Laura P. Stabile

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.

Combined Analysis of Estrogen Receptor β-1 and Progesterone Receptor Expression Identifies Lung Cancer Patients with Poor Outcome

Purpose: Steroid hormones and growth factors affect lung cancer, and it is possible they act in concert to influence patient outcome. Experimental Design: Primary lung tumors and normal lung tissue were analyzed for expression and localization of estrogen receptor α and β-1 (ERα and ERβ), aromatase, progesterone receptor (PR), and epidermal growth factor receptor (EGFR). Results: Tumors expressed higher levels of ERβ compared to matched normal lung, whereas the reverse was true of PR. High cytoplasmic ERβ expression was identified as an independent negative prognostic predictor of overall survival (OS; HR = 1.67), and low total PR was identified as an independent negative predictor of time to progression (TTP; HR = 1.59). After adjusting for stage, age, sex, and smoking, combined high cytoplasmic ERβ and low total PR showed enhanced effects on OS (HR = 2.64) and on TTP (HR = 6.02). Further effects on OS were observed when EGFR expression was included (HR = 5.32). Patients with low cytoplasmic ERβ, low aromatase, low EGFR, and high total PR had shorter OS than patients with the opposite pattern (HR = 6.60). Contribution of these markers to survival showed no significant sex differences in a multivariable model. ERα was elevated in tumors but was not predictive of survival, and appears to represent a variant ERα protein that is only recognized by a C-terminal antibody. Conclusions: Hormonal and EGFR pathways together may contribute to lung cancer prognosis. Lung tumors with high ERβ-1/low PR may define patients with aggressive biology. A validation study is necessary to fully assess the predictive value of these markers. Clin Cancer Res; 17(1); 154–64. ©2010 AACR.

Estrogen Receptor Signaling in Lung Cancer

Lung cancer has long been thought of as a cancer that mainly affects men, but over the past several decades, because of the high increase in tobacco use by women, there has been a corresponding dramatic increase in lung cancer among women. Since 1998, lung cancer deaths in women have surpassed those caused by breast cancer in the United States. Annual lung cancer deaths among US women currently surpass those caused by breast, ovarian, and cervical cancers combined. Women are more likely than men to be diagnosed with adenocarcinoma and small cell carcinoma of the lung compared to squamous cell carcinoma, and never-smokers diagnosed with lung cancer are almost three times more likely to be female than male. These observations in the population, coupled to the findings that both estrogen receptors (ERs) and aromatase, the enzyme that synthesizes 17beta-estradiol, are expressed by lung tumors, suggest a role for female steroid hormones in control of lung cancer growth. Preclinical data and clinical data are increasingly emerging to support this concept, and to suggest that a local production of estrogen and expression of ERs occurs in lung tumors that arise in men as well as in women. An additional protein that recognizes 17beta-estradiol with high affinity, GPR30, also is expressed in lung tumors at high levels and may be responsible for some of the proliferation signals induced by estrogen.

HGF-independent Potentiation of EGFR Action by c-Met

The c-Met receptor is a potential therapeutic target for non-small cell lung cancer (NSCLC). Signaling interactions between c-Met and the mutant epidermal growth factor receptor (EGFR) have been studied extensively, but signaling intermediates and biological consequences of lateral signaling to c-Met in EGFR wild-type tumors are minimally understood. Our observations indicate that delayed c-Met activation in NSCLC cell lines is initiated by wild-type EGFR, the receptor most often found in NSCLC tumors. EGFR ligands induce accumulation of activated c-Met, which begins at 8 h and continues for 48 h. This effect is accompanied by an increase in c-Met expression and phosphorylation of critical c-Met tyrosine residues without activation of mitogen-activated protein kinase (MAPK) or Akt. Gene transcription is required for delayed c-Met activation; however, phosphorylation of c-Met by EGFR occurs without production of hepatocyte growth factor (HGF) or another secreted factor, supporting a ligand-independent mechanism. Lateral signaling is blocked by two selective c-Met tyrosine kinase inhibitors (TKIs), PF2341066 and SU11274, or with gefitinib, an EGFR TKI, suggesting kinase activity of both receptors is required for this effect. Prolonged c-Src phosphorylation is observed, and c-Src pathway is essential for EGFR to c-Met communication. Pretreatment with pan-Src family kinase inhibitors, PP2 and dasatinib, abolishes delayed c-Met phosphorylation. A c-Src dominant-negative construct reduces EGF-induced c-Met phosphorylation compared with control, further confirming a c-Src requirement. Inhibition of c-Met with PF2341066 and siRNA decreases EGF-induced phenotypes of invasion by ∼86% and motility by ∼81%, suggesting that a novel form of c-Met activation is utilized by EGFR to maximize these biological effects. Combined targeting of c-Met and EGFR leads to increased xenograft antitumor activity, demonstrating that inhibition of downstream and lateral signaling from the EGFR-c-Src–c-Met axis might be effective in treatment of NSCLC.

