DAMD 1794-J-4118 TITLE : Biologic Effects of HER-2 / Neu Gene Overexpression and Agonists and Antagonists to the Receptor in Human Breast Cancer

The management of human breast cancer frequently includes radiation therapy as an important intervention, and improvement in the clinical efficacy of radiation is desirable. Overexpression of the HER-2 growth factor receptor occurs in 25-30% of human breast cancers and correlates with poor clinical outcome, including earlier local relapse following conservative surgery accompanied by radiation therapy. In breast cancer cells with overexpression of HER-2 receptor, recombinant humanized monoclonal antibodies (rhuMAbs) to HER-2 receptors (rhuMAb HER-2) decrease cell proliferation in vitro and reduce tumor formation in nude mice. Therapy with rhuMAb HER-2 enhances tumor sensitivity to radiation at doses of 1-5 Gy, exceeding remission rates obtained with radiation alone. This benefit is specific to cells with HER-2 overexpression and does not occur in cells without overexpression. Treatment of cells with radiation (2-4 Gy) alone provokes a marked increase in unscheduled DNA synthesis, a measure of DNA repair, but HER-2-overexpressing cells treated with a combination of rhuMAb HER-2 and radiation demonstrate a decrease of unscheduled DNA synthesis to 25-44% of controls. Using an alternate test of DNA repair, i.e., radiation-damaged or undamaged reporter DNA, we introduced a cytomegalovirus-driven ß-galactosidase into HER-2-overexpressing breast cancer cells that had been treated with rhuMAb HER-2 or control. At 24 h posttransfection, the extent of repair assayed by measuring reporter DNA expression was high after exposure to radiation alone but significantly lower in cells treated with combined radiation and rhuMAb HER-2 therapy. To further characterize effects of rhuMAb HER-2 and the combination of antibody and radiation on cell growth, analyses of cell cycle phase distribution were performed. Antibody reduces the fraction of HER-2-overexpressirig breast cancer cells in S phase at 24 and 48 h. Radiation treatment is also known to promote cell cycle arrest, predominantly at Gj, with low S-phase fraction at 24 and 48 h. In the presence of rhuMAb HER-2, radiation elicits a similar reduction in S phase at 24 h, but a significant reversal of this arrest appears to begin 48 h postradiation exposure. The level of S-phase fraction at 48 h is significantly greater than that found at 24 h with the combined antibody-radiation therapy, suggesting that early escape from cell cycle arrest in the presence of antireceptor antibody may not allow sufficient time for completion of DNA repair in HER-2-overexpressirig cells. Because it is well known that failure of adequate p21WAFl induction after DNA damage is associated with failure of cell cycle arrest, we also assessed the activity of this critical mediator of the cellular response to DNA damage. The results show induction of p21WAFl transcripts and protein product at 6,12, and 24 h after radiation treatment; however, increased levels of p21WAFl transcript and protein are not sustained in HER-2overexpressing cells exposed to radiation in the presence of rhuMAb HER-2. Although transcript and protein levels increase at 6-12 h, they are both diminished by 24 h. Levels of p21WAFl transcript and protein at Received 7/24/98; accepted 1/15/99. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by a Stop Cancer Award (to R. J. P.); National Cancer Institute Awards R29-CA60835 (to R. J. P.), R01-CA36827 (to D. J. S.), and P01-CA32737 (to D. J. S.); United States Army Grants MRMC DAMD17-94-J4370 (to R. J. P.) and DAMD17-94J4118 (to D. J. S.); and funds from the Revlon/UCLA Women's Cancer Research Program (to D. J. S.). 2 To whom requests for reprints should be addressed, at Division of HematologyOncology, UCLA School of Medicine, 11-934 Factor Building, Los Angeles, CA 90095. Phone: (310) 825-5193; Fax: (310) 825-6192. 24 h are significantly lower than in cells treated by radiation without antibody. A reduction in the basal level of p21WAFl transcript also occurred after 12-24 h exposure to antibody alone. The effect of HER-2 antibody may be related to tyrosine phosphorylation of p21WAFl protein. Tyrosine phosphorylation of p21WAFl is increased after treatment with radiation alone, but phosphorylation is blocked by combined treatment with antireceptor antibody and radiation. This dysregulation of p21WAFl in HER-2-overexpressing breast cells after treatment with rhuMAb HER-2 and radiation appears to be independent of p53 expression levels but does correlate with reduced levels of mdm2 protein. These data indicate that human breast cancer cells damaged by radiation may be especially vulnerable to injury if they are also deprived of essential signal transduction pathways provided by the HER-2 growth factor receptor pathway.