TYROSINE KINASE INHIBITORS IN ENDOMETRIAL CANCER: 0020

Introduction: The EGFR family and relationship to carcinogenesis The epidermal growth factor family is comprised of EGFR (ErbB1), HER-2/neu (ErbB2), HER-3 (ErbB3), and HER-4 (ErbB4). All are present in reproductive tissues. Growth factors and their receptors are known to play critical roles in cell growth and differentiation(1). Growth factors such as EGF are potent mitogens for several human epithelial cell types including the endometrium(2,3) and have been implicated in cancer development(4). EGFR has been shown to be highly expressed in normal endometrium and overexpressed in endometrial cancer specimens(5), where it has been associated with a poor prognosis(6–8). Increased expression of EGF related protein and EGFR may contribute to a drug resistant phenotype(9). Inhibition of EGFR with monoclonal antibodies leads to growth arrest(10), and a similar, and potentially synergistic effect is anticipated with inhibition of EGFR tyrosine kinase activity(11–14). New therapeutic molecules which block EGFR are under study in tumors from many sites, now including the endometrium. Agents in phase II clinical trials include Iressa (ZD1839), Herceptin, and Lapatinib (GW572016). The studies and discussion that follow outline the biologic rationale and suggest potential clinical applications for these agents in endometrial cancer. Methods and study results: EGFR signaling in endometrial cancer cells EGFR has intrinsic tyrosine kinase activity which is activated upon ligand binding. Downstream serine/threonine kinase cascades are then induced, leading to cell proliferation and the inhibition of apoptosis(15,16). We have now identified multiple downstream targets of the EGFR pathway in Hec50co poorly differentiated and Ishikawa well differentiated endometrial cancer cells. Phosphoproteomic profiling of downstream targets of EGFR was carried out using Kinetworks Phospho-Site Screen 1.3 (Kinexus, Victoria, BC) multilane immunoblotting that determines the phosphorylation state of 31 downstream signaling molecules. These experiments demonstrate that EGFR activation upregulates and tyrosine kinase inhibitors prevent the phosphorylation of MAP kinase Erk1/2, MAP kinase kinase MEK1/2, cyclin dependent kinase 1, protein kinase B (Akt), oncogene Raf 1, retinoblastoma 1, and signal transducer and activator of transcription 3. These findings define the principal EGFR signaling pathways activated in two representative endometrial cancer cell lines. Potential EGFR cross-talk with steroid hormone receptors: implications in type I and type II endometrial tumors The EGF/EGFR pathway is induced by estrogen and is arguably the principal growth promoting mechanism in the endometrium(2,3). Estrogen and progesterone are the most important steroid hormones that modulate endometrial cell proliferation and differentiation, respectively, and their receptors (ER and PR) are expressed in many endometrial tumors. However, ER and PR are frequently down-regulated in advanced endometrial cancers. Type I endometrial tumors are those that arise in a hormone-dependent manner and express normally-functioning and regulated ER, PR, and EGFR. Type II endometrial tumors are not responsive to hormones and may not express ER and PR(17). Type II tumors are proposed to exhibit constitutive activation of alternative growth pathways such as those controlled by EGFR. We propose that the loss of ER and PR and the constitutive activation of EGFR may be functionally linked. Indeed, ER and PR are downstream phosphorylation targets of EGFR. Following work in breast cancer cells(18–21), we have now shown in endometrial cancer cells that in the presence of progesterone, phosphorylation of PR occurs via this pathway, leading to receptor ubiquitination and the targeting of PR to the proteasome for destruction. Clinical trials The tyrosine kinase inhibitor ZD1839 (Iressa™) has been studied as a single agent in a phase II clinical trial (GOG 229C) of women with advanced endometrial cancer. Preliminary data analysis indicates that of 29 patients enrolled, 1 patient experienced a complete response and several others had stable disease at 6 months. These findings await official confirmation and are presented at this time for illustrative purposes only. Another GOG study is now underway, GOG 229D, which will evaluate the effectiveness of the dual tyrosine kinase inhibitor, GW572016 (Lapatinib™), as a single agent in a similar cohort of patients. As in other organ sites, it is possible that tyrosine kinase inhibitors will show activity in only a small subset of patients, if at all. It remains to be determined whether response will be linked to mutations in the ATP binding pocket of EGFR, as has been demonstrated in lung cancer patients(22,23). However, based upon the studies below, we propose that tyrosine kinase inhibitors are likely to provide the most benefit for the general population of patients with endometrial tumors when used in conjunction with other traditional therapies. Effects of Iressa alone and in combination with chemotherapy in experimental models Using in vitro cell lines Hec50co, KLE, and Ishikawa, we have evaluated the concentration of Iressa, Taxol, and Iressa + Taxol that kills 50% of the cells after 24 hours of treatment (the IC50). The IC50 for Iressa as a single agent is 11 μM in well differentiated Ishikawa cells, 63 μM in poorly differentiated Hec50co cells, and 100 μM in poorly differentiated KLE cells. In Hec50co and KLE cells, Taxol alone exhibits an IC50 of 14.7 nM. However, the addition of 10 μM Iressa to Taxol substantially reduces the IC50 of Taxol by 10-fold, and results in a synergistic inhibition of cell survival. These studies are meant to serve as preliminary data upon which to base the choice of combination regimens in future clinical trials. The synergistic action of Iressa added to Taxol was seen at all doses, with maximal activity at 10 μM. As Iressa is concentrated within cells, it is likely that this drug concentration is achievable even when circulating levels of Iressa are much lower, in the range of 1 μM (personal communication, AstraZeneca Pharmaceuticals, Inc.), making it feasible that levels of Iressa can be reached in patients that could potentiate Taxol's effects. In addition, our laboratory has used two xenografted animal models, the athymic mouse and the nude rat, for confirmatory preclinical studies. These further demonstrate the effectiveness of combining a biologic agent with chemotherapy. We conclude that combinatorial strategies should be further evaluated in the laboratory and incorporated into future clinical trials if the preclinical findings are encouraging. Acknowledgments: Funding for these studies was provided by NIH CAR01 99908-01 (KL), The University of New Mexico Cancer Research and Treatment Center (SD, KL), the Gynecologic Oncology Group Core Laboratory for Receptors (KL), The Department of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine (JB, LL, LA, AH, TN), The Barbara Beach Family Fund for Endometrial Cancer Research (KL, SD, DD), Dean and Alice Irvin, and Shirley Leslie.