Dual blockade of EGFR and c-Met abrogates redundant signaling and proliferation in head and neck carcinoma cells

Purpose: Head and neck squamous cell carcinoma (HNSCC) is usually fatal, and innovative approaches targeting growth pathways are necessary to effectively treat this disease. Both the epidermal growth factor receptor (EGFR) and the hepatocyte growth factor (HGF)/c-Met pathways are overexpressed in HNSCC and initiate similar downstream signaling pathways. c-Met may act in consort with EGFR and/or be activated as a compensatory pathway in the presence of EGFR blockade. Experimental Design: Expression levels of EGFR and c-Met were determined by Western analysis in HNSCC cell lines and correlated with antitumor responses to inhibitors of these pathways. Results: Combining the c-Met inhibitor PF2341066 with the EGFR inhibitor gefitinib abrogated HNSCC cell proliferation, invasion, and wound healing significantly more than inhibition of each pathway alone in HNSCC cell lines. When both HGF and the EGFR ligand, TGF-α, were present in vitro, P-AKT and P-MAPK expression were maximally inhibited by targeting both EGFR and c-Met pathways, suggesting that c-Met or EGFR can compensate when phosphorylation of the other receptor is inhibited. We also showed that TGF-α can induce phosphorylation of c-Met over sixfold by 8 hours in the absence of HGF, supporting a ligand-independent mechanism. Combined targeting of c-Met and EGFR resulted in an enhanced inhibition of tumor volumes accompanied by a decreased number of proliferating cells and increased apoptosis compared with single agent treatment in vivo. Conclusions: Together, these results suggest that dual blockade of c-Met and EGFR may be a promising clinical therapeutic strategy for treating HNSCC. Clin Cancer Res; 17(13); 4425–38. ©2011 AACR.

Pilot study of gefitinib and fulvestrant in the treatment of post-menopausal women with advanced non-small cell lung cancer

INTRODUCTION Estrogen receptor beta (ERbeta) has been detected in non-small cell lung cancer (NSCLC) cell lines and tumor specimens. The ER down-regulator, fulvestrant, blocked estradiol-stimulation of tumor growth and gene transcription in NSCLC preclinical models and showed additive effects with the epidermal growth factor receptor (EGFR) inhibitor gefitinib. The safety and tolerability of combination therapy with the EGFR inhibitor, gefitinib, and fulvestrant was explored. METHODS Post-menopausal women with advanced NSCLC received gefitinib 250 mg po daily and fulvestrant 250 mg IM monthly. RESULTS Twenty-two patients were enrolled. Eight patients had adenocarcinoma, six NSCLC-NOS, four squamous cell, and four BAC. Seven patients were never-smokers. Eight patients received > or =2 lines of prior chemotherapy, six received one prior chemotherapy, and eight were treatment-naïve. One patient experienced grade 4 dyspnea possibly related to treatment; all other grade 3/4 toxicities were unrelated to treatment. Twenty patients were evaluable for response: three partial responses (PRs) were confirmed (response rate of 15%, 95% CI: 5-36%). The median progression-free survival (PFS), overall survival (OS), and estimated 1-year OS were 12 weeks (3-112 weeks), 38.5 weeks (7-135 weeks), and 41% (95% CI: 20-62%), respectively. Survival outcomes did not differ by prior lines of therapy. A subset analysis revealed that OS in the eight patients whose tumors exhibited at least 60% ERbeta nuclear IHC staining measured 65.5 weeks, while that of the five patients with ERbeta staining of less than 60% was 21 weeks. One patient with bronchioalveolar carcinoma (BAC) and a PR had an EGFR L858R mutation in exon 21. There was no correlation between ERbeta IHC expression and histology or smoking history. CONCLUSIONS Combination therapy with gefitinib and fulvestrant in this population was well tolerated and demonstrated disease activity.

