Previous reports have shown that certain anti-HER2
antibodies and
heregulin can inhibit clonogenic growth of breast and
ovarian cancers that overexpress HER2. Anti-HER2
antibodies bind to HER2 directly, whereas
heregulin does not bind to HER2 alone, but rather interacts with HER2 through the formation of heterodimers with HER3 or HER4. The purpose of the present study was to elucidate the mechanisms by which anti-HER2 antibody and
heregulin inhibit
tumor growth. The anti-HER2
monoclonal antibody (mAb) ID5 was found to block G1-S progression of the cell cycle, whereas
heregulin inhibited passage through G2-M. Compatible with the effects on the cell cycle, treatment with mAb ID5 decreased levels of
cyclin-dependent kinase (CDK) 2,
cyclin E, and CDK6
proteins and reduced
cyclin E-CDK2-associated
kinase activity; mAb HD5-treated cells had increased p27Kip1 expression and an increased association of p27Kip1 with CDK2. In contrast, treatment with
heregulin increased
protein levels of CDK2, CDK6, CDC2, and
cyclin B1. More
Retinoblastoma protein was found in the hypophosphorylated state in the cells treated with mAb ID5, whereas more
retinoblastoma protein was in the hyperphosphorylated state in
heregulin-treated cells.
Heregulin was able to induce cell differentiation as assessed by
Oil Red O staining and apoptosis as assessed by sub-G1 peak on flow cytometry and the presence of DNA fragmentation in ApopTag histochemistry staining. Neither differentiation nor apoptosis was observed in the cells treated with mAb ID5. We conclude that anti-HER-2 mAb ID5 and
heregulin exert growth inhibition through different mechanisms. In mammary cells overexpressing HER2, anti-HER2 mAb ID5 induces G1 arrest, whereas
heregulin induces G2-M arrest, cell differentiation, and apoptosis.