HER-2 overexpression differentially alters transforming growth factor-β responses in luminal versus mesenchymal human breast cancer cells

INTRODUCTION: Amplification of the HER-2 receptor tyrosine kinase has been implicated in the pathogenesis and aggressive behavior of approximately 25% of invasive human breast cancers. Clinical and experimental evidence suggest that aberrant HER-2 signaling contributes to tumor initiation and disease progression. Transforming growth factor beta (TGF-β) is the dominant factor opposing growth stimulatory factors and early oncogene activation in many tissues, including the mammary gland. Thus, to better understand the mechanisms by which HER-2 overexpression promotes the early stages of breast cancer, we directly assayed the cellular and molecular effects of TGF-β1 on breast cancer cells in the presence or absence of overexpressed HER-2. METHODS: Cell proliferation assays were used to determine the effect of TGF-β on the growth of breast cancer cells with normal or high level expression of HER-2. Affymetrix microarrays combined with Northern and western blot analysis were used to monitor the transcriptional responses to exogenous TGF-β1 in luminal and mesenchymal-like breast cancer cells. The activity of the core TGF-β signaling pathway was assessed using TGF-β1 binding assays, phospho-specific Smad antibodies, immunofluorescent staining of Smad and Smad DNA binding assays. RESULTS: We demonstrate that cells engineered to over-express HER-2 are resistant to the anti-proliferative effect of TGF-β1. HER-2 overexpression profoundly diminishes the transcriptional responses induced by TGF-β in the luminal MCF-7 breast cancer cell line and prevents target gene induction by a novel mechanism that does not involve the abrogation of Smad nuclear accumulation, DNA binding or changes in c-myc repression. Conversely, HER-2 overexpression in the context of the mesenchymal MDA-MB-231 breast cell line potentiated the TGF-β induced pro-invasive and pro-metastatic gene signature. CONCLUSION: HER-2 overexpression promotes the growth and malignancy of mammary epithelial cells, in part, by conferring resistance to the growth inhibitory effects of TGF-β. In contrast, HER-2 and TGF-β signaling pathways can cooperate to promote especially aggressive disease behavior in the context of a highly invasive breast tumor model

MAPK pathway inhibition induces MET and GAB1 levels, priming BRAF mutant melanoma for rescue by hepatocyte growth factor.

Therapeutic resistance is a major obstacle to achieving durable clinical responses with targeted therapies, highlighting a need to elucidate the underlying mechanisms responsible for resistance and identify strategies to overcome this challenge. An emerging body of data implicates the tyrosine kinase MET in mediating resistance to BRAF inhibitors in BRAFV600E mutant melanoma. In this study we observed a dominant role for the HGF/MET axis in mediating resistance to BRAF and MEK inhibitors in models of BRAFV600E and NRAS mutant melanoma. In addition, we showed that MAPK pathway inhibition induced rapid increases in MET and GAB1 levels, providing novel mechanistic insight into how BRAFV600E mutant melanoma is primed for HGF-mediated rescue. We also determined that tumor-derived HGF, not systemic HGF, may be required to convey resistance to BRAF inhibition in vivo and that resistance could be reversed following treatment with AMG 337, a selective MET inhibitor. In summary, these findings support the clinical evaluation of MET-directed targeted therapy to circumvent resistance to BRAF and MEK inhibitors in BRAFV600E mutant melanoma. In addition, the induction of MET following treatment with BRAF and MEK inhibitors has the potential to serve as a predictive biomarker for identifying patients best suited for MET inhibitor combination therapy

Ganitumab (AMG 479) inhibits IGF-II-dependent ovarian cancer growth and potentiates platinum-based chemotherapy.

PurposeInsulin-like growth factor 1 receptor (IGF-IR) has been implicated in the pathogenesis of ovarian cancer. Ganitumab is an investigational, fully human monoclonal antibody against IGF-IR. Here, we explore the therapeutic potential of ganitumab for the treatment of ovarian cancer.Experimental designThe effects of ganitumab were tested in vitro against a panel of 23 established ovarian cancer cell lines. The ability of ganitumab to inhibit IGF-I-, IGF-II-, and insulin-mediated signaling was examined in vitro and in tumor xenografts using ovarian cancer models displaying IGF-IR/PI3K/AKT pathway activation by two distinct mechanisms, PTEN loss and IGF-II overexpression. Drug interactions between ganitumab and cisplatin, carboplatin, or paclitaxel were studied in vitro and in vivo.ResultsIn vitro, growth inhibition varied significantly among individual ovarian cancer cell lines. IGF-II mRNA and phospho-IGF-IR protein expression were quantitatively correlated with response to ganitumab, and PTEN mutations conferred resistance to ganitumab. Ganitumab potently inhibited baseline and IGF-I-, IGF-II-, and insulin-induced IGF-IR and IGF-IR/insulin hybrid receptor signaling in vitro and in vivo. Synergistic and additive drug interactions were seen for ganitumab and carboplatin or paclitaxel in vitro. Furthermore, ganitumab significantly increased the efficacy of cisplatin in ovarian cancer xenograft models in vivo.ConclusionsThese observations provide a biologic rationale to test ganitumab as a single agent or in combination with carboplatin/cisplatin and paclitaxel in patients with ovarian cancer. Moreover, assessment of tumor expression of IGF-II, phospho-IGF-IR, or PTEN status may help select patients with ovarian cancer who are most likely to benefit from ganitumab. Clin Cancer Res; 20(11); 2947-58. ©2014 AACR

