P2-01-25: Truncated p110 ERBB2 (CTF611) Increases Migration and Invasion of Breast Epithelial Cells by Inhibiting STAT5b Activation

Abstract Background: Truncated ERBB2 receptors are present in a subset of human ERBB2+ amplified/overexpressing breast tumors, and are associated with trastuzumab resistance, metastasis, and poor clinical prognosis. However, whether truncated ERBB2 receptors are drivers of metastasis has not been well defined. In this study, we examined effects of full-length (p185) and truncated (p110) ERBB2 on the migration and invasion of human mammary epithelial cells, including HMLE and MCF10A cells. Material and Methods: Recombinant p185 and p110 ERBB2 were stably expressed in human mammary epithelial cells (HMLE) and MCF10A cells via retroviral vector. Expression of comparable levels of p185 and p110 in cells was confirmed by western blot. The phosphorylation states of downstream signaling proteins including STAT5 were assayed via phosphoproteomics and Collaborative Enzyme Enhanced Reactive (CEER™) immunoassay. The effects of the p110 constructs on cell migration and invasion were investigated by transwell assays. shRNA-encoding lentivirus was used for specific silencing of STAT5b in HMLE cells, and STAT5b silencing was confirmed at the protein level using western blot. Results and Discussion: Expression of p110 ERBB2 increased cell migration (HMLE, p = 0.04; MCF10A, p&amp;lt; 0.01) and invasion (HMLE, p= 0.03) when compared to expression of p185. Furthermore, expression of p110 in HMLE cells was associated with reduced phosphorylation of STAT5b. shRNA mediated silencing of STAT5b was sufficient to increase the migration (p &amp;lt; 0.01) and invasion of HMLE cells, phenocopying the p110 driven effects on HMLE cells. In clinical studies, loss of activated STAT5 protein correlates with breast cancer progression and is a negative predictor of survival. By analyzing publicly available gene expression datasets, we found that STAT5b mRNA expression is also significantly decreased in breast cancer compared to normal breast tissues in several studies, as well as in ERBB2 amplified vs. nonamplified samples. To our knowledge, this is the first reported perturbation of STAT signaling by truncated ERBB2 receptor, and suggests a mechanism by which truncated p110 ERBB2 (CTF611) increases migration and invasion of breast epithelial cells. This study extends the available data regarding STAT5 loss in breast cancer progression. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-01-25.</jats:p

Abstract P5-05-02: Whole Genome In Vivo RNA Interference Screening Identifies the Leukemia Inhibitory Factor Receptor as a Novel Breast Tumor Suppressor

Abstract Background: Cancer is caused by mutations in oncogenes and tumor suppressor genes resulting in the deregulation of processes fundamental to the normal behavior of cells. The identification and characterization of oncogenes and tumor suppressors has led to new treatment strategies that have significantly improved cancer outcome. The advent of next generation sequencing has allowed the elucidation of the fine structure of cancer genomes, however, the identification of pathogenic changes is complicated by the inherent genomic instability of cancer cells. Therefore, functional approaches for the identification of novel genes involved in the initiation and development of tumors are critical. Methods: In order to identify functionally important tumor suppressor genes we have conducted the first human whole genome in vivo RNA interference (RNAi) screen. Partially transformed human mammary epithelial cells (HMLEs), which do not form tumors in immunodeficient mice, were infected with the Expression Arrest™ GIPZ lentiviral shRNA library consisting of 62,000 shRNAs targeting the whole human genome, and injected into the mammary fat pad of immunodeficient mice. shRNAs that silenced tumor suppressor genes fully transformed the mammary epithelial cells resulting in tumor formation. Candidate tumor suppressor genes were identified by PCR amplification and sequencing of tumor integrated shRNAs. For validation, candidate tumor suppressor genes were silenced in HMLEs and ectopically expressed in fully transformed breast cancer cells. The effect of modifying gene expression on the transformed phenotype was assessed using soft agar colony formation assays. Clinical significance was determined by comparing expression in normal and cancerous human breast tissue using Oncomine Research. Results and Discussion: Using our novel approach, we identify previously validated tumor suppressor genes including TP53 and MNT, as well as several novel candidate tumor suppressor genes including leukemia inhibitory factor receptor (LIFR). Silencing LIFR expression with multiple shRNA constructs fully transformed human mammary epithelial cells resulting in enhanced colony formation in soft agar (P&amp;lt;0.05). Furthermore, overexpression of LIFR significantly inhibited colony formation in soft agar of fully transformed MDA231 and MCF7 breast cancer cells (P&amp;lt;0.01). In addition, our analysis of clinical data revealed that LIFR expression is significantly decreased in a large percentage of human cancers including breast (P&amp;lt;0.0001), lung (P&amp;lt;0.0001), hepatocellular (P&amp;lt;0.0001) and gastrointestinal tumors (P&amp;lt;0.0001). These results validate LIFR as a previously unidentified highly significant tumor suppressor, and also demonstrate the power of whole genome in vivo RNAi screens as a method for identifying novel genes regulating tumorigenesis. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-05-02.</jats:p

Macrophage depletion induces edema through release of matrix-degrading proteases and proteoglycan deposition.

