Monique B. Nilsson

An Epithelial–Mesenchymal Transition Gene Signature Predicts Resistance to EGFR and PI3K Inhibitors and Identifies Axl as a Therapeutic Target for Overcoming EGFR Inhibitor Resistance

Purpose: Epithelial–mesenchymal transition (EMT) has been associated with metastatic spread and EGF receptor (EGFR) inhibitor resistance. We developed and validated a robust 76-gene EMT signature using gene expression profiles from four platforms using non–small cell lung carcinoma (NSCLC) cell lines and patients treated in the Biomarker-Integrated Approaches of Targeted Therapy for Lung Cancer Elimination (BATTLE) study. Experimental Design: We conducted an integrated gene expression, proteomic, and drug response analysis using cell lines and tumors from patients with NSCLC. A 76-gene EMT signature was developed and validated using gene expression profiles from four microarray platforms of NSCLC cell lines and patients treated in the BATTLE study, and potential therapeutic targets associated with EMT were identified. Results: Compared with epithelial cells, mesenchymal cells showed significantly greater resistance to EGFR and PI3K/Akt pathway inhibitors, independent of EGFR mutation status, but more sensitivity to certain chemotherapies. Mesenchymal cells also expressed increased levels of the receptor tyrosine kinase Axl and showed a trend toward greater sensitivity to the Axl inhibitor SGI-7079, whereas the combination of SGI-7079 with erlotinib reversed erlotinib resistance in mesenchymal lines expressing Axl and in a xenograft model of mesenchymal NSCLC. In patients with NSCLC, the EMT signature predicted 8-week disease control in patients receiving erlotinib but not other therapies. Conclusion: We have developed a robust EMT signature that predicts resistance to EGFR and PI3K/Akt inhibitors, highlights different patterns of drug responsiveness for epithelial and mesenchymal cells, and identifies Axl as a potential therapeutic target for overcoming EGFR inhibitor resistance associated with the mesenchymal phenotype. Clin Cancer Res; 19(1); 279–90. ©2012 AACR.

Interleukin-6, secreted by human ovarian carcinoma cells, is a potent proangiogenic cytokine.

Angiogenesis, a key rate-limiting step in the growth and dissemination of malignant tumors, is regulated by the balance between positive and negative effectors. Recent studies indicate that the pleiotropic cytokine interleukin-6 (IL-6) may contribute to the vascularization of some tumors by disrupting the equilibrium between positive and negative angiogenic regulatory molecules. We determined whether IL-6 participates in the angiogenesis observed during the progression of ovarian carcinoma. We measured IL-6 production by human ovarian cancer cell lines in vitro and in vivo. Not all cell lines secreted IL-6 in vitro; however, when the cell lines were implanted into the peritoneal cavity of female nude mice, every line secreted IL-6. Most human ovarian carcinoma cell lines tested secreted significant levels of the soluble IL-6 receptor (sIL-6R). Endothelial cell lines established from the ovary and mesentery of female H-2K(b)-tsA58 mice were tested for response to IL-6. Both endothelial cell lines expressed the IL-6R and their stimulation with the exogenous ligand significantly enhanced cell migration and activated the downstream signaling molecule signal transducers and activators of transcription 3. Dual immunohistochemical staining for IL-6R and CD31 revealed IL-6R expression on human endothelial cells within normal ovary and carcinoma specimens. Gelfoam sponges containing 0.4% agarose and IL-6 or basic fibroblast growth factor and implanted into the subcutis of BALB/c mice were vascularized to the same extent. Collectively, the data indicate that ovarian tumor cells secreted IL-6, a highly angiogenic cytokine that supports progression of disease.

