This project will focus on the role of activation of polyamine biosynthesis and MAPK signaling cascade (by either oncogenic ras or HER-2neu) in breast cancer development. The rationale for these studies is provided by (1) Dr. Manni's preliminary data suggestive of a role for polyamines in mammary carcinogenesis, (2) the demonstrated cooperativity between ras signalling and polyamines in several models of malignant transformation, (3) the preliminary data showing cooperativity between HER-2neu signaling and polyamines in mammary carcinogenesis, and (4) the recent finding by the applicant that ODC overexpression increases MAPK activity in mammary epithelial cells. Furthermore, the investigator will test the importance of expression of a functional estrogen receptor in ras/HER-2neu and/or polyamine-mediated mammary carcinogenesis. He will conduct experiments in the spontaneously immortalized but non-tumorigenic MCF-10A and HMT-3522 human breast epithelial cell lines. Specifically: (1) He will determine the influence of overexpression of ornithine decarboxylase (ODC) (the first rate-limiting enzyme in polyamine biosynthesis) individually and in combination with activation of the MAPK cascade (by either ras or HER2-neu) on in vitro and in vivo features of transformation. (2) He will determine the role of the MAPK cascade pathway in mediating polyamines and/or ras/HER-2neu mediated effects by transfecting our cells with constitutively active and dominant negative mutants of several kinases belonging to this signalling cascade (e.g. Raf-1, MEK, ERK-1, ERK-2). (3) He will take advantage of the recent generation of ER transfected MCF-10A and HMT-3522 cell lines which express a functional ER to test the possible cooperativity between hormonal and oncogenic signals in the malignant transformation of human breast epithelial cells. (4) He will evaluate the """"""""cross-talk"""""""" at the transcriptional level between the estrogen receptor pathway and ras/HER-2neu/ polyamine-mediated signaling as it relates to mammary carcinogenesis. It is anticipated that the results of the proposed experiments will provide new insights into the mechanisms subserving the cooperativity between estrogens and classic oncogenic signals in inducing transformation of normal human breast epithelial cells.