Colony-stimulating factor 1 receptor (CSF1R) blockade abates tumor-associated macrophage (TAM) infiltrates and provides marked clinical benefits in diffuse-type tenosynovial giant cell tumors. However, facial edema is a common adverse event associated with TAM elimination in patients. In this study, we examined molecular and cellular events associated with edema formation in mice and human patients with cancer treated with a CSF1R blocking antibody. Extended antibody treatment of mice caused marked body weight gain, an indicator of enhanced body fluid retention. This was associated with an increase of extracellular matrix-remodeling metalloproteinases (MMPs), namely MMP2 and MMP3, and enhanced deposition of hyaluronan (HA) and proteoglycans, leading to skin thickening. Discontinuation of anti-CSF1R treatment or blockade of MMP activity restored unaltered body weight and normal skin morphology in the mice. In patients, edema developed at doses well below the established optimal biological dose for emactuzumab, a CSF1R dimerization inhibitor. Patients who developed edema in response to emactuzumab had elevated HA in peripheral blood. Our findings indicate that an early increase of peripheral HA can serve as a pharmacodynamic marker for edema development and suggest potential interventions based on MMP inhibition for relieving periorbital edema in patients treated with CSF1R inhibitors

Truncated p110 ERBB2 induces mammary epithelial cell migration, invasion and orthotopic xenograft formation, and is associated with loss of phosphorylated STAT5

Truncated-ERBB2 isoforms (t-ERBB2s), resulting from receptor proteolysis or alternative translation of the ERBB2 mRNA, exist in a subset of human breast tumors. t-ERBB2s lack the receptor extracellular domain targeted by therapeutic anti-ERBB2 antibodies and antibody–drug conjugates, including trastuzumab, trastuzumab-DM1 and pertuzumab. In clinical studies, expression of t-ERBB2 in breast tumors correlates with metastasis as well as trastuzumab resistance. By using a novel immuno-microarray method, we detect a significant t-ERBB2 fraction in 18 of 31 (58%) of immunohistochemistry (IHC)3+ ERBB2+ human tumor specimens, and further show that t-ERBB2 isoforms are phosphorylated in a subset of IHC3+ samples (10 of 31, 32%). We investigated t-ERBB2 biological activity via engineered expression of full-length and truncated ERBB2 isoforms in human mammary epithelial cells (HMECs), including HMEC and MCF10A cells. Expression of p110 t-ERBB2, but not p95m (m=membrane, also 648CTF) or intracellular ERBB2s, significantly enhanced cell migration and invasion in multiple cell types. In addition, only expression of the p110 isoform led to human breast epithelial cell (HMLE) xenograft formation in vivo. Expression of t-ERBB2s did not result in hyperactivation of the phosphoinositide kinase-3/AKT or mitogen-activated protein kinase signaling pathways in these cells; rather, phosphoproteomic array profiling revealed attenuation of phosphorylated signal transducer and activator of transcription 5 (STAT5) in p110-t-ERBB2-expressing cells compared to controls. Short hairpin-mediated silencing of STAT5 phenocopied p110-t-ERBB2-driven cell migration and invasion, while expression of constitutively active STAT5 reversed these effects. Thus, we provide novel evidence that (1) expression of p110 t-ERBB2 is sufficient for full transformation of HMEC, yielding in vivo xenograft formation, and (2) truncated p110 t-ERBB2 expression is associated with decreased phosphorylation of STAT5

Whole genome in vivo RNAi screening identifies the leukemia inhibitory factor receptor as a novel breast tumor suppressor

Cancer is caused by mutations in oncogenes and tumor suppressor genes, resulting in the deregulation of processes fundamental to the normal behavior of cells. The identification and characterization of oncogenes and tumor suppressors has led to new treatment strategies that have significantly improved cancer outcome. The advent of next generation sequencing has allowed the elucidation of the fine structure of cancer genomes, however, the identification of pathogenic changes is complicated by the inherent genomic instability of cancer cells. Therefore, functional approaches for the identification of novel genes involved in the initiation and development of tumors are critical. Here we report the first whole human genome in vivo RNA interference screen to identify functionally important tumor suppressor genes. Using our novel approach, we identify previously validated tumor suppressor genes including TP53 and MNT, as well as several novel candidate tumor suppressor genes including leukemia inhibitory factor receptor (LIFR). We show that LIFR is a key novel tumor suppressor, whose deregulation may drive the transformation of a significant proportion of human breast cancers. These results demonstrate the power of genome wide in vivo RNAi screens as a method for identifying novel genes regulating tumorigenesis

PI3K pathway inhibitors for the treatment of brain metastases with a focus on HER2+ breast cancer

The incidence of breast cancer brain metastases has increased in recent years, largely due to improved control of systemic disease with human epidermal growth factor receptor 2 (HER2)-targeted agents and the inability of most of these agents to efficiently cross the blood–blood barrier (BBB) and control central nervous system disease. There is, therefore, an urgent unmet need for treatments to prevent and treat HER2+ breast cancer brain metastases (BCBMs). Aberrant activation of the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway is frequently observed in many cancers, including primary breast tumors and BCBMs. Agents targeting key components of this pathway have demonstrated antitumor activity in diverse cancers, and may represent a new treatment strategy for BCBMs. In preclinical studies, several inhibitors of PI3K and mTOR have demonstrated an ability to penetrate the BBB and down-regulate PI3K signaling, indicating that these agents may be potential therapies for brain metastatic disease. The PI3K inhibitor buparlisib (BKM120) and the mTOR inhibitor everolimus (RAD001) are currently under evaluation in combination with trastuzumab in patients with HER2+ BCBMs