Lina Albitar

EGFR isoforms and gene regulation in human endometrial cancer cells

BackgroundEpidermal growth factor (EGF) and its receptor (EGFR) constitute a principal growth-promoting pathway in endometrial cancer cells. Pre-clinical studies were undertaken to compare the expression of EGFR isoforms and the downstream effects of activating or blocking EGFR function in Ishikawa H cells, derived from a moderately differentiated type I endometrioid adenocarcinoma, or in Hec50co cells, derived from a poorly differentiated type II adenocarcinoma with papillary serous sub-differentiation.ResultsWe investigated whether EGFR mutations are present in the tyrosine kinase domain (exons 18-22) of EGFR and also whether EGFR isoforms are expressed in the Ishikawa H or Hec50co cell lines. Sequence of the EGFR tyrosine kinase domain proved to be wild type in both cell lines. While both cell lines expressed full-length EGFR (isoform A), EGFR and sEGFR (isoform D) were expressed at significantly lower levels in Hec50co cells compared to Ishikawa H cells. Analysis of gene expression following EGF vs. gefitinib treatment (a small molecule EGFR tyrosine kinase inhibitor) was performed. Early growth response 1, sphingosine kinase 2, dual specificity phosphatase 6, and glucocorticoid receptor DNA binding factor 1 are members of a cluster of genes downstream of EGFR that are differentially regulated by treatment with EGF compared to gefitinib in Ishikawa H cells, but not in Hec50co cells.ConclusionsType I Ishikawa H and type II Hec50co endometrial carcinoma cells both express EGFR and sEGFR, but differ markedly in their responsiveness to the EGFR inhibitor gefitinib. This difference is paralleled by differences in the expression of sEGFR and EGFR, as well as in their transcriptional response following treatment with either EGF or gefitinib. The small cluster of differently regulated genes reported here in these type I vs. type II endometrial cancer-derived cell lines may identify candidate biomarkers useful for predicting sensitivity to EGFR blockade.

Regulation of signaling phosphoproteins by epidermal growth factor and Iressa (ZD1839) in human endometrial cancer cells that model type I and II tumors

To understand how type I and II endometrial tumors uniquely respond to tyrosine kinase inhibitor treatments, we evaluated the signaling pathways of epidermal growth factor (EGF) receptor (EGFR) under the effects of EGF and Iressa (ZD1839, gefitinib) using Ishikawa H and Hec50co cells that model type I and II endometrial carcinomas, respectively. The cells were assayed for the expression of EGFR and both cell lines express an average of 100,000 EGFR per cell; however, Ishikawa H cells express higher levels of HER-2/neu compared with Hec50co cells (1.38 × 105 compared with 2.04 × 104, respectively). Using the Kinetworks multi-immunoblotting approach, which profiles 31 signaling phosphoproteins, the most striking result was that Hec50co cells show a higher number of basal phosphorylated sites compared with Ishikawa H cells. Furthermore, we identified targets of Iressa treatment in both cell lines. Iressa, at a dose of 1 μmol/L, blocked the autophosphorylation of EGFR in Ishikawa H and Hec50co cells with some distinctive effects on downstream effectors. Nevertheless, in both cell lines, EGF stimulated and Iressa blocked the major EGFR target mitogen-activated protein kinases extracellular signal-regulated kinase 1 and 2 equally. The high basal phosphorylation of numerous signaling molecules in Hec50co cells that were not inhibited by Iressa indicates that other growth factor pathways are active in addition to EGFR. We conclude that endometrial cancer cells that model type I and II carcinomas have the capacity to respond to EGFR inhibition as a therapeutic strategy; however, the response of the more aggressive type II tumors may be limited by the constitutive activation of other signaling pathways. [Mol Cancer Ther 2005;4(12):1891–9]

Blocking Epidermal Growth Factor Receptor Signaling in HTR-8/SVneo First Trimester Trophoblast Cells Results in Dephosphorylation of PKBα/AKT and Induces Apoptosis.

We identified a major peptide signaling target of EGF/EGFR pathway and explored the consequences of blocking or activating this pathway in the first trimester extravillous trophoblast cells, HTR-8/SVneo. A global analysis of protein phosphorylation was undertaken using novel technology (Kinexus Kinetworks) that utilizes SDS-polyacrylamide minigel electrophoresis and multi-lane immunoblotting to permit specific and semiquantitative detection of multiple phosphoproteins. Forty-seven protein phosphorylation sites were queried, and the results reported based on relative phosphorylation at each site. EGF- and Iressa-(gefitinib, ZD1839, an inhibitor of EGFR) treated HTR-8/SVneo cells were subjected to immunoblotting and flow cytometry to confirm the phosphoprotein screen and to assess the effects of EGF versus Iressa on cell cycle and apoptosis. EGFR mediates the phosphorylation of important signaling proteins, including PKBα/AKT. This pathway is likely to be central to EGFR-mediated trophoblast survival. Furthermore, EGF treatment induces proliferation and inhibits apoptosis, while Iressa induces apoptosis.

The combination of Paclitaxel and Gefitinib inhibits endometrial cancer cells by inducing mitotic catastrophe: proof of principle for dual therapy in endometrial cancer

This is an Open Access article distributed under the terms of the Creative Commons Attribution 3.0 Unported License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The combination of Paclitaxel and Gefitinib inhibits endometrial cancer cells by inducing mitotic catastrophe: proof of principle for dual therapy in endometrial cancer

Abstract 1795: The Combination of Paclitaxel and Gefitinib Inhibits Endometrial Cancer Cells by Inducing Mitotic Catastrophe: Proof of Principle for Dual Therapy in Endometrial Cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Serous uterine endometrial cancer is a lethal disease for which new therapeutic regimens are urgently needed. Combinations of chemotherapeutic agents and small molecule growth factor inhibitors have demonstrated activity in cancers from other sites. Our objective was to determine whether such a combination could be active in serous endometrial cancer cells. The effects of the EGFR inhibitor gefitinib (ZD1839, Iressa) alone, paclitaxel alone, and the combination of both agents on the cell cycle and on cell proliferation were studied using Hec50co cells, a validated model for type II serous endometrial cancer with absent p53. First, we established the IC50 for paclitaxel alone (14 nM) compared to that of paclitaxel and gefitinib in combination (1.3 nM). This 10-fold reduction in the IC50 with dual therapy yielded a combination index of 0.25, strongly suggesting that the paclitaxel and gefitinib combination resulted in synergistic growth inhibition. Sixty-seven percent of the cells treated with paclitaxel and gefitinib arrested in the G2/M phase. This arrest of the cell cycle at G2/M with combination therapy was significantly greater than with single agent treatment alone (p < 0.001) and was associated with inhibition of p38 phosphorylation and the induction of CDC25C phosphatase and Cyclin B1. When induced, these factors abrogate the G2/M checkpoint and result in rapid, premature progression to M phase; this mechanism is particularly active in the setting of absent p53. With rapid progression into M, the cells were highly sensitive to paclitaxel and were killed by the mechanism of mitotic catastrophe. Similar findings were observed in the same cells treated with the combination of paclitaxel and a specific inhibitor of p38, SB203580. Our study suggests that inhibition of EGFR or downstream p38 pathway can abrogate the mitotic stress checkpoint induced by cytotoxic agents targeting microtubules such as paclitaxel. These findings suggest a new therapeutic strategy for the treatment of serous endometrial carcinoma worthy of clinical confirmation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1795.