Combined Vascular Endothelial Growth Factor Receptor and Epidermal Growth Factor Receptor (EGFR) Blockade Inhibits Tumor Growth in Xenograft Models of EGFR Inhibitor Resistance

Purpose: The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) gefitinib and erlotinib benefit some non–small cell lung cancer (NSCLC) patients, but most do not respond (primary resistance) and those who initially respond eventually progress (acquired resistance). EGFR TKI resistance is not completely understood and has been associated with certain EGFR and K-RAS mutations and MET amplification. Experimental Design: We hypothesized that dual inhibition of the vascular endothelial growth factor (VEGF) and EGFR pathways may overcome primary and acquired resistance. We investigated the VEGF receptor/EGFR TKI vandetanib, and the combination of bevacizumab and erlotinib in vivo using xenograft models of EGFR TKI sensitivity, primary resistance, and three models of acquired resistance, including models with mutated K-RAS and secondary EGFR T790M mutation. Results: Vandetanib, gefitinib, and erlotinib had similar profiles of in vitro activity and caused sustained tumor regressions in vivo in the sensitive HCC827 model. In all four resistant models, vandetanib and bevacizumab/erlotinib were significantly more effective than erlotinib or gefitinib alone. Erlotinib resistance was associated with a rise in both host and tumor-derived VEGF but not EGFR secondary mutations in the KRAS mutant-bearing A549 xenografts. Dual inhibition reduced tumor endothelial proliferation compared with VEGF or EGFR blockade alone, suggesting that the enhanced activity of dual inhibition is due at least in part to antiendothelial effects. Conclusion: These studies suggest that erlotinib resistance may be associated with a rise in both tumor cell and host stromal VEGF and that combined blockade of the VEGFR and EGFR pathways can abrogate primary or acquired resistance to EGFR TKIs. This approach merits further evaluation in NSCLC patients.

Proteomic Profiling Identifies Dysregulated Pathways in Small Cell Lung Cancer and Novel Therapeutic Targets Including PARP1

UNLABELLED Small cell lung cancer (SCLC) is an aggressive malignancy distinct from non-small cell lung cancer (NSCLC) in its metastatic potential and treatment response. Using an integrative proteomic and transcriptomic analysis, we investigated molecular differences contributing to the distinct clinical behavior of SCLCs and NSCLCs. SCLCs showed lower levels of several receptor tyrosine kinases and decreased activation of phosphoinositide 3-kinase (PI3K) and Ras/mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK) pathways but significantly increased levels of E2F1-regulated factors including enhancer of zeste homolog 2 (EZH2), thymidylate synthase, apoptosis mediators, and DNA repair proteins. In addition, PARP1, a DNA repair protein and E2F1 co-activator, was highly expressed at the mRNA and protein levels in SCLCs. SCLC growth was inhibited by PARP1 and EZH2 knockdown. Furthermore, SCLC was significantly more sensitive to PARP inhibitors than were NSCLCs, and PARP inhibition downregulated key components of the DNA repair machinery and enhanced the efficacy of chemotherapy. SIGNIFICANCE SCLC is a highly lethal cancer with a 5-year survival rate of less than 10%. To date, no molecularly targeted agents have prolonged survival in patients with SCLCs. As a step toward identifying new targets, we systematically profiled SCLCs with a focus on therapeutically relevant signaling pathways. Our data reveal fundamental differences in the patterns of pathway activation in SCLCs and NSCLCs and identify several potential therapeutic targets for SCLCs, including PARP1 and EZH2. On the basis of these results, clinical studies evaluating PARP and EZH2 inhibition, together with chemotherapy or other agents, warrant further investigation.

Upregulated stromal EGFR and vascular remodeling in mouse xenograft models of angiogenesis inhibitor–resistant human lung adenocarcinoma

