Monoclonal antibody to HER-2/neu receptor modulates repair of radiation-induced DNA damage and enhances radiosensitivity of human breast cancer cellsoverexpressing this oncogene

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 acc ompanied by radiation therapy, In breast cancer cells with overexpression o f HER-2 receptor, recombinant humanized monoclonal antibodies (rhuMAbs) to HER-2 receptors (rhuMAb HER-2) decrease cell proliferation in vitro and red uce tumor formation in nude mice. Therapy with rhuMAb HER-2 enhances tumor sensitivity to radiation at doses of 1-5 Gy, exceeding remission rates obta ined with radiation alone. This benefit is specific to cells with HER-2, ov erexpression and does not occur in cells without overexpression. Treatment of cells with radiation (2-4 Gy) alone provokes a marked increase in unsche duled DNA synthesis, a measure of DNA repair, but HER-2-overexpressing cell s treated with a combination of rhuMAb HER-2 and radiation demonstrate a de crease of unscheduled DNA synthesis to 25-44% of controls. Using an alterna te test of DNA repair, i.e., radiation-damaged or undamaged reporter DNA, w e introduced a cytomegalovirus-driven beta-galactosidase into HER-2-overexp ressing breast cancer cells that had been treated with rhuMAb HER-2 or cont rol. At 24 h posttransfection, the extent of repair assayed by measuring re porter DNA expression was high after exposure to radiation alone but signif icantly lower in cells treated with combined radiation and rhuMAb HER-2 the rapy. To further characterize effects of rhuMAb HER-2 and the combination o f antibody and radiation on cell growth, analyses of cell cycle phase distr ibution were performed. Antibody reduces the fraction of HER-2-overexpressi ng breast cancer cells in S phase at 24 and 48 h, Radiation treatment is al so known to promote cell cycle arrest, predominantly at G(1), with low S-ph ase fraction at 24 and 48 h. In the presence of rhuMAb HER-2, radiation eli cits 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-ph ase 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 su fficient time for completion of DNA repair in HER-2-overexpressing cells. B ecause it is well known that failure of adequate p21WAF1 induction after DN A damage is associated with failure of cell cycle arrest, we also assessed the activity of this critical mediator of the cellular response to DNA dama ge. The results show induction of p21WAF1 transcripts and protein product at 6, 12, and 24 h after radiation treatment; however, increased levels of p21WA F1 transcript and protein are not sustained in HER-2-overexpressing cells e xposed to radiation in the presence of rhuMAb HER-2. Although transcript an d protein levels increase at 6-12 h, they are both diminished by 24 h. Leve ls of p21WAF1 transcript and protein at 24 h are significantly lower than i n cells treated by radiation without antibody. A reduction in the basal lev el of p21WAF1 transcript also occurred after 12-24 h exposure to antibody a lone. The effect of HER-2 antibody may be related to tyrosine phosphorylati on of p21WAF1 protein. Tyrosine phosphorylation of p21WAF1 is increased aft er treatment with radiation alone, but phosphorylation is blocked by combin ed treatment with antireceptor antibody and radiation. This dysregulation o f p21WAF1 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 tha t human breast cancer cells damaged by radiation may be especially vulnerab le to injury if they are also deprived of essential signal transduction pat hways provided by the HER-2 growth factor receptor pathway.