possibility involves a therapeutic strategy employing single-agent tyrosine kinase inhibitors to disengage the functionally coupled oncogenic HER2Delta16 and Src tyrosine kinase pathways.

Role of the HER2 [Ile655Val] genetic polymorphism in tumorogenesis and in the risk of trastuzumab-related cardiotoxicity

To examine the impact of a frequent her2 gene polymorphism (Ile655Val) on tumor growth and on the pharmacodynamics of treatment by trastuzumab. Experimental study: The growth characteristics of cells expressing the Ile or Val isoform were examined in vitro and after injection into nude mice. The effect of trastuzumab was determined in both experimental models. Clinical study: 61 patients with advanced breast cancers and treated by trastuzumab were genotyped for HER2 by PCR-RFLP. The influence of HER2 genotype on the trastuzumab treatment was examined. Experimental study: HER2-expressing cells acquired the characteristics of tumor cells. The Val isoform-expressing cells showed the highest growth capacity and developed aggressive tumors sensitive to trastuzumab. Clinical study: There was no link between tumor response or survival and HER2 genotype. All cases of treatment-related cardiotoxicity were found in the Ile/Val group and there was no cardiac toxicity in the Val/Val and Ile/Ile patients. This study establishes a clear-cut difference between the two HER2 isoforms regarding their tumorogenic potential with an advantage for the Val/HER2 isoform. In breast cancer patients treated with trastuzumab, the presence of a Val allele may constitute a risk factor for cardiac toxicity.