The HER2/neu oncogene is an important diagnostic and prognostic factor in breast and other cancers and also serves as a therapeutic target. We developed and characterized a breast cancer cell line, designated Bam1a, from a lobular carcinoma that developed in \. a BALB-NeuT transgenic mouse expressing the activated rat HER2/neu oncogene under the MMTV promoter. This cell line, like the parental tumor, over-expresses the activated HER2/neu, EGFR and ErbB-3. By microarray analysis we detect co-over expression of cyclin D1, MEK, β-catenin, Akt and Rb in Bam1a cells, similar to the genetic profile of HER2 over-expressing breast cancers. We evaluated the effects of the EGFR/HER2 inhibitor, Iressa, on this breast tumor line in vitro and in vivo. We characterized the effects of Iressa on EGFR, HER2 and ErbB-3 phosphorylation by Western Blot and whole-cell ELISA and determined the effects on downstream signaling through growth, survival and stress pathways and the impact on proliferation, cell cycle and apoptosis. Iressa treatment diminished phosphorylation of the ErbB-3> EGFR> HER2/neu in a dose-dependent fashion. Decreased phosphorylation of STAT-3 (Tyr-705 and Ser-727) occurred with similar kinetics. Mitogenic signaling through MEK, p44/42 MAPK and stress signaling through JNK1 and c-Jun was greatly impaired at 1μM Iressa within 4h and remained suppressed over 24h. Cell proliferation ceased in the presence of 250-500 nM Iressa. Cytostasis was observed within 24h at 1 μM Iressa. Cell cycle arrest was characterized by accumulation and retention of cells in G0 (from 49 to 71%), decreased cells in S-phase (from 23% to 1%) and failure of cells to traverse the G2M (static at 28%) checkpoint. These events coincided with hypo-phosphorylation of Rb and cdc2, loss/degradation of Cyclin D1, Cyclin B1 and p21, reduced transcript levels of PCNA, chk2 and p21, and increased levels of p27. Phosphorylation and nuclear translocation of SAPK 2/3 occurred within 4 h. At which time, levels of the pro-apoptotic BH-3 only proteins, bim and bad (hypo-phosphorylated) increased dramatically. Iressa was a potent inducer of apoptosis, and impaired survival signaling through the Akt pathway. Cleavage of caspase-3 and PARP was readily detected in cells treated for 24 h with 2μM. In vivo, oral administration of Iressa was able to prevent the outgrowth of Bam1a tumor cells from palpable lesions and shrink established tumors in syngeneic female BALB/c mice. The effects of Iressa in vivo appear to involve the direct effects of Iressa on tumor cells and the ability of Iressa to impair signaling between tumor cells and the microenvironment. Understanding the signal transduction pathways that modulate the growth and survival of HER2/neu over-expressing breast cancers in vitro and in vivo will improve the development and translation of HER2 targeted therapies.
[Proc Amer Assoc Cancer Res, Volume 47, 2006]