Early biomarkers of response of mammary tumors to preventive/therapeutic agents

B33 In most preclinical breast cancer therapy and prevention studies xenograft models in mice, mammary carcinogenesis models in rats and transgenic mouse models which spontaneously develop breast cancer, have been used. The efficacy of agents has been mostly assessed by reduction in tumor incidence and multiplicity, which occur after several weeks or months of treatment and requires substantial amounts of test agent(s). We have been examined the ability of short-term biomarker assays (7 - 10 days treatment of palpable mammary tumors) employing limited amounts of the test agents to predict the long-term preventive efficacy of various drugs. The biomarkers we assessed included: the proliferation index (PI) estimated by in vivo labeling with BrdU, 100 mg/kg bw., 2 hrs before sacrifice of the animals, apoptotic index (AI) (TUNEL assay), and the cell cycle distribution (S-phase) by FACS analysis of tumor cells generated from frozen samples. We compared the data for the long term inhibition of mammary carcinogenesis (tumor multiplicity) with short term effects on PI and AI by various agents including by tamoxifen at two dose levels, 2.5 and 1.0 mg/kg diet; vorozole, an aromatase inhibitor, at 2.5, 0.32 and 0.08 mg/kg/bw i.g.; the farnesyltransferase inhibitor, R115777 (FTI), at 50 mg/kg/bw; 9-cis retinoic acid (9cRA) at 50 mg/kg diet; the RXR agonists targretin at 150 or 15 mg/kg diet and the newly synthesized rexenoid agonist UAB30 at 200 mg/kg diet; sulindac at 800mg/kg diet; phenyl butyric acid (PBA) at 4000 mg/kg diet; meclezine at 1000 mg/kg diet and 5,6-benzoflavone at 165mg/kg diet were compared with effects on proliferative activity(BrdU-LI) and apoptosis (AI) index in tumors treated short term with the agents. The above agents decreased tumor multiplicity by: tamoxifen 100 and 74%; vorozole 100, 85, and 48%; FTI 73%; 9cRA 70%; targretin 85 and 18%; UAB30 62%, sulindac 9%; PBA14%; meclizine 48%; and 5,6-benzoflavone 53%. A decrease in PI (BrdU-labeled cells) and increase in AI was found in tumors following short-term treatment with effective agents. The values for PI and AI in tumors of control animals were respectively: 12.8∓3.2% and 0.6∓0.2%. PI was decreased: by Tamoxifen 80 and 50% for the high and low doses and AI increased 7 and 3.5 fold respectively; by vorozole ↓ PI 90, 70 and 65%, and with apoptosis↑ AI increased 6, 4 and 3 fold respectively; by FTI ↓ PI - 79% and ↑ AI a 6 fold increase; by 9cRA ↓ PI - 55% and an ↑ AI 3 fold increase; by targretin ↓ PI 70 and 35% and ↑ AI 3 and 2 fold increase; by UAB30 ↓ PI 60%, but had with no effect on apoptosis; by 4,5,6-benzoflavone ↓ PI 30%, with no effect on apoptosis; by meclizine ↓ PI 50% and ↑ AI a 3 fold increase. The ineffective agents PBA and sulindac did not change PI and AI in mammary tumors. In general highly effective agents, which decreased tumor multiplicty > 66%, tended to significantly decrease proliferation and increase the apoptotic index, while ineffective agents had insignificant effects on either parameter. Finally moderately effective agents which decreased tumor multiplicty 25-65% routinely decreased proliferation but had more variable effects on AI. The preliminary data for S-phase fraction from FACS analysis correlate with those of BrdU-labeled cells particularly at high dose levels. Currently we are evaluating the feasibility of a second line of biomarkers mostly related to cell cycle regulation (cyclin D1 and E, p21, p27) and apoptosis (caspase 3, Bcl-2, Bax, Bed, Bcl-Xl).This study was supported by NCI, N0-CN-95023, NO-CN-05115 and NO-CN-15112