Proliferation Of SKBR3 Cells Research Articles

Abstract B123: Preclinical evaluation of FS102: A HER2-specific Fcab with a novel mechanism of action.

Abstract HER2 is a validated oncogenic target which is overexpressed in many cancers. Although the approved HER2-specific biologics have improved the standard of care of HER2-positive breast and gastric cancers, a significant unmet medical need exists due to intrinsic or acquired resistance. By randomizing two loops in the CH3 domain of an IgG1 Fc fragment, we generated a HER2-specific Fcab™ (Fc fragment with antigen binding), called FS102, which binds HER2 with high affinity (2 nM), and recognizes an epitope that does not overlap with the epitopes of trastuzumab or pertuzumab. FS102 has a favorable t1/2 of ∼ 60 hours in mice. Biophysical characterization has shown the Fcab to have suitable properties to be developed as a therapeutic. To assess if interactions with the unique HER2 epitope could translate into clinically relevant pharmacological activities, we conducted in vitro and in vivo characterization of FS102 in comparison to trastuzumab monotherapy or trastuzumab-pertuzumab combination therapy. Using the HER2-positive breast cell line SKBr3 as an in vitro model system, we determined the effect of FS102 on HER2 downregulation, cellular proliferation, cell cycle progression, and apoptosis. Both FS102 and trastuzumab were internalized efficiently by SKBr3 cells, but only the former induced profound reduction in cell surface and total HER2 protein levels. Trastuzumab and FS102 both reduced SKBr3 cell proliferation by approximately 50% upon 5 days treatment. However the two agents achieved this inhibition via distinct mechanisms of action (MOA). Consistent with published data, trastuzumab induced G1 cell cycle arrest, while FS102 showed no effect on cell cycle progression. Instead, FS102 significantly induced apoptosis as measured by caspase activity and a loss of membrane integrity, an effect not observed upon exposure to trastuzumab. FS102 displays potent anti-tumor effects in HER2 overexpressing patient-derived xenograft (PDX) murine models, which are positive for a clinically relevant biomarker. Significant tumor inhibition has been observed in gastric, colorectal and breast PDX models. This includes complete tumor regression in 3 out of 8 models evaluated. Moreover, FS102 is significantly more effective than trastuzumab when tested in the biomarker-positive subset of PDX tumors. In one of these PDX model systems, where the tumors were refractory to trastuzumab-pertuzumab combination treatment, subsequent FS102 treatment reversed tumor growth and ultimately led to complete regression of the tumor. The results indicate that FS102 may have the potential to overcome resistance to conventional HER2 therapy in a biomarker-defined patient subpopulation, and that the Fcab technology is an attractive platform to generate novel biologics with unique antigen recognition and MOA. Additional studies are underway to further elucidate the FS102-induced ErbB receptor degradation pathway. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B123. Citation Format: Sarah Batey, Kinmei Leung, Robert Rowlands, Samine Isaac, Joana Carvalho, Sarah Weller, Mateusz Wydro, Miguel Gaspar, Melanie Medcalf, Robert Pegram, Vicky Drewett, Mihriban Tuna, John Haurum, Haijun H. Sun. Preclinical evaluation of FS102: A HER2-specific Fcab with a novel mechanism of action. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B123.

Abstract C6: EMC1/KIAA0090 overexpression enhances proliferation and motility in breast cancer cells

