Mary E. Rothstein

HGF and c-Met Participate in Paracrine Tumorigenic Pathways in Head and Neck Squamous Cell Cancer

Purpose: We determined hepatocyte growth factor (HGF) and c-Met expression and signaling in human head and neck squamous cell carcinoma (HNSCC) cells and primary tissues and tested the ability of c-Met tyrosine kinase inhibitors (TKI) to block HGF-induced biological signaling. Experimental Design: Expression and signaling were determined using immunoblotting, ELISA, and immunohistochemistry. Biological end points included wound healing, cell proliferation, and invasion. c-Met TKIs were tested for their ability to block HGF-induced signaling and biological effects in vitro and in xenografts established in nude mice. Results: c-Met was expressed and functional in HNSCC cells. HGF was secreted by HNSCC tumor-derived fibroblasts, but not by HNSCC cells. Activation of c-Met promoted phosphorylation of AKT and mitogen-activated protein kinase as well as release of the inflammatory cytokine interleukin-8. Cell growth and wound healing were also stimulated by HGF. c-Met TKIs blocked HGF-induced signaling, interleukin-8 release, and wound healing. Enhanced invasion of HNSCC cells induced by the presence of tumor-derived fibroblasts was completely blocked with a HGF-neutralizing antibody. PF-2341066, a c-Met TKI, caused a 50% inhibition of HNSCC tumor growth in vivo with decreased proliferation and increased apoptosis within the tumors. In HNSCC tumor tissues, both HGF and c-Met protein were increased compared with expression in normal mucosa. Conclusions: These results show that HGF acts mainly as a paracrine factor in HNSCC cells, the HGF/c-Met pathway is frequently up-regulated and functional in HNSCC, and a clinically relevant c-Met TKI shows antitumor activity in vivo. Blocking the HGF/c-Met pathway may be clinically useful for the treatment of HNSCC.

Cross-Talk between Estrogen Receptor and Epidermal Growth Factor Receptor in Head and Neck Squamous Cell Carcinoma

Purpose: This study aimed to characterize estrogen receptor expression and signaling in head and neck squamous cell carcinoma (HNSCC) cell lines and patient tissues, and to evaluate estrogen receptor and epidermal growth factor (EGF) receptor (EGFR) cross-activation in HNSCC. Experimental Design: Estrogen receptor expression and signaling in HNSCC cell lines were assessed by immunoblotting. In vitro proliferation and invasion were evaluated in HNSCC cell lines in response to estrogen receptor and EGFR ligands or inhibitors. Estrogen receptor and EGFR protein expression in patient tissues was assessed by immunohistochemical staining. Results: Phospho–mitogen-activated protein kinase (P-MAPK) levels were significantly increased following combined estrogen and EGF treatment. Treatment of HNSCC cells with estrogen and EGF significantly increased cell invasion compared with either treatment alone, whereas inhibiting these two pathways resulted in reduced invasion compared with inhibiting either pathway alone. EGFR (P = 0.008) and nuclear estrogen receptor α (ERαnuc; P < 0.001) levels were significantly increased in HNSCC tumors (n = 56) compared with adjacent mucosa (n = 30), whereas nuclear estrogen receptor β (ERβnuc) levels did not differ (P = 0.67). Patients with high ERαnuc and EGFR tumor levels had significantly reduced progression-free survival compared with patients with low tumor ERαnuc and EGFR levels (hazards ratio, 4.09; P = 0.01; Cox proportional hazards). In contrast, high ERβnuc tumor levels were not associated with reduced progression-free survival alone or when combined with EGFR. Conclusions: ERα and ERβ were expressed in HNSCC, and stimulation with estrogen receptor ligands resulted in both cytoplasmic signal transduction and transcriptional activation. Estrogen receptor and EGFR cross-talk was observed. Collectively, these studies indicate that estrogen receptor and EGFR together may contribute to HNSCC development and disease progression. (Clin Cancer Res 2009;15(21):6529–40)

Therapeutic targeting of human hepatocyte growth factor with a single neutralizing monoclonal antibody reduces lung tumorigenesis