Angiogenesis is critical for tumor growth and metastasis, and several inhibitors of angiogenesis are currently in clinical use for the treatment of cancer. However, not all patients benefit from antiangiogenic therapy, and those tumors that initially respond to treatment ultimately become resistant. The mechanisms underlying this, and the relative contributions of tumor cells and stroma to resistance, are not completely understood. Here, using species-specific profiling of mouse xenograft models of human lung adenocarcinoma, we have shown that gene expression changes associated with acquired resistance to the VEGF inhibitor bevacizumab occurred predominantly in stromal and not tumor cells. In particular, components of the EGFR and FGFR pathways were upregulated in stroma, but not in tumor cells. Increased activated EGFR was detected on pericytes of xenografts that acquired resistance and on endothelium of tumors with relative primary resistance. Acquired resistance was associated with a pattern of pericyte-covered, normalized revascularization, whereas tortuous, uncovered vessels were observed in relative primary resistance. Importantly, dual targeting of the VEGF and EGFR pathways reduced pericyte coverage and increased progression-free survival. These findings demonstrated that alterations in tumor stromal pathways, including the EGFR and FGFR pathways, are associated with, and may contribute to, resistance to VEGF inhibitors and that targeting these pathways may improve therapeutic efficacy. Understanding stromal signaling may be critical for developing biomarkers for angiogenesis inhibitors and improving combination regimens.

Stress Hormones Regulate Interleukin-6 Expression by Human Ovarian Carcinoma Cells through a Src-dependent Mechanism

Recent studies have demonstrated that chronic stress promotes tumor growth, angiogenesis, and metastasis. In ovarian cancer, levels of the pro-angiogenic cytokine, interleukin 6 (IL-6), are known to be elevated in individuals experiencing chronic stress, but the mechanism(s) by which this cytokine is regulated and its role in tumor growth remain under investigation. Here we show that stress hormones such as norepinephrine lead to increased expression of IL-6 mRNA and protein levels in ovarian carcinoma cells. Furthermore, we demonstrate that norepinephrine stimulation activates Src tyrosine kinase and this activation is required for increased IL-6 expression. These results demonstrate that stress hormones activate signaling pathways known to be critical in ovarian tumor progression.

Epidermal Growth Factor Receptor Inhibitors in Development for the Treatment of Non–Small Cell Lung Cancer

The epidermal growth factor receptor (EGFR) inhibitors erlotinib, gefitinib, and cetuximab have undergone extensive clinical testing and have established clinical activity in non–small cell lung cancer and other types of solid tumors. A number of newer inhibitors are currently in clinical development with different spectra of activity or mechanisms of receptor inhibition. These include monoclonal antibodies, such as panitumumab and matuzumab; dual inhibitors of EGFR and vascular endothelial growth factor receptor, such as ZD6474 and AEE788; inhibitors of multiple EGFR family members, such as lapatinib; and irreversible inhibitors, such as canertinib and HKI272. Preclinical studies suggest that several of these agents may have activity in tumors refractory to erlotinib or gefitinib. Among these agents, ZD6474 has undergone the most extensive clinical testing. The antitumor activity of ZD6474 in these two randomized phase II clinical trials in patients with non–small cell lung cancer was felt to be sufficiently promising to warrant phase III clinical testing. Several of the other EGFR inhibitors are also undergoing advanced clinical testing, either alone or in combination with other agents. EGFR has now been validated as a clinically relevant target, and several different types of agents inhibiting this receptor are currently in development. Future research will be needed to elucidate the role of these agents in patients with EGFR inhibitor–naive and EGFR inhibitor–refractory disease, to define the molecular characteristics that predict response, and to determine whether these drugs should be used in combination with other targeted agents or chemotherapy.

Antivascular Therapy for Orthotopic Human Ovarian Carcinoma through Blockade of the Vascular Endothelial Growth Factor and Epidermal Growth Factor Receptors

