Targeting the estrogen receptor for lung cancer therapy
Authors: Jill M. Siegfried, Laura P. Stabile, Pamela A. Hershberger, Mark Nichols, Joan H. Schiller
Journal: Cancer Research (2005)

Proc Amer Assoc Cancer Res, Volume 46, 2005

SY14-3 

Identifying new therapeutic treatments for lung cancer is critical to improving overall survival. We have recently determined that non-small lung cancer (NSCLC) cell lines and tumor tissues frequently express the Estrogen Receptor β (ERβ), and that estrogens act as mitogens for NSCLC cells in vitro and in vivo . ERβ expression was found in 102 of 106 (96%) of primary NSCLC lung tumors. Both nuclear and cytoplasmic ERβ staining was uniformly observed (101/106 cases, 95%), suggesting that both non-genomic and genomic signaling occurs from the ERβ in lung cancer. ERβ protein was also uniformly observed by immunoblot of lung tumor cell lines. ERα antibodies to the hinge region or to N-terminal regions were completely negative by immunohistochemistry in lung tumor tissues and by immunoblotting in cell lines, suggesting the form of ERα present in breast tumors is not expressed in lung tumors. ERα staining using an antibody that can detect truncated forms of ERα (HC20, raised against the carboxyterminus) was positive in 76/106 cases (72%). ERα staining using the HC20 antibody was found in both the nucleus and cytoplasm in 68/106 cases (64% of cases). Level of nuclear ERα with HC20 antibody was higher in lung tumors from females than in males ( P < 0.05), and these patients were more likely to die of disease than those with lower ERα nuclear staining ( P < 0.05). DNA synthesis was induced over 20-fold by estrogen in serum-deprived cells, and cell proliferation induced by estrogen was blocked by the estrogen receptor antagonist fulvestrant (Faslodex). Growth of lung tumors as xenografts in immunocompromised mice was stimulated 2 to 3-fold by estrogen pellets, and this effect could also be blocked by fulvestrant. Transcription from an ERE element was stimulated 2-fold by estrogen and blocked by fulvestrant in NSCLC cells. Further, NSCLC cell lines express the ER p160 co-activator GRIP, and evidence was found for a physical interaction between ERβ and GRIP. GRIP can also function as a transcriptional co-activator with ERβ in lung cancer cells. Estrogen and fulvestrant modulate mRNA expression of genes in NSCLC that are important for control of cell proliferation and adhesion, such as cyclin D, Id2, and E-cadherin. Id2 and E-cadherin protein were also induced after exposure to estrogens, and were significantly blocked by fulvestrant. Since NSCLCs also commonly express EGFR, we have begun targeting both the estrogen receptor (ER) pathway and the epidermal growth factor receptor (EGFR) pathway using fulvestrant and the selective EGFR tyrosine kinase inhibitor gefitinib (Iressa) in lung cancer cells. EGFR protein expression is down-regulated in response to estrogen and up-regulated in response to fulvestrant in vitro , suggesting that the EGFR pathway is activated when estrogen is depleted in lung cancer cells. Treatment of lung cancer with antiestrogenic drugs could give rise to a population of cells with increased levels of EGFR, or increased dependence on EGFR signaling. Therefore, targeting EGFR in combination with hormone therapy could potentially be of benefit by increasing lung tumor cell death and preventing signaling through this alternate growth pathway. We examined three non-small cell lung cancer cell lines, A549, 201T, and 273T, each expressing different levels of EGFR protein, and all expressing ERβ. We examined cell growth using a standard MTS assay and apoptosis using the Apo-ONE™ Homogeneous Caspase-3/7 Assay. Lung cancer cells were exposed to 10 nM estrogen and 1ng/ml EGF and either 1μM fulvestrant, 1μM gefitinib or a combination of these drugs. In all cell lines examined, a 90% or higher decrease in cell proliferation with the combination was observed. This was accompanied by a 2-fold increase in apoptosis. To determine the involvement of bidirectional signaling between these two receptors in the lung, levels of phospho-p44/42 MAPK protein were measured over time in response to estrogen and EGF treatment. Rapid signaling responses were observed within five minutes in the lung cancer cell lines using either EGF or estrogen. MAPK activation was greatly enhanced in presence of both stimuli, indicating the possible involvement of membrane or cytoplasmic ER transactivation of EGFR. Gefitinib and fulvestrant treatment inhibited this response. In an in vivo lung tumor xenograft model, the combination of gefitinib and fulvestrant therapy decreased tumor growth in scid mice by approximately 70%, an increased effect compared to either treatment alone. The malignant cell density and status of MAPK activation in tumors were significantly reduced by combination therapy compared to either drug alone. Ki67 labeling was also significantly decreased by combination therapy compared to single therapy. The cross-signaling between the EGFR/ER pathways in the lung along with the emerging role of estrogens in lung tumorigenesis provides a rationale to combine EGFR tyrosine kinase inhibitors with antiestrogen therapy for lung cancer treatment. In a Phase I clinical trial examining toxicity of a combined regimen of fulvestrant administered IM as a depot along with daily dosing of oral 250 mg gefitinib in post-menopausal women with Stage IIIB or IV lung cancer, the combination was well-tolerated and no serious or unexpected side effects were observed. Of 14 evaluable patients to date, 2 partial responses were observed, both in never smokers. Stable disease was observed as best response in 9 patients, including smokers and non-smokers, and disease progression was observed in 3 patients. Four patients with stable disease have since progressed. This trial is ongoing and median overall survival time has not been reached but is greater than 28 weeks. Median progression-free survival is 21 weeks (range 4-37+ weeks). Variation in ER or EGFR status has not correlated with therapeutic response in this small group of patients, and information on EGFR mutant status in responders is pending. The combination of gefitinib and fulvestrant has activity and is well-tolerated, and further study of lung cancer therapy with anti-estrogens is warranted. We have also determined that aromatase, the terminal enzyme in estrogen synthesis, is expressed by both lung tumor cell lines and cultured lung fibroblasts. NSCLC cells have the capacity to convert testosterone to estrogen, suggesting that aromatase inhibitors also have promise for lung cancer therapy. Since both ER and aromatase expression were observed in males as well as females, anti-estrogen therapy may have activity against lung cancer in both sexes. Chemoprevention with relatively non-toxic anti-estrogens that have shown activity against breast cancer may also have potential for individuals at high risk for lung cancer. This work was supported by NIH P50CA90440, SPORE in Lung Cancer to the University of Pittsburgh.