The hepatocyte growth factor (HGF)/c-Met signaling pathway is involved in lung tumor growth and progression, and agents that target this pathway have clinical potential for lung cancer treatment. L2G7, a single potent anti-human HGF neutralizing monoclonal antibody, showed profound inhibition of human HGF-induced phosphorylated mitogen-activated protein kinase induction, wound healing, and invasion in lung tumor cells in vitro. Transgenic mice that overexpress human HGF in the airways were used to study the therapeutic efficacy of L2G7 for lung cancer prevention. Mice were treated with the tobacco carcinogen, nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, over 4 weeks. Beginning at week 3, i.p. treatment with 100 μg L2G7 or isotype-matched antibody control, 5G8, was initiated and continued through week 15. The mean number of tumors per mouse in the L2G7-treated group was significantly lower than in the control group (1.58 versus 3.19; P = 0.0005). Proliferative index was decreased by 48% (P = 0.013) in tumors from L2G7-treated mice versus 5G8-treated mice, whereas extent of apoptosis was increased in these same tumors by 5-fold (P = 0.0013). Phosphorylated mitogen-activated protein kinase expression was also significantly decreased by 84% in tumors from L2G7-treated mice versus 5G8-treated mice (P = 0.0003). Tumors that arose in HGF transgenic animals despite L2G7 treatment were more likely to contain mutant K-ras, suggesting that targeting the HGF/c-Met pathway may not be as effective if downstream signaling is activated by a K-ras mutation. These preclinical results show that blocking the HGF/c-Met interaction with a single monoclonal antibody delivered systemically can have profound inhibitory effects on development of lung tumors. [Mol Cancer Ther 2008;7(7):1913–22]

Targeting of Both the c-Met and EGFR Pathways Results in Additive Inhibition of Lung Tumorigenesis in Transgenic Mice.

EGFR and c-Met are both overexpressed in lung cancer and initiate similar downstream signaling, which may be redundant. To determine how frequently ligands that initiate signaling of both pathways are found in lung cancer, we analyzed serum for hepatocyte growth factor (HGF), transforming growth factor-alpha, and amphiregulin (AREG) in lung cancer cases and tobacco-exposed controls. HGF and AREG were both significantly elevated in cases compared to controls, suggesting that both HGF/c-Met and AREG/EGFR pathways are frequently active. When both HGF and AREG are present in vitro, downstream signaling to MAPK and Akt in non-small cell lung cancer (NSCLC) cells can only be completely inhibited by targeting both pathways. To test if dual blockade of the pathways could better suppress lung tumorigenesis in an animal model than single blockade, mice transgenic for airway expression of human HGF were treated with inhibitors of both pathways alone and in combination after exposure to a tobacco carcinogen. Mean tumor number in the group using both the HGF neutralizing antibody L2G7 and the EGFR inhibitor gefitinib was significantly lower than with single agents. A higher tumor K-ras mutation rate was observed with L2G7 alone compared to controls, suggesting that agents targeting HGF may be less effective against mutated K-ras lung tumors. This was not observed with combination treatment. A small molecule c-Met inhibitor decreased formation of both K-ras wild-type and mutant tumors and showed additive anti-tumor effects when combined with gefitinib. Dual targeting of c-Met/EGFR may have clinical benefit for lung cancer.

Signaling pathways involved in cyclooxygenase-2 induction by hepatocyte growth factor in non small-cell lung cancer.