Abstract Human KIAA0090 (EMC1) is an evolutionarily conserved and ubiquitously expressed gene that maps to a chromosomal region (1p36.13) with frequent aberrations in several types of cancer. The RefSeq mRNA (isoform 1) of human EMC1 encodes a highly conserved protein of 993 amino acids whose yeast ortholog was recently proposed to function in unfolded protein response (UPR) and/or endoplasmic reticulum-associated degradation (ERAD). Deletion of the gene in yeast and C. elegans leads to slow growth (reduced fitness), and studies using genetic approaches or affinity capture in yeast, human and flies have revealed interactors of EMC1 involved in multiple pathways, including cell cycle, secretory pathway, UPR, ERAD, ion transport, cytoskeleton, transcription factors, cell signaling and mitochondrial electron transfer. Accumulated evidence on the requirement of UPR components for tumor growth and maintenance besides potential involvement of human EMC1 in cancer as suggested by global gene-expression analyses prompted us to investigate the role of this protein in breast cancer progression. Using antibodies raised, in our laboratory, against the N-terminal half of human EMC1, we showed staining mostly restricted to the basal layer of mammary epithelium in normal tissues, whereas in breast cancer tissues strong staining especially of what appears to be invasive cells and associated vasculature was observed. Additionally, using a tissue microarray with 96 samples of invasive primary breast tumor, we observed staining for EMC1 predominantly in zones of invasion and of variable levels of intensity, that we classified in weak, moderate and strong. Most cases (80/96, 83%) showed moderate to strong intensity, and strong expression was more frequent in tumors of grade II. Also, we observed a positive correlation of EMC1 with estrogen and progesterone receptor expression. Furthermore, we stably transfected SKBR-3 cells, which bear HER-2 gene amplification, with full length EMC1 cloned into the pcDNA3 vector, and protein overexpression was confirmed by immunostaining and qRT-PCR. SKBR-3 cells, which have a typical epithelial feature of cuboidal shape and grow grouped together with one another, underwent a noticeable morphological change after EMC1 overexpression, characterized by a more flattened, mesenchymal-like morphology and sparse distribution. Prominent filopodia, revealed by phalloidin labeling, was also observed after EMC1 exogenous expression. Additionally, EMC1 overexpression also enhanced motility and proliferation of SKBR-3 cells through ERK1/2 activation. Citation Format: Roberto Augusto Silva Molina, Rodrigo Ribeiro Silva, Josane Freitas Souza, Milene Mantovani Lopes, Daniel Guimaraes Tiezzi, Enilza Maria Espreafico. EMC1/KIAA0090 overexpression enhances proliferation and motility in breast cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C6.

The HER2-Binding Affibody Molecule (ZHER2∶342)2 Increases Radiosensitivity in SKBR-3 Cells

We have previously shown that the HER2-specific affibody molecule (ZHER2∶342)2 inhibits proliferation of SKBR-3 cells. Here, we continue to investigate its biological effects in vitro by studying receptor dimerization and clonogenic survival following irradiation. We found that (ZHER2∶342)2 sensitizes the HER2-overexpressing cell line SKBR-3 to ionizing radiation. The survival after exposure to (ZHER2∶342)2 and 8 Gy (S8Gy 0.006) was decreased by a factor four compared to the untreated (S8Gy 0.023). The low HER2-expressing cell line MCF-7 was more radiosensitive than SKBR-3 but did not respond to (ZHER2∶342)2. Treatment by (ZHER2∶342)2 strongly increased the levels of dimerized and phosphorylated HER2 even after 5 minutes of stimulation. The monomeric ZHER2∶342 does not seem to be able to induce receptor phosphorylation and dimerization or sensitize cells to irradiation.

Potential role of HER2‐overexpressing exosomes in countering trastuzumab‐based therapy

Exosomes are endosome-derived nanovesicles actively released into the extracellular environment and biological fluids, both under physiological and pathological conditions, by different cell types. We characterized exosomes constitutively secreted by HER2-overexpressing breast carcinoma cell lines and analyzed in vitro and in vivo their potential role in interfering with the therapeutic activity of the humanized antibody Trastuzumab and the dual tyrosine kinase inhibitor (TKI) Lapatinib anti-HER2 biodrugs. We show that exosomes released by the HER2-overexpressing tumor cell lines SKBR3 and BT474 express a full-length HER2 molecule that is also activated, although to a lesser extent than in the originating cells. Release of these exosomes was significantly modulated by the growth factors EGF and heregulin, two of the known HER2 receptor-activating ligands and naturally present in the surrounding tumor microenvironment. Exosomes secreted either in HER2-positive tumor cell-conditioned supernatants or in breast cancer patients' serum bound to Trastuzumab. Functional assays revealed that both xenogeneic and autologous HER2-positive nanovesicles, but not HER2-negative ones, inhibited Trastuzumab activity on SKBR3 cell proliferation. By contrast, Lapatinib activity on SKBR3 cell proliferation was unaffected by the presence of autologous exosomes. Together, these findings point to the role of HER2-positive exosomes in modulating sensitivity to Trastuzumab, and, consequently, to HER2-driven tumor aggressiveness.

