Lung Cancer SPORE

Projects

Project 1: Intersection of Estrogen Receptor Signaling and Epidermal Growth Factor Receptor Signaling in Lung Cancer

Jill M. Siegfried, PhD Co-Project Leader
Jennifer R. Grandis, MD, Co-Project Leader

Lung cancer incidence is increasing in women worldwide and it is apparent from epidemiological studies that sex differences exist in the presentation of lung cancer. The proportion of patients diagnosed with lung cancer under age 50 is significantly higher for women compared to men (1). Women also are diagnosed to a greater extent than men with adenocarcinoma and small cell carcinoma (2, 3), both of which are secretory-type tumors. Never smokers diagnosed with lung cancer are also predominantly female (4). These differences in presentation suggest there are sex differences in the development of lung cancer. We hypothesize that one component of these sex differences is related to estrogen and its receptors. Evidence from our laboratory obtained during the first SPORE grant period shows that both known estrogen receptors (ERs), ER alpha (ERα) and ER beta (ERβ), are commonly expressed in non-small cell lung cancers (NSCLCs) of different histologic types. ERα appears to be mainly present as a variant protein of smaller molecular weight than full-length ERα, while ERβ protein is present at the expected size. These receptors are localized in both the nucleus and the cytoplasm, as well as some membrane localization, in NSCLC tissues and in normal lung. Genomic signaling through ERβ has been clearly demonstrated by us in NSCLC, as well as non-genomic signaling involving activation of the epidermal growth factor receptor (EGFR). The non-genomic signaling may involve both ERα and ERβ. We have evidence that combined targeting of the ER and the EGFR produces enhanced anti-proliferative effects in preclinical models. We also discovered that tumors from males contain ERs and can respond to estrogens. Many male lung tumors also appear to contain aromatase. This suggests that although some of the hormonal effects due to estrogen may be more pronounced in women compared to men due to a greater lifetime production of ligand, the ER pathway might also be targeted in males with NSCLC, especially if local estrogen production in lung tissues is present via aromatase, or if ligand-independent signaling plays a role in ER action in the lung. The hypothesis under investigation in Project One of the UPCI Lung Cancer SPORE renewal is that ER expression and signaling have functional significance in NSCLC. Based on results obtained in the first SPORE grant period, we hypothesize that ER and EGFR both activate proliferative signaling pathways in NSCLC; these pathways overlap and interact. Co-inhibition of ER and EGFR may show greater anti-tumor activity in NSCLC than inhibition of either pathway alone.

The Specific Aims are:

  1. Determine signaling molecules involved in ER-EGFR pathway interactions in NSCLC cell lines. We will ask the following questions in this aim: (a) Does EGFR cause ligand–independent activation of ERα and ERβ in NSCLC cells, resulting in genomic signaling in the absence of estrogen? (b) Does the ligand-dependent activation of ER that causes EGFR activation (non-genomic signaling) depend on Src, and can EGFR activation by ER be interrupted by Src inhibitors? We will examine cell lines that have both wild-type EGFR and EGFR with mutation in the tyrosine kinase domain.
  2. Examine effectiveness of joint inhibition of the ER-EGFR pathways on tumor growth in NSCLC compared to single therapy in NSCLC, using clinically relevant agents. We will ask the following question in this aim: Is the anti-tumor efficacy of combined ER/EGFR and/or Src targeting using clinically relevant agents superior to targeting individual pathways in preclinical models?
  3. Determine if aromatase is present and functional in normal and malignant lung cells and if an aromatase inhibitor has anti-tumor activity. We will ask the following questions in this aim: (a) How common is the expression of aromatase, the enzyme that synthesizes estrogen, in normal and malignant cells from the human airway? (b) Does aromatase function sufficiently in cultured normal and malignant cells from human airway to result in measurable production of estrogen? (c) Do primary NSCLC tissues from males and females contain aromatase, which cell types within tumors express it, and is aromatase related to other parameters such as ER expression, sex, histology, or outcome? (d) Does an aromatase inhibitor have anti-tumor effects against NSCLC in preclinical models?
  4. Analysis of ER and EGFR status in tissues obtained from NSCLC patients treated on clinical trials of combination therapy (anti-estrogen and EGFR TKI). We will ask the following questions in this aim: (a) Does combination therapy produce anti-tumor effects against late-stage NSCLC? (b) Is combination therapy superior to targeting the EGFR pathway alone? (c) What is the relationship between response to therapy and ER and EGFR pathway signaling in NSCLC patients? (d) Are variations in the EGFR gene observed in association with therapeutic response to combination therapy, and what are the characteristics of patients who show clinical benefit?

