Baruch Erez

Metastasis is regulated via microRNA-200/ZEB1 axis control of tumour cell PD-L1 expression and intratumoral immunosuppression

Immunosuppression of tumor-infiltrating lymphocytes (TIL) is a common feature of advanced cancer, but its biological basis has remained obscure. We demonstrate here a molecular link between epithelial-to-mesenchymal transition (EMT) and CD8+ TIL immunosuppression, two key drivers of cancer progression. We show that microRNA-200 (miR-200), a cell-autonomous suppressor of EMT and metastasis, targets PD-L1. Moreover, ZEB1, an EMT activator and transcriptional repressor of miR-200, relieves miR-200 repression of PD-L1 on tumor cells, leading to CD8+ T cell immunosuppression and metastasis. These findings are supported by robust correlations between the EMT score, miR-200 levels and PD-L1 expression in multiple human lung cancer datasets. In addition to revealing a link between EMT and T cell dysfunction, these findings also show that ZEB1 promotes metastasis through a heretofore unappreciated cell non-autonomous mechanism, and suggest that subgroups of patients in whom malignant progression is driven by EMT activators may respond to treatment with PD-L1 antagonists.

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.

KDM2A promotes lung tumorigenesis by epigenetically enhancing ERK1/2 signaling

Epigenetic dysregulation has emerged as a major contributor to tumorigenesis. Histone methylation is a well-established mechanism of epigenetic regulation that is dynamically modulated by histone methyltransferases and demethylases. The pathogenic role of histone methylation modifiers in non-small cell lung cancer (NSCLC), which is the leading cause of cancer deaths worldwide, remains largely unknown. Here, we found that the histone H3 lysine 36 (H3K36) demethylase KDM2A (also called FBXL11 and JHDM1A) is frequently overexpressed in NSCLC tumors and cell lines. KDM2A and its catalytic activity were required for in vitro proliferation and invasion of KDM2A-overexpressing NSCLC cells. KDM2A overexpression in NSCLC cells with low KDM2A levels increased cell proliferation and invasiveness. KDM2A knockdown abrogated tumor growth and invasive abilities of NSCLC cells in mouse xenograft models. We identified dual-specificity phosphatase 3 (DUSP3) as a key KDM2A target gene and found that DUSP3 dephosphorylates ERK1/2 in NSCLC cells. KDM2A activated ERK1/2 through epigenetic repression of DUSP3 expression via demethylation of dimethylated H3K36 at the DUSP3 locus. High KDM2A levels correlated with poor prognosis in NSCLC patients. These findings uncover an unexpected role for a histone methylation modifier in activating ERK1/2 in lung tumorigenesis and metastasis, suggesting that KDM2A may be a promising therapeutic target in NSCLC.

CXCR2 expression in tumor cells is a poor prognostic factor and promotes invasion and metastasis in lung adenocarcinoma

CXCR2 in non-small cell lung cancer (NSCLC) has been studied mainly in stromal cells and is known to increase tumor inflammation and angiogenesis. Here, we examined the prognostic importance of CXCR2 in NSCLC and the role of CXCR2 and its ligands in lung cancer cells. The effect of CXCR2 expression on tumor cells was studied using stable knockdown clones derived from a murine KRAS/p53-mutant lung adenocarcinoma cell line with high metastatic potential and an orthotopic syngeneic mouse model and in vitro using a CXCR2 small-molecule antagonist (SB225002). CXCR2 protein expression was analyzed in tumor cells from 262 NSCLC. Gene expression profiles for CXCR2 and its ligands (CXCR2 axis) were analyzed in 52 human NSCLC cell lines and 442 human lung adenocarcinomas. Methylation of CXCR2 axis promoters was determined in 70 human NSCLC cell lines. Invasion and metastasis were decreased in CXCR2 knockdown clones in vitro and in vivo. SB225002 decreased invasion in vitro. In lung adenocarcinomas, CXCR2 expression in tumor cells was associated with smoking and poor prognosis. CXCR2 axis gene expression profiles in human NSCLC cell lines and lung adenocarcinomas defined a cluster driven by CXCL5 and associated with smoking, poor prognosis, and RAS pathway activation. Expression of CXCL5 was regulated by promoter methylation. The CXCR2 axis may be an important target in smoking-related lung adenocarcinoma.