Ganitumab (AMG 479) Inhibits IGF-II–Dependent Ovarian Cancer Growth and Potentiates Platinum-Based Chemotherapy

PURPOSE: Insulin-like growth factor 1 receptor (IGF-IR) has been implicated in the pathogenesis of ovarian cancer. Ganitumab is an investigational, fully human monoclonal antibody against IGF-IR. Here, we explore the therapeutic potential of ganitumab for the treatment of ovarian cancer. EXPERIMENTAL DESIGN: The effects of ganitumab were tested in vitro against a panel of 23 established ovarian cancer cell lines. The ability of ganitumab to inhibit IGF-I–, IGF-II–, and insulin-mediated signaling was examined in vitro and in tumor xenografts using ovarian cancer models displaying IGF-IR/PI3K/AKT pathway activation by two distinct mechanisms, PTEN loss and IGF-II overexpression. Drug interactions between ganitumab and cisplatin, carboplatin, or paclitaxel were studied in vitro and in vivo. RESULTS: In vitro, growth inhibition varied significantly among individual ovarian cancer cell lines. IGF-II mRNA and phospho–IGF-IR protein expression were quantitatively correlated with response to ganitumab, and PTEN mutations conferred resistance to ganitumab. Ganitumab potently inhibited baseline and IGF-I–, IGF-II–, and insulin-induced IGF-IR and IGF-IR/insulin hybrid receptor signaling in vitro and in vivo. Synergistic and additive drug interactions were seen for ganitumab and carboplatin or paclitaxel in vitro. Furthermore, ganitumab significantly increased the efficacy of cisplatin in ovarian cancer xenograft models in vivo. CONCLUSIONS: These observations provide a biologic rationale to test ganitumab as a single agent or in combination with carboplatin/cisplatin and paclitaxel in patients with ovarian cancer. Moreover, assessment of tumor expression of IGF-II, phospho–IGF-IR, or PTEN status may help select patients with ovarian cancer who are most likely to benefit from ganitumab

Context specific effects of HER-2 overexpression on the biological responses and transcription program induced by transforming growth factor (TGF)-β in breast cancer cells

<p><b>Copyright information:</b></p><p>Taken from "HER-2 overexpression differentially alters transforming growth factor-β responses in luminal versus mesenchymal human breast cancer cells"</p><p>Breast Cancer Research 2005;7(6):R1058-R1079.</p><p>Published online 8 Nov 2005</p><p>PMCID:PMC1410754.</p><p>Copyright © 2005 Wilson et al.; licensee BioMed Central Ltd.</p> Phenotypes of the luminal MCF-7 and mesenchymal MDA-MB-231 cells with and without engineered HER-2 overexpression. The epithelial growth pattern of MCF-7 cells is characteristically altered by HER-2 overexpression, which promotes an elongated morphology and increased proliferation rates as well as faster growing tumor xenografts [12,13]. The MDA-MB-231 cells are heterogeneous with the majority of cells, having a spindle shaped morphology. The effect of HER-2 overexpression on the morphological appearance was not dramatic except that significantly more (approximately three times) of the large, flattened round cells were observed. The MDA-MB-231 H2 cells have also been shown to be more metastatic than the MDA-MB-231 CN or parental lines (data not shown). Summary of the TGF-β transcriptional program obtained by Affymetrix microarray profiling of cells treated with recombinant TGF-β1. The number of elements significantly affected (p < 0.01 and fold change greater than ± 1.7 using Rosetta Resolver) are graphed. Light and dark red indicate genes upregulated after 6 h and 24 h, respectively, and light and dark green represent genes downregulated after 6 and 24 h, respectively. Highlighted genes from the dominant functional gene signatures as determined by the gene ontology information found in Source [105] and GeneCards [106]. Red and green indicate TGF-β stimulated and repressed genes, respectively. ECM, extracellular matrix

