An abundant 24 - 29 kDa polypeptide associated with the presence of estrogen receptor (BR) in human breast tumors and tumor derived cell lines has recently been identified as the small heat shock or stress protein, hsp27. Preliminary clinical studies suggest that the presence of this protein in breast tumors is an indicator of functional ER and that elevated levels of hsp27 correlate with aggressive metastasis and poor prognosis. In ER+ breast tumor cells and uterine endometrium, hsp27 gene expression is regulated by both stress and estrogen. The first goal of this proposal is to learn how estrogen regulates expression of the hsp27 gene. The effects of estrogen stimulation on hsp27 gene transcription, mRNA abundance, mRNA stability, and hsp27 protein accumulation will be studied. Regulatory elements controlling hsp27 gene transcription in response to stress and estrogen stimulation will be identified by mutational analysis, gel band retardation assays, and in vivo DNA footprinting. The question of whether ER directly activates transcription of the hsp27 gene will be addressed in mammary tumor lines expressing mutant forms of the receptor. The second objective of this project is to determine the influence of hsp27 on cell growth parameters and survivaL The ER+ cell line normally expressing high levels of hsp27 will be used to develop variants containing copies of the hsp27 gene in the antisense orientation. Clonal cell lines expressing different levels of hsp27 will be compared with respect to proliferation rate, motility, chemotaxis and drug resistance. An ER negative breast tumor cell line that normally expresses low levels of hsp27 will be transformed with a plasmid containing an hsp27 gene under control of a metallothionein promoter. After establishing conditions for hsp27 induction by low levels of cadmium, the effect of elevated hsp27 levels on cell growth parameters will determined. The interaction of hsp27 with other cellular constituents will be compared in mammary tumor cells and fibroblasts where the protein may have distinct physiological effects on proliferation. Results obtained from this study should clarify the relationship between ER and expression of hsp27 in mammary tumors and could provide a conceptual framework for the role of this stress protein in tumor progression and metastasis.
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