Many studies have suggested a role for the hepatocyte growth factor (HGF)/c-Met pathway in tumorigenesis. Some actions of HGF are believed to be mediated by cyclooxygenase-2 (COX-2), resulting in the production of prostaglandin E2 (PGE2). We examined four c-Met-positive non–small-cell lung cancer (NSCLC) cell lines for effects of HGF on COX-2. HGF increased COX-2 protein expression 3-fold over basal levels. Induction of COX-2 occurred through both the extracellular signal-regulated kinase 1/2 and p38 pathways. HGF treatment caused activation of the activator protein-1, CCAAT/enhancer-binding protein, and cAMP response element-binding protein transcription factors, and COX-2 induction was blocked by actinomycin D. The half-life of COX-2 mRNA was also increased by HGF. HGF stimulation resulted in a 4-fold increase in PGE2 secretion, and treatment of NSCLC cells with exogenous PGE2 significantly increased cell proliferation. The addition of PGE2 to NSCLC cells also led to rapid phosphorylation of c-Met in the absence of HGF, which was blocked by epidermal growth factor receptor (EGFR) inhibition. EGFR ligands were released in response to PGE2. This suggests that secretion of PGE2 induced by HGF/c-Met pathway activation can further activate the c-Met pathway via EGFR in a reinforcing loop that is independent of HGF. HGF and PGE2 each significantly stimulated invasion in NSCLC cells. Cells transiently transfected with c-Met antisense plasmid showed a significant decrease in HGF- or PGE2-induced invasion. PGE2-induced invasion was EGFR-dependent, confirming a link between PGE2, EGFR, and c-Met. Targeting of both the HGF/c-Met and PGE2 pathways with a neutralizing antibody to HGF and celecoxib resulted in enhanced anti-invasion effects in response to HGF.

Prevention of tobacco carcinogen-induced lung cancer in female mice using antiestrogens

Increasing evidence shows that estrogens are involved in lung cancer proliferation and progression, and most human lung tumors express estrogen receptor β (ERβ) as well as aromatase. To determine if the aromatase inhibitor anastrozole prevents development of lung tumors induced by a tobacco carcinogen, alone or in combination with the ER antagonist fulvestrant, ovariectomized female mice received treatments with the tobacco carcinogen 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) along with daily supplements of androstenedione, the substrate for aromatase. Placebo, anastrozole and/or fulvestrant were administered in both an initiation and a promotion protocol of lung tumorigenesis. The combination of fulvestrant and anastrozole given during NNK exposure resulted in significantly fewer NNK-induced lung tumors (mean = 0.5) compared with placebo (mean = 4.6, P < 0.001), fulvestrant alone (mean = 3.4, P < 0.001) or anastrozole alone (mean = 2.8, P = 0.002). A significantly lower Ki67 cell proliferation index was also observed compared with single agent and control treatment groups. Beginning antiestrogen treatment after NNK exposure, when preneoplastic lesions had already formed, also yielded maximum antitumor effects with the combination. Aromatase expression was found mainly in macrophages infiltrating preneoplastic and tumorous areas of the lungs, whereas ERβ was found in both macrophages and tumor cells. Antiestrogens, especially in combination, effectively inhibited tobacco carcinogen-induced murine lung tumorigenesis and may have application for lung cancer prevention. An important source of estrogen synthesis may be inflammatory cells that infiltrate the lungs in response to carcinogens, beginning early in the carcinogenesis process. ERβ expressed by inflammatory and neoplastic epithelial cells in the lung may signal in response to local estrogen production.

Combining the multitargeted tyrosine kinase inhibitor vandetanib with the antiestrogen fulvestrant enhances its antitumor effect in non-small cell lung cancer.

Introduction: Estrogen is known to promote proliferation and to activate the epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC). Vascular endothelial growth factor (VEGF) is a known estrogen responsive gene in breast cancer. We sought to determine whether the VEGF pathway is also regulated by estrogen in lung cancer cells, and whether combining an inhibitor of the ER pathway with a dual vascular endothelial growth factor receptor (VEGFR)/EGFR inhibitor would show enhanced antitumor effects. Methods: We examined activation of EGFR and expression of VEGF in response to &bgr;-estradiol, and the antitumor activity of the multitargeted VEGFR/EGFR/RET inhibitor, vandetanib, when combined with the antiestrogen fulvestrant both in vitro and in vivo. Results: NSCLC cells expressed VEGFR-3 and EGFR. Vandetanib treatment of NSCLC cells resulted in inhibition of EGFR and VEGFR-3 and inhibition of &bgr;-estradiol-induced P-MAPK activation, demonstrating that vandetanib blocks &bgr;-estradiol-induced EGFR signaling. Treatment with &bgr;-estradiol stimulated VEGFA mRNA and protein (p < 0.0001 over baseline), suggesting estrogenic signaling causes heightened VEGFA pathway activation. This estrogenic induction of VEGFA mRNA seems largely dependent on cross-talk with EGFR. Long-term vandetanib treatment also significantly increased ER&bgr; protein expression. The combination of vandetanib with fulvestrant maximally inhibited cell growth compared with single agents (p < 0.0001) and decreased tumor xenograft volume by 64%, compared with 51% for vandetanib (p < 0.05) and 23% for fulvestrant (p < 0.005). Antitumor effects of combination therapy were accompanied by a significant increase in apoptotic cells compared with single agents. Conclusions: Fulvestrant may enhance effects of vandetanib in NSCLC by blocking estrogen-driven activation of the EGFR pathway.