Combined MEK and VEGFR Inhibition in Orthotopic Human Lung Cancer Models Results in Enhanced Inhibition of Tumor Angiogenesis, Growth, and Metastasis

Purpose: Ras/Raf/mitogen-activated protein–extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK signaling is critical for tumor cell proliferation and survival. Selumetinib is a potent, selective, and orally available MEK1/2 inhibitor. In this study, we evaluated the therapeutic efficacy of selumetinib alone or with cediranib, an orally available potent inhibitor of all three VEGF receptor (VEGFR) tyrosine kinases, in murine orthotopic non–small cell lung carcinoma (NSCLC) models. Experimental Design: NCI-H441 or NCI-H460 KRAS-mutant human NSCLC cells were injected into the lungs of mice. Mice were randomly assigned to treatment with selumetinib, cediranib, paclitaxel, selumetinib plus cediranib, or control. When controls became moribund, all animals were sacrificed and assessed for lung tumor burden and locoregional metastasis. Lung tumors and adjacent normal tissues were subjected to immunohistochemical analyses. Results: Selumetinib inhibited lung tumor growth and, particularly at higher dose, reduced locoregional metastasis, as did cediranib. Combining selumetinib and cediranib markedly enhanced their antitumor effects, with near complete suppression of metastasis. Immunohistochemistry of tumor tissues revealed that selumetinib alone or with cediranib reduced ERK phosphorylation, angiogenesis, and tumor cell proliferation and increased apoptosis. The antiangiogenic and apoptotic effects were substantially enhanced when the agents were combined. Selumetinib also inhibited lung tumor VEGF production and VEGFR signaling. Conclusions: In this study, we evaluated therapy directed against MEK combined with antiangiogenic therapy in distinct orthotopic NSCLC models. MEK inhibition resulted in potent antiangiogenic effects with decreased VEGF expression and signaling. Combining selumetinib with cediranib enhanced their antitumor and antiangiogenic effects. We conclude that combining selumetinib and cediranib represents a promising strategy for the treatment of NSCLC. Clin Cancer Res; 18(6); 1641–54. ©2012 AACR.

Treatment with HIF-1α Antagonist PX-478 Inhibits Progression and Spread of Orthotopic Human Small Cell Lung Cancer and Lung Adenocarcinoma in Mice

Introduction: PX-478 is a potent small-molecule inhibitor of hypoxia-inducible factor 1&agr; (HIF-1&agr;). In prior preclinical studies, it had antitumor activity against various solid tumors in subcutaneous xenografts but had no measurable activity against a non-small cell lung cancer (NSCLC) xenograft. To determine the effectiveness of PX-478 against lung tumors, we investigated HIF-1&agr; expression in several lung cancer cell lines, both in vitro and in vivo, and treated orthotopic mouse models of human lung cancer with PX-478. Methods: Cells from two human lung adenocarcinoma cell models (PC14-PE6 and NCI-H441) or two human small cell lung cancer (SCLC) models (NCI-H187 and NCI-N417) were injected into the left lungs of nude mice and were randomized 16 to 18 days after injection with daily oral treatment with PX-478 or vehicle for 5 days. Results: In the PC14-PE6 NSCLC model, treatment with 20 mg/kg PX-478 significantly reduced the median primary lung tumor volume by 87% (p = 0.005) compared with the vehicle-treated group. PX-478 treatment also markedly reduced mediastinal metastasis and prolonged survival. Similar results were obtained in a second NSCLC model. In SCLC models, PX-478 was even more effective. In the NCI-H187 model, the median primary lung tumor volume was reduced by 99% (p = 0.0001). The median survival duration was increased by 132%. In the NCI-N417 model, the median primary lung tumor volume was reduced by 97% (p = 0.008). Conclusions: We demonstrated that the PX-478, HIF-1&agr; inhibitor, had significant antitumor activity against two orthotopic models of lung adenocarcinomas and two models of SCLC. These results suggest the inclusion of lung cancer patients in phase I clinical trials of PX-478.

Fibulin-2 is a driver of malignant progression in lung adenocarcinoma.

The extracellular matrix of epithelial tumors undergoes structural remodeling during periods of uncontrolled growth, creating regional heterogeneity and torsional stress. How matrix integrity is maintained in the face of dynamic biophysical forces is largely undefined. Here we investigated the role of fibulin-2, a matrix glycoprotein that functions biomechanically as an inter-molecular clasp and thereby facilitates supra-molecular assembly. Fibulin-2 was abundant in the extracellular matrix of human lung adenocarcinomas and was highly expressed in tumor cell lines derived from mice that develop metastatic lung adenocarcinoma from co-expression of mutant K-ras and p53. Loss-of-function experiments in tumor cells revealed that fibulin-2 was required for tumor cells to grow and metastasize in syngeneic mice, a surprising finding given that other intra-tumoral cell types are known to secrete fibulin-2. However, tumor cells grew and metastasized equally well in Fbln2-null and -wild-type littermates, implying that malignant progression was dependent specifically upon tumor cell-derived fibulin-2, which could not be offset by other cellular sources of fibulin-2. Fibulin-2 deficiency impaired the ability of tumor cells to migrate and invade in Boyden chambers, to create a stiff extracellular matrix in mice, to cross-link secreted collagen, and to adhere to collagen. We conclude that fibulin-2 is a driver of malignant progression in lung adenocarcinoma and plays an unexpected role in collagen cross-linking and tumor cell adherence to collagen.