Translational Value of Project One

Role of Inter-SPORE Collaborations: Aims 3 and 4 of this project are collaborations with the UCLA Lung Cancer SPORE (Steven Dubinett, MD, PI). Regarding Aim 3, the UCLA SPORE is also examining aromatase in NSCLC and finds expression of this enzyme in both lung tumor cell lines and in tissues from lung cancer patients. The UCLA SPORE has also documented conversion of testosterone to estrogen in NSCLC cell lines and has shown anti-tumor effects of the aromatase inhibitor, anastrazole, in the NSCLC cell lines A549 and H23. We plan in our Aim 3 to utilize cell lines (including NSCLC cell lines, normal lung fibroblasts and human bronchial epithelial cells) and NSCLC tissues not being studied at UCLA, to demonstrate how widely aromatase protein and function is present in lung cancer. We will also examine a different set of NSCLC patients’ tumor and normal tissues for aromatase activity by immunohistochemistry. Since the role of aromatase in NSCLC is an entirely novel area of investigation, this joint endeavor will help to increase new knowledge and to confirm each SPORE’s findings. We plan to share our preclinical data and compare results with different aromatase antibodies in Aim 3 as well as to share the data from the other aims with the UCLA group, thus allowing the work to proceed at a faster pace. In Aim 4, the Phase II clinical trial involving erlotinib alone versus erlotinib plus the anti-estrogen fulvestrant will be conducted jointly at UCLA and Pittsburgh. We intend to enroll patients at each institution and to carry out the analysis of clinical response and signaling molecules in the patients’ paraffin-embedded tumors jointly. We will analyze the data from the clinical trial together and present and publish it together.

Aims 1 and 2 are also collaborations with the University of Pittsburgh Head and Neck Cancer SPORE (Jennifer Grandis, M.D., PI). Drs. Siegfried, Stabile, and Grandis are undertaking a developmental research project in the Pittsburgh Head and Neck Cancer SPORE to determine the extent of ER expression in head and neck cancers, and the signaling pathways activated by estrogen. The preliminary data suggest that ERβ is expressed by all head and neck cancer cell lines examined, and estrogens increase cell proliferation and ERE transcriptional activity in head and neck cancer cell lines, while the anti-estrogen fulvestrant can inhibit these responses. Knowledge from each tumor type will be shared between our two SPOREs, and if the preclinical data from head and neck cancers provides a rationale for treatment with anti-estrogens (alone or in combination with an anti-EGFR agent), Drs. Grandis and Siegfried will propose a clinical trial for head and neck cancer patients in the Head and Neck SPORE.

Translational Value of Project One

Targeting the EGFR through small molecule tyrosine kinase inhibitors (TKIs) to date is of limited utility in the absence of an EGFR mutation, which occurs in a minority of lung cancer patients. Even with an EGFR mutation, duration of response to EGFR TKIs may be short and resistance also develops through selection of further EGFR mutation (T790M) (5). Understanding how ER and EGFR interact in NSCLC will provide a rational basis for therapies targeting these two pathways to increase the effectiveness of EGFR targeted therapy. Combination therapy may increase duration of response in the presence of mutant EGFR, as well as improve therapeutic response in patients with wild-type EGFR.

Results to date of a Phase I clinical trial being carried out in the first SPORE grant period (described below) targeting both pathways together with a gefitinib/fulvestrant combination showed that combination therapy is safe and has anti-tumor activity in Stage IIIB/IV female post-menopausal NSCLC patients. Further testing of combination therapy (erlotinib/fulvestrant) compared to single therapy in animals and humans, proposed in this project, will determine whether combination therapy gives superior anti-tumor effects compared to EGFR TKI monotherapy. Since erlotinib increased survival in NSCLC patients in a Phase III trial vs. placebo/best supportive care, while gefitinib did not, erlotinib will be substituted for gefitinib in this project.

Increased understanding of the role of estrogen, estrogen synthesis, and ER in lung cancer will provide a rationale for future targeting of this pathway for therapy earlier in the course of disease as well as for future