Inhibition of human non-small cell lung tumors by a c-Met antisense/U6 expression plasmid strategy

c-Met is a receptor tyrosine kinase whose activation by hepatocyte growth factor (HGF) can lead to transformation and tumorigenicity in a variety of tumors. We investigated the effects of suppressing c-Met protein expression in human non-small cell lung tumors. Expression plasmids containing either sense or antisense sequences of the human c-met gene were constructed under control of the U6 snRNA promoter. A U6 control plasmid was also constructed that did not contain any c-met sequence. These constructs have been examined both in vitro and in an in vivo tumor xenograft model. The c-Met protein was downregulated by 50–60% in two lung cancer cell lines that were transiently transfected with the c-Met antisense versus U6 control. Tumor cells treated with the c-Met antisense construct also show decreased phosphorylation of c-Met and MAP kinase when exposed to exogenous HGF. Lung cancer cells were grown as xenografts in mice and treated by intratumoral liposome-mediated transfer of the c-Met sense, antisense or U6 control plasmids. The treatment of lung tumors with c-Met antisense versus U6 control plasmid resulted in the downregulation of the c-Met protein expression, a 50% decrease in tumor growth over a 5-week treatment period and an increased rate of apoptosis. These results suggest that targeting the HGF/c-Met pathway may be an effective novel strategy to treat lung cancer patients.

c-Src Activation Mediates Erlotinib Resistance in Head and Neck Cancer by Stimulating c-Met

Purpose: Strategies to inhibit the EGF receptor (EGFR) using the tyrosine kinase inhibitor erlotinib have been associated with limited clinical efficacy in head and neck squamous cell carcinoma (HNSCC). Co-activation of alternative kinases may contribute to erlotinib resistance. Experimental Design: We generated HNSCC cells expressing dominant-active c-Src (DA-Src) to determine the contribution of c-Src activation to erlotinib response. Results: Expression of DA-Src conferred resistance to erlotinib in vitro and in vivo compared with vector-transfected control cells. Phospho-Met was strongly upregulated by DA-Src, and DA-Src cells did not produce hepatocyte growth factor (HGF). Knockdown of c-Met enhanced sensitivity to erlotinib in DA-Src cells in vitro, as did combining a c-Met or c-Src inhibitor with erlotinib. Inhibiting EGFR resulted in minimal reduction of phospho-Met in DA-Src cells, whereas complete phospho-Met inhibition was achieved by inhibiting c-Src. A c-Met inhibitor significantly sensitized DA-Src tumors to erlotinib in vivo, resulting in reduced Ki67 labeling and increased apoptosis. In parental cells, knockdown of endogenous c-Src enhanced sensitivity to erlotinib, whereas treatment with HGF to directly induce phospho-Met resulted in erlotinib resistance. The level of endogenous phospho-c-Src in HNSCC cell lines was also significantly correlated with erlotinib resistance. Conclusions: Ligand-independent activation of c-Met contributes specifically to erlotinib resistance, not cetuximab resistance, in HNSCC with activated c-Src, where c-Met activation is more dependent on c-Src than on EGFR, providing an alternate survival pathway. Addition of a c-Met or c-Src inhibitor to erlotinib may increase efficacy of EGFR inhibition in patients with activated c-Src. Clin Cancer Res; 19(2); 380–92. ©2012 AACR.

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)

Estrogenic Steroid Hormones in Lung Cancer

It is becoming increasingly clear that steroid hormones are involved in the biology of many organs outside the reproductive system. Evidence has been accumulating since the mid 1990s that the lung contains receptors for both estrogen and progesterone and that these hormones have some role in lung development, pulmonary inflammation, and lung cancer. The estrogen receptor β (ERβ) is the major ER expressed in lung tissues, while inflammatory cells capable of infiltrating the lung are reported to express both ERα and ERβ. Although there is evidence in animals of preferential effects of ERβ in the lungs of females, human lung tumors from males also contain ERβ-positive cells and express aromatase, the enzyme that converts testosterone to estrogens. This review will discuss current literature findings on the role of the ERs and the progesterone receptor (PR), as well CYP19 (aromatase), the rate-limiting enzyme in the synthesis of estrogen, in lung cancer.