Purpose: We determined whether the administration of the tyrosine kinase inhibitor, AEE788, which targets the epidermal growth factor receptor and the vascular endothelial growth factor receptor, alone or in combination with paclitaxel, can inhibit progressive growth of human ovarian carcinoma in the peritoneal cavity of female nude mice. Experimental Design: Western blot analysis and immunohistochemical analysis identified the optimal dose and schedule of AEE788 therapy. In several different experiments, paclitaxel-sensitive and paclitaxel-resistant human ovarian carcinoma cells were injected into the peritoneal cavity of nude mice. Seven days later, treatment with saline (control), AEE788 alone, paclitaxel alone, or a combination of AEE788 and paclitaxel began and continued for 45 days when the mice were necropsied. In independent survival experiments, the mice were necropsied when they became moribund. Results: Oral administration of AEE788 inhibited phosphorylation of the epidermal growth factor receptor and vascular endothelial growth factor receptor for up to 48 hours. Treatment with AEE788 plus paclitaxel significantly reduced tumor weight and increased survival of mice implanted with paclitaxel-sensitive cell lines compared with control mice or mice treated with AEE788 alone or paclitaxel alone. In mice implanted with paclitaxel-resistant cells, the combination therapy also significantly reduced tumor weight but did not prolong survival. The combination therapy induced apoptosis of both tumor cells and tumor-associated endothelial cells. Conclusions: The administration of AEE788 and paclitaxel inhibits the progression of human ovarian carcinoma in the peritoneal cavity of female nude mice, in part, by inducing apoptosis of tumor-associated endothelial cells.

Multiple receptor tyrosine kinases regulate HIF-1α and HIF-2α in normoxia and hypoxia in neuroblastoma: implications for antiangiogenic mechanisms of multikinase inhibitors

Novel treatment approaches are needed for children with advanced neuroblastoma. Studies with neuroblastoma cells have indicated the presence of a hypoxia-driven vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR)-1 autocrine loop modulating hypoxia-inducible factor-1alpha (HIF-1α). Whether other receptor tyrosine kinases (RTKs) are capable of modulating HIF-1α levels and whether RTKs can regulate HIF-2α as well is largely unknown. We evaluated neuroblastoma cell lines for expression of various RTKs. Although cell lines were heterogeneous in the expression of VEGFR-1, -3, c-Kit and RET, most cells expressed PDGFR-α and -β. Ligand-induced activation of multiple RTKs upregulated HIF-1α levels, whereas activation of VEGFR-1 alone upregulated HIF-2α. Multitargeted tyrosine kinase inhibitor sunitinib reduced hypoxia-induced rises in HIF-1α and HIF-2α through mechanisms involving effects on both mRNA levels and protein stability. In addition, sunitinib and sorafenib had direct effects on tumor cell viability in vitro. In a neuroblastoma xenograft model, tumor growth inhibition by sunitinib was associated with inhibition of angiogenesis and reduced HIF-1α levels. These findings show that multiple RTKs may regulate the HIF axis in normoxia and hypoxia and suggest that multikinase inhibitors may exert antiangiogenic effects not only by direct effects on endothelial cells, but also by blocking compensatory hypoxia- and ligand-induced changes in HIF-1α and HIF-2α.

Epidermal growth factor receptor regulates MET levels and invasiveness through hypoxia-inducible factor-1α in non-small cell lung cancer cells

Recent studies have established that amplification of the MET proto-oncogene can cause resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) cell lines with EGFR-activating mutations. The role of non-amplified MET in EGFR-dependent signaling before TKI resistance, however, is not well understood. Using NSCLC cell lines and transgenic models, we demonstrate here that EGFR activation by either mutation or ligand binding increases MET gene expression and protein levels. Our analysis of 202 NSCLC patient specimens was consistent with these observations: levels of MET were significantly higher in NSCLC with EGFR mutations than in NSCLC with wild-type EGFR. EGFR regulation of MET levels in cell lines occurred through the hypoxia-inducible factor (HIF)-1α pathway in a hypoxia-independent manner. This regulation was lost, however, after MET gene amplification or overexpression of a constitutively active form of HIF-1α. EGFR- and hypoxia-induced invasiveness of NSCLC cells, but not cell survival, were found to be MET dependent. These findings establish that, absent MET amplification, EGFR signaling can regulate MET levels through HIF-1α and that MET is a key downstream mediator of EGFR-induced invasiveness in EGFR-dependent NSCLC cells.