Co-targeting c-Met and COX-2 leads to enhanced inhibition of lung tumorigenesis in a murine model with heightened airway HGF.

Background: The hepatocyte growth factor (HGF)/c-Met pathway is often dysregulated in non–small-cell lung cancer (NSCLC). HGF activation of c-Met induces cyclooxygenase-2 (COX-2), resulting in downstream stimulation by prostaglandin E2 of additional pathways. Targeting both c-Met and COX-2 might lead to enhanced antitumor effects by blocking signaling upstream and downstream of c-Met. Methods: Effects of crizotinib or celecoxib alone or in combination were tested in NSCLC cells in vitro and in mice transgenic for airway expression of human HGF. Results: Proliferation and invasion of NSCLC cells treated with a combination of crizotinib and celecoxib were significantly lower compared with single treatments. Transgenic mice showed enhanced COX-2 expression localized to preneoplastic areas following exposure to the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, which was not present without carcinogen exposure. This shows that COX-2 activity is present during lung tumor development in a high HGF environment. After 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone treatment, a significant decrease in the number of lung tumors per animal was observed after 13-week treatments of crizotinib, celecoxib, or the combination compared with placebo (p < 0.001). With combination treatment, the number of tumors was also significantly lower than single agent treatment (p < 0.001). In the resulting lung tumors, P-c-Met, COX-2, prostaglandin E2, and P-MAPK were significantly downmodulated by combination treatment compared with single treatment. Expression of the epithelial-mesenchymal transition markers E-cadherin and snail was also modulated by combination treatment. Conclusions: In the presence of high HGF, dual inhibition of c-Met and COX-2 may enhance antitumor effects. This combination may have clinical potential in NSCLCs with high HGF/c-Met expression or epithelial-mesenchymal transition phenotype.

Abstract LB-079: Effect of diet-induced obesity on tobacco carcinogen-induced lung carcinogenesis in mice

Obesity has been shown to increase breast cancer risk by causing inflammation through altered estrogen signaling. Whether obesity plays a similar role in lung carcinogenesis is currently not known. However, the role of estrogen signaling in the development and progression of lung cancer is well-established and the inflammation-estrogen signaling axis may underlie the link between obesity and lung cancer risk as well. We used a preclinical animal model to determine the role of diet-induced obesity in promoting lung carcinogenesis and inflammation caused by tobacco carcinogen exposure [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NNK] and the ability of estrogen pathway and/or inflammatory inhibitors to reduce this effect. Female FVB/N mice were administered a high fat (HF; 60kcal% fat; N = 67) or low fat (LF; 10kcal% fat; N = 66) diet ad libitum starting 2 weeks before NNK exposure until sacrifice. NNK was administered for 4 weeks, followed by a 4 week holding period for preneoplasia development. Subsequently, placebo, the aromatase inhibitor anastrozole (0.1mg/kg; p.o. daily) and/or aspirin (25mg/kg; p.o. daily) treatment was administered for 13 weeks after which lung tumors and blood biomarkers were evaluated. Poisson regression was used to assess differences in tumor number and mixed-effects models for differences in tumor size. Overall, the LF diet resulted in more tumors per animal (HF mean number of tumors 8.8, range 1-16; LF mean number of tumors 11.5, range 5-20), but signficantly larger tumors were observed in the HF fed mice compared to LF fed mice in all four treatment groups (HF mean tumor size 0.60 mm2, range 0.05-12.24 mm2; LF mean tumor size 0.37 mm2, range 0.06-2.14 mm2) which correlated with increased tumor Ki67 expression. Anastrozole and aspirin as single agents were significantly more effective at decreasing tumor number in HF fed mice compared to LF fed mice; however, the combined treatment was equally effective in both dietary groups. Inflammatory cell count in bronchoalveolar lavage fluid, tumor infiltrating inflammatory cells and tumor aromatase, COX-2 and nuclear factor kappa-B expression were all increased in HF compared to LF fed mice. This increased inflammatory response may be responsible for the observed increase in tumor size in the HF fed mice. In addition, levels of circulating inflammatory cytokines such as IL-6, IL-10 and TNF-α were significantly down-modulated by anastrozole and also by combined treatment in LF fed mice but not in HF fed mice. These results suggest that while dietary fat may not affect lung cancer incidence, a HF diet may contribute to lung tumor progression potentially through dysregulation of the inflammation-estrogen signaling axis. Supported by R21 CA184611. Citation Format: Brenda Diergaarde, Beatriz Kanterewicz, Mary E. Rothstein, Autumn Gaither-Davis, Shira R. Abberbock, Brenda F. Kurland, Laura P. Stabile. Effect of diet-induced obesity on tobacco carcinogen-induced lung carcinogenesis in mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-079.