Divergent transforming growth factor (TGF)-β gene activation and biological responses in MCF-7 and MDA-MB-231 cell lines

<p><b>Copyright information:</b></p><p>Taken from "HER-2 overexpression differentially alters transforming growth factor-β responses in luminal versus mesenchymal human breast cancer cells"</p><p>Breast Cancer Research 2005;7(6):R1058-R1079.</p><p>Published online 8 Nov 2005</p><p>PMCID:PMC1410754.</p><p>Copyright © 2005 Wilson et al.; licensee BioMed Central Ltd.</p> The TGF-β induced transcriptional program is almost entirely non-overlapping in the luminal MCF-7 and the mesenchymal MDA-MB-231 breast cancer cell lines. The number of genes induced and repressed greater than 1.5-fold with a p-value < 0.01 after a 24 h exposure to TGF-β are shown. All of the individual genes commonly affected in both control cell lines (MCF-7 CN and MDA-MB-231 CN) are listed below the diagram. Model for the differential outcomes of TGF-β stimulation in estrogen receptor (ER) positive luminal breast cells compared to ER-negative mesenchymal cells. Model for context specific effects of HER-2 overexpression on TGF-β signaling in ER-positive luminal cells versus ER-negative mesenchymal cells. EMT, epithelial to mesenchymal transition

HER-2 overexpression inhibits transforming growth factor (TGF)-β1 target gene induction in the luminal MCF-7 and ZR-75-1 breast cancer cells

<p><b>Copyright information:</b></p><p>Taken from "HER-2 overexpression differentially alters transforming growth factor-β responses in luminal versus mesenchymal human breast cancer cells"</p><p>Breast Cancer Research 2005;7(6):R1058-R1079.</p><p>Published online 8 Nov 2005</p><p>PMCID:PMC1410754.</p><p>Copyright © 2005 Wilson et al.; licensee BioMed Central Ltd.</p> Northern blot analysis of and expression. The membrane contained total RNA from the indicated CN and H2 cells treated with either diluent control (0 h) or 2 ng/ml TGF-β1 (8 or 24 h). HER-2 overexpression prevents p15induction in MCF-7 and ZR-75-1 cells by TGF-β1. Western blot analysis of total cellular extracts prepared after treatment with diluent control (0 h) or 2 ng/ml TGF-β1 (8, 24 or 52 h). Bottom panels are the same membranes re-probed with an antibody to cdk4

HER-2 overexpression modulates transforming growth factor (TGF)-β responses in a cell context dependent manner

<p><b>Copyright information:</b></p><p>Taken from "HER-2 overexpression differentially alters transforming growth factor-β responses in luminal versus mesenchymal human breast cancer cells"</p><p>Breast Cancer Research 2005;7(6):R1058-R1079.</p><p>Published online 8 Nov 2005</p><p>PMCID:PMC1410754.</p><p>Copyright © 2005 Wilson et al.; licensee BioMed Central Ltd.</p> HER-2 overexpression in MCF-7 breast cancer cells blocks TGF-β mediated growth arrest. CN and H2 cells were treated with diluent control (diamonds) or TGF-β1 (0.2 ng/ml (black circles); 0.4 ng/ml (white circles); or 0.8 ng/ml (triangles)) and counted on the indicated days after treatment. Each point is the average of triplicate wells (± standard deviation for error bars). The growth of MCF-7 CN cells is severely inhibited by TGF-β1. MCF-7 H2 cells are resistant to growth inhibition by TGF-β1. MCF-7 CN (diamonds) versus MCF-7 H2 (white circles) cells treated with 0.2 ng/ml TGF-β1. ZR-75-1 cells are resistant to growth inhibition by TGF-β1 without HER-2 overexpression (ZR-75-1/CN (diamonds) versus ZR-75-1/H2 (white circles)) treated with 0.2 ng/ml TGF-β1. TGF-β1 stimulates the growth of MDA-MB-231 H2 cells. MDA MB-231 CN (diamonds) and H2 cells (white cirlcles) were grown for 6 days in the presence of TGF-β1, β2 or β3 (0.01 to 100 ng/ml) or a diluent control. Cells were pulsed with [H] thymidine for the final 24 h of assay and thymidine incorporation was measured. The average counts of triplicate wells for each data point are represented as % of diluent control. TGF-β induces a 'piling' phenotype in MDA-MB-231 that is dependent on HER-2 overexpression. MDA-MB-231 CN and H2 cells were grown for 5 days in the presence of 10 ng/ml TGF-β1 or diluent control. Cells were stained with crystal violet dye and photographed with a 20× (top four panels) or a 60× (bottom two panels) objective