Abstract 3632: Addition of the HIF-1α inhibitor PX-478 enhances the therapeutic efficacy of EGFR inhibitors in an orthotopic human lung adenocarcinoma model

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Introduction: Multiple studies demonstrate that EGFR signaling confers a survival advantage under hypoxic conditions by inducing VEGF expression through HIF-1α dependent and independent mechanisms. However, clinically EGFR inhibition alone is not always effective in NSCLC tumors particularly for those tumors that express a mutant K-Ras gene. We therefore studied the effects of PX-478, a small molecule inhibitor of HIF-1α, in combination with the EGFR inhibitors erlotinib and cetuximab in human lung adenocarcinomas that carry an activating K-Ras mutation growing orthotopically in mice. Methods: NCI-H441 human lung adenocarcinoma cells (1×106), were injected into the left lungs of nude mice. Mice were randomized (10/group) 20 days after injection, when lung tumors were visible in a subset of 4 mice, to treatment with vehicle, PX-478 (20 mg/kg/day for 5 days), erlotinib (50mg/kg/day), cetuximab (1ug/mouse, every third day) or any combination of the drugs. The experiment was terminated when control mice became moribund 63 days after injection. Mice were sacrificed and assessed for lung tumor burden, pleural effusion and metastasis. Lung tumors and adjacent normal tissues were collected for immunochistochemical analyses. Results: Treatment of mice with either cetuximab or PX-478 as monotherapy reduced the median primary lung tumor volume by 44% (p=0.006) and 77% (p<0.0001), respectively, whilst treatment with erlotinib was only marginally effective in this model. The combination of PX-478 with erlotinib or cetuximab was significantly superior to monotherapy with a reduction of lung tumor volume by 95% (p<0.0001) and 90% (p<0.0001) respectively, as compared to control. PX-478 monotherapy reduced the incidence of mediastinal metastasis by 80% (p=0.0001) while EGFR inhibition had no significant effect. Combined therapy with PX-478 and erlotinib or cetuximab further reduced metastasis. Trimodality therapy completely prevented mediastinal metastasis and reduced tumor volume by 97% (p<0.0001). Immuno-histochemical studies of lung tumors are now being completed to assess tumor and endothelial cell proliferation and apoptosis, microvessel density, and the expression of EGFR and HIF-1α. Conclusions: We have shown that the inclusion of the HIF-1α inhibitor PX-478 enhances the therapeutic efficacy of EGFR inhibitors in a human lung adenocarcinoma harboring a K-Ras mutation. These data suggest that HIF-1α antagonism could be an effective strategy to overcome the relative resistance to EGFR inhibition that has been observed in K-Ras mutated NSCLC tumors in the clinic. Our data provide the basis for a combined modality treatment approach with these two agents for lung cancer. 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 3632.