Abstract 608: Targeting the estrogen pathway in a male mouse model of lung tumor prevention

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA A hormonal role in the pathology of lung cancer is well documented. We have shown that the aromatase inhibitor anastrozole and the anti-estrogen fulvestrant effectively inhibited tobacco carcinogen-induced lung tumorigenesis in a female mouse model of lung cancer prevention. Additionally, we have shown that inflammatory cells that infiltrate the lungs in response to carcinogens may be a source of estrogen synthesis, and have confirmed aromatase and estrogen receptor expression in a pure macrophage population of differentiated THP-1 cells. These results suggest that targeting the estrogen pathway may be beneficial for both treatment and prevention of lung cancer. There is no sex difference in expression of estrogen-related markers or in relation of these markers to survival in lung cancer patients, suggesting that both men and women may benefit from hormonal therapy. We hypothesized that aromatase activity is a factor in lung cancer development regardless of sex. Since testosterone can serve as an estrogen precursor, we determined whether blocking estrogen action is a feasible lung tumor prevention strategy in male mice. To standardize the amount of estrogen in each animal, orchiectomized male mice were utilized and exogenous testosterone was administered via slow release pellets or daily androstendione injections. Under these conditions, testosterone or androstendione is converted to estrogen through aromatase. The tobacco carcinogen NNK (24mg) was administered in weeks 1-4 followed by a holding period for preneoplasia development. Placebo or anastrozole (0.1mg/kg; p.o. daily) treatment was administered in weeks 9-21. Treatment group (10-11 mice per group) differences were assessed by Poisson regression for number of tumors, and by linear mixed models for tumor size. Anastrozole inhibited the mean number of NNK induced lung tumors per animal by 23% in orchiectomized male mice without hormonal supplementation (placebo treatment mean= 11; range= 8-13 vs anastrozole mean= 8; range= 6-10; p=0.11), by 62.5% in mice supplemented with testosterone (placebo treatment mean= 8; range= 6-11 vs anastrozole mean= 3; range= 2-4; p<0.001) and by 55% in mice supplemented with androstendione (placebo treatment mean= 12, range= 9-16 vs anastrozole mean= 5; range= 4-7; p<0.001). In intact male mice with no hormonal manipulation, anastrozole inhibited lung tumor formation by 33% (p=0.001). Fulvestrant also showed a significant decrease in lung tumors regardless of hormonal status. Tumor number was decreased in general by testosterone suggesting that testosterone may have an inhibitory effect on lung tumor formation. Tumor size was also decreased by anastrozole and fulvestrant under all experimental conditions. Tumor Ki67 and serum β-estradiol levels at sacrifice were significantly decreased by anastrozole. These results suggest that hormonal therapies may benefit male lung cancer patients. Supported by P50CA090440 and the Lung Cancer Research Foundation. Citation Format: Laura P. Stabile, Mary E. Rothstein, Brenda F. Kurland, Diana Cunningham, Matthew Orlowski, Jill M. Siegfried. Targeting the estrogen pathway in a male mouse model of lung tumor prevention. [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 608. doi:10.1158/1538-7445.AM2014-608