Abstract 612: FOXO1A is a target for HER2 overexpressing breast tumors

Abstract Trastuzumab treatment has improved the overall survival of HER2 overexpressing breast cancer patients. However, many of these patients will eventually become resistant to treatment. It is unclear what mechanisms may contribute to resistance to Trastuzumab. In this study we have tested the hypothesis that targeting of the FKHR transcription factor FOXO1A in HER2 overexpressing breast tumor cells, can overcome the Trastuzumab resistance in vitro. We have demonstrated that overexpression of HER2 leads to activation of PI3K/Akt pathway and subsequent inactivation of FOXO1A in HER2 overexpressing breast cancer cells, SKBR3. In wildtype SKBR3 cells, Trastuzumab downregulates active Akt (pAkt) and increases FOXO1A expression that leads to increase in p27kip1, and decrease in cyclin D1, and finally inhibits SKBR3 cell proliferation. In contrast, the effect of Trastuzumab was eliminated by the reduction of FOXO1A in HER2 overexpressing cells with constitutively active Akt1 (Clone SKBR3/AA28). The down-regulation of FOXO1A in SKBR3/AA28 resulted in nuclear export of p27kip1. Blocking the constitutively active Akt by a specific Akt/protein kinase B signaling inhibitor-2 (API-2) significantly increased FOXO1A expression and rendered the SKBR3/AA28 cells more responsive to Trastuzumab induced growth inhibition. Re-activation of FOXO1A by stable transfection or transient transfection also restored the growth inhibitory effects of Trastuzumab in SKBR3/AA28 cells. In summary, Trastuzumab can inhibit proliferation of HER2 overexpressing breast cancer cells by re-activating FOXO1A through inhibition the PI3K/Akt pathway. FOXO1A may therefore serve as a target for HER2 overexpressing breast tumors, and its activation or increased expression may overcome resistance to Trastuzumab treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 612.

Stable RNA interference of ErbB-2 gene synergistic with epirubicin suppresses breast cancer growth in vitro and in vivo

Overexpression of human epidermal growth factor receptor-2 (Her2, ErbB-2) contributes to the progression and metastasis of breast cancer, implying that Her2 gene is a suitable target of RNA interference (RNAi) for breast cancer therapy. Here, we employed plasmid-mediated expression of 2 different Her2-shRNAs (pU6-Her2shRNAs) efficiently silenced the target gene expression on Her2 expressing SKBR-3 breast cancer cells in both mRNA and protein levels. Consequently, pU6-Her2shRNA increased apoptosis and reduced proliferation of SKBR-3 cells assayed by TUNEL and MTT, respectively. In vivo, intra-tumor injection of pU6-Her2shRNA inhibited the growth of SKBR-3 tumors inoculated subcutaneously in nude mice. Furthermore, pU6-Her2shRNA synergized the tumor suppression effect of epirubicin to SKBR-3 cells in vitro and implanted subcutaneously in nude mice. Therefore, we concluded that stable silencing of Her2 gene expression with plasmid expressing shRNA may hold great promise as a novel therapy for Her2 expressing breast cancers alone or in combination with anthracycline chemotherapy.

Herceptin‐induced inhibition of ErbB2 signaling involves reduced phosphorylation of Akt but not endocytic down‐regulation of ErbB2

The anti-proliferative effect of the ErbB2 specific antibody Herceptin in cells overexpressing ErbB2 has previously been explained by endocytic downregulation of ErbB2. However, in the following, we demonstrate that Herceptin inhibited proliferation of ErbB2 overexpressing cells without downregulating ErbB2. Herceptin did also not induce endocytosis of ErbB2. Herceptin was found to blunt proliferation of SKBr3 cells overexpressing EGFR, ErbB2, and ErbB3 and expressing functional PTEN, probably by recruiting PTEN to the plasma membrane. Akt was found to be constitutively phosphorylated both in SKBr3 cells overexpressing EGFR, ErbB2 and ErbB3, and in SKOv3 cells, overexpressing EGFR and ErbB2. However, phosphorylation of Akt was inhibited by Herceptin only in SKBr3 cells. SKOv3 cells, which lack the tumour suppressor protein Ras homolog member I, was found to have constitutively phosphorylated mitogen activated protein kinase and functionally increased Ras activity. SKOv3 cells further had low expression levels of PTEN. We thus confirm that the anti-proliferative effect of Herceptin in SKBr3 cells is due to recruitment of PTEN to the plasma membrane and conclude that Herceptin does not blunt phosphatidyl inositol 3 kinase-induced growth in cells with constitutive Ras activity. We further conclude that endocytic downregulation of ErbB2 does not contribute to Herceptin's antiproliferative effect.