Abstract 5146: The histone demethylase KDM2A is a new promoter of tumorigenesis, drug target and negative prognostic biomarker for non-small cell lung cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The molecular etiology of non-small cell lung cancer (NSCLC) is heterogeneous and mostly dominated by alterations in kinase signaling pathways (i.e. KRAS, EGFR, EML4-ALK, PI3K, MEK1). Epigenetic modifiers, including histone methyltransferase and demethylases, have emerged as important regulators of oncogenic phenotypes in a small but growing number of tumor types, but the pathogenic role of histone methylation modifiers in NSCLC is largely unknown. We found that the histone H3 lysine 36 (H3K36) demethylase KDM2A is a novel oncogenic promoter of NSCLC. In our analysis KDM2A is frequently overexpressed in NSCLC cell lines and patient samples (≥14% in NSCLC tumors), and high expression levels of KDM2A correlate with poor prognosis in three independent patient populations from the USA and Asia. KDM2A knockdown by RNAi in KDM2A overexpressing cell lines inhibits proliferation and invasiveness of NSCLC cells in vitro and in three mouse xenograft models (subcutaneous, intravenous, and orthotopic models). Consistently, KDM2A overexpression promotes these cellular characteristics in NSCLC cell lines with low endogenous KDM2A levels. Rescue experiments using KDM2A-depleted cells showed that ectopic expression of wild-type KDM2A, but not its catalytic mutant mKDM2A, restored proliferation and invasion. These results indicate that the effect of KDM2A on oncogenic phenotypes is largely dependent on its catalytic activity and validate it as drug target for development of small molecule inhibitors. Mechanistically, we uncovered, that KDM2A activates the MAPK signaling pathway (ERK1/2) by transcriptionally repressing the MAPK phosphatase DUSP3. In summary, these novel findings indicate that KDM2A overexpression promotes NSCLC tumor growth and invasion. Our results provide new insights into how the dysregulation of an epigenetic enzyme can be coupled to activation of the ERK1/2 signaling pathway to promote NSCLC tumorigenesis and suggest that KDM2A is a promising anti-cancer therapeutic drug target for KDM2A-overexpressing NSCLC patients. Citation Format: Klaus W. Wagner, Hunain Alam, Shilpa S. Dhar, Uma Giri, Na Li, Yongkun Wei, Tina Cascone, Dipak Giri, Jae-Hwan Kim, Yuanqing Ye, Asha Multani, Chia-Hsin Chan, Baruch Erez, Babita Saigal, Hui-Kuan Lin, Xifeng Wu, Mien-Chie Hung, John Heymach, MinGyu Lee. The histone demethylase KDM2A is a new promoter of tumorigenesis, drug target and negative prognostic biomarker for non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5146. doi:10.1158/1538-7445.AM2014-5146

Abstract 3269: VEGF inhibitor resistance is associated with stromal EGFR activation and normalized revascularization in an orthotopic model of lung adenocarcinoma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Therapeutic resistance to angiogenesis inhibitors represents a major obstacle in the treatment of non-small cell lung cancer (NSCLC). Previously, we reported that acquired resistance of subcutaneous murine models of NSCLC to the anti-vascular endothelial growth factor (VEGF) antibody bevacizumab (BV) was mediated by upregulation and activation of the epidermal growth factor receptor (EGFR) signaling pathway in stromal cells. Here, we investigated the effects of BV and dual VEGFR/EGFR inhibition, and the mechanisms of therapeutic resistance in H441 NSCLC orthotopic tumors. Short-term BV treatment (2 weeks) resulted in a significant tumor volume reduction compared with vehicle-treated tumors (p=0.026). For survival analysis, tumor-bearing mice were randomized to receive vehicle, erlotinib (E), BV, erlotinib + BV (E + B), or the VEGFR/EGFR inhibitor vandetanib (V), until moribund. All therapies significantly prolonged survival compared with vehicle (p<0.0001; E vs. vehicle p<0.05). Long-term BV administration improved survival compared with E (median survival 77 vs. 58 days, p=0.00015); however, both E + BV and V treatments (median survival 101 and 91 days, respectively) prolonged survival compared with E or BV alone (p=0.0001 E + BV vs. E or BV; p=0.0004 V vs. E; p=0.022 V vs. BV). Microvessel density (MVD) was strongly decreased in BV-sensitive tumors compared with 2 week vehicle treatment (p=0.0008); however, tumors resistant to BV or dual VEGFR/EGFR inhibition showed revascularization with increased MVD compared with BV-sensitive tumors (p=0.045). In erlotinib-resistant group MVD was significantly lower than in BV-resistant tumors (p=0.034). Levels of p-EGFR increased in BV-resistant tumors compared with controls (p=0.039) and co-localized with the stroma supporting large, normalized vessels. This signaling was suppressed in tumors resistant to VEGFR/EGFR targeting compared with both controls and BV-resistant tumors (p=0.0001 E + BV vs. vehicle; p=0.0008 E + BV vs. BV; p = 0.011 V vs. vehicle; p=0.009 V vs. BV), demonstrating persistent EGFR blockade with treatment. Pericyte coverage increased in BV-resistant tumors compared with controls and BV-sensitive tumors (p=0.003 BV vs. vehicle; p<0.0001 BV progression vs. BV 2 weeks). In tumors resistant to erlotinib or VEGFR/EGFR inhibition, pericyte coverage was reduced to levels comparable to controls (p=0.001 E vs. BV; p=0.054 E + BV vs. BV; p=0.007 V vs. BV). These findings demonstrate that in an orthotopic NSCLC model, resistance to BV is associated with tumor revascularization, featuring large, pericyte-covered vessels with increased perivascular EGFR activation. Dual VEGFR/EGFR blockade abrogates the BV-induced increase in pericyte coverage and delays the emergence of resistance. Stromal EGFR may contribute to VEGF inhibitor resistance through activation on perivascular cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3269. doi:10.1158/1538-7445.AM2011-3269