Aromatase and breast cancer

Abstract

Several aromatase inhibitors and also new antiestrogens are now available for treating breast cancer. We have developed a model to compare the antitumor efficacy of these agents and to explore strategies for their optimal use. Results from the model have been predictive of clinical outcome. In this model, tumors are grown in ovariectomized, immunodeficient mice from MCF-7 human breast cancer cells transfected with the aromatase gene (MCF-7Ca). The possibility that blockade of estrogen action and estrogen synthesis may be synergistic was explored by treating mice with the aromatase inhibitor letrozole and the antiestrogen tamoxifen alone and in combination. The results indicated that letrozole alone was better than all other treatments. In addition, when tamoxifen treatment was no longer effective, tumor growth was significantly reduced in mice switched to letrozole treatment. However, tumors ultimately began to grow during continued treatment. To investigate the mechanisms by which tumors eventually adapt and grow during letrozole treatment, we determined the expression of signaling proteins in tumors during the course of letrozole treatment compared to the tumors of control mice. Tumors initially up-regulated the ER while responding to treatment, but subsequently receptor levels decreased in tumors unresponsive to letrozole. Also, Her-2 and adapter proteins (p-Shc and Grb-2) as well as all of the signaling proteins in the MAPK cascade (p-Raf, p-Mekl/2, and p-MAPK), but not in the Pl3/Akt pathway, were increased in tumors no longer responsive to letrozole. To investigate whether sensitivity to letrozole could be regained, cells were isolated from the letrozole resistant tumors (LTLT) and treated with inhibitors of the MAPKinase pathway (PD98059 and UO126). These compounds reduced MAPK activity and increased ER expression. EGFR/Her-2 inhibitors, gefitinib and AEE78S although not effective in the parental MCF-70a cells, restored the sensitivity of LTLT cells to letrozole. In xenografts, beginning treatment with letrozole and faslodex to down regulate the ER prevented increases in Her-2 and activation of MAPK and was highly effective in inhibiting tumor growth throughout 29 weeks of treatment. These results suggest that blocking both ER- and growth factor-mediated transcription may delay development of resistance and maintain growth inhibition of ER+ breast cancer.

Figure 1. The Effect of Letrozole Treatment on Grb2, p-MAPK, and p-ERα¿ Expression in MCF-7Ca Tumor Xenografts

Tumors were collected from letrozole-treated mice at 4 weeks (when they were responding to letrozole), 28 and 56 weeks (when they were growing on letrozole), analyzed by Western immunoblotting, and were compared with tumors of vehicle-treated mice collected at week 4 (control). Tumors were homogenized in lysis buffer, and equal amounts of protein (60 Ag) were separated on a denaturating polyacrylamide gel and transferred to a nitrocellulose membrane. After blocking nonspecific binding with 5% nonfat milk in PBS-T, the membranes were incubated with respective primary antibodies, and specific binding was visualized by using species-specific immunoglobulin G followed by ECL detection (ECL kit) and exposure to ECL X-ray film. After exposure to X-ray film, the membranes were stripped and probed for β-actin to confirm that equal amount of proteins were loaded in each lane. Numbers below the blots represent fold change in protein expression compared with the control obtained by densitometric analysis. (From Jelovac et al., Cancer Res., 2005).

Figure 2. The Effect of MAPKinase Inhibitor, PD98059 on the Growth of MCF-7Ca and LTLT-Ca Cells

MCF-7Ca and LTLT-Ca cells were transferred into steroid-free medium for 3 days before plating (1 × 10⁴ cells per well) into 24-well plates. The next day, cells were washed with DPBS and treated with steroid-free medium containing androstenedione (25nmol/L) and the indicated concentrations of PD98059. The medium was changed every 3 days, and the cells were counted 9 days later using the MTT assay. Percentage of the PD98059 cells compared with the vehicle-treated cells (control). Expression of pMAPK is shown in the insert. (From Jelovac et al., Cancer Res., 2005).

Figure 3. The Expression of Estrogen Receptor Protein and Signaling Proteins p-Raf, p-MEK1/2, p-MAPK, p-p90RSK, and p-Elk in LTLT-Ca Cells Compared with the Parental MCF-7Ca Cells

Cell lysates were prepared as described in Methods. Equal amounts of protein (60 Ag) were separated on a denaturating polyacrylamide gel and transferred to a nitrocellulose membrane. After blocking nonspecific binding with 5% nonfat milk in PBS-T, the membranes were incubated with the respective primary antibodies, and specific binding was visualized by using species-specific immunoglobulin G followed by ECL detection (ECL kit) and exposure to ECL X-ray film. After exposure to X-ray film, the membranes were stripped and probed for h-actin to confirm that equal amount of proteins were loaded in each lane. Numbers below the blots represent fold change in protein expression compared with the control obtained by densitometric analysis. For MAPK activity, assay cell lysates were prepared as described in Methods. Proteins (200 Ag) were subjected to immunoprecipitation using specific MAPK antibody. Next day, after adding kinase reaction buffer and GST fusion protein of Elk-1 (MAPK substrate), samples were incubated at 30°C for 30 minutes. Samples were analyzed by Western blotting using phospho-Elk-1 antibody. (From Jelovac et al., Cancer Res., 2005).