HORMONAL REGULATION OF C-HA-RAS AND ROLE IN METASTASIS

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Oncogenes and suppressor genes that can affect tumor progression may control important points in the regulation of genes that are ultimately responsible for alteration seen in highly metastatic cells. Hence, understanding of the mechanisms regulating expression of the genes capable of influencing tumor development and progression is crucial for providing insights leading to more effective prognosis or therapy. The long term goal of this project is to identify and characterize genes that re involved in mammary tumor development and tumor progression. Elevated levels of nonmutated c-Ha-ras proteins have been reported to play a crucial role in tumorigenesis in the mammary tissue. We have shown a direct correlation between endogenous c-Ha-ras protein levels and metastatic behavior of mouse mammary tumor subpopulations, and have identified an novel hormone responsive transcriptional regulatory element in the intron-1 of the mouse Ha-ras gene. The enhancer activity of intron-1 element in the intron-1 element is induced strongly by glucocorticoids (edamethasone, Dx), and progesterone (Pg) in nonmetastatic sublines. IN metastatic mammary sublines it is moderately induce by Dx, Pg, and E2. Two specific complexes , A1 and A2, are formed when nuclear extract s form metastatic and nonmetastatic cells are incubated with the intron-1 element. Complex B is formed only when the intron-1 element is reacted with nuclear proteins of metastatic cells. We show significant differences in c-Has-ras protein levels, complex formation with intron-1 element, and hormonal responsiveness of the ras intron-1 element between metastatic and nonmetastatic mammary sublines used in our study. The hypothesis to be tested is that in the mouse mammary system tumor progression and expression of metastatic behavior is linked to the mechanisms regulating/deregulating Ha-ras gene expression. Our specific aims are: 1) To determine if hormonal responsiveness of ras expression is related to a mammary tumor phenotype; i.e., do cells with low p21ras levels exhibit greater hormone mapping whether there are other regulatory sites in intron-1 of the mouse Ha-ras gene and how they and regulatory sites 5-of intron-1 interact with the intron-1 of the mouse Ha-ras gene and how they and regulatory since similar motifs containing the ERE-and GRE-half sites are present in intron-1 element, since exon-1 of mouse, rat and human c-Ha-ras genes. 3) To define and characterize the transacting factors interacting with the ras intron-1 element in metastatic and nonmetastatic cells. 4) To determine whether the intron-1 element can confer hormonal responsiveness in in vitro transcription systems. 5) To determine whether E3 and glucocorticoid regulates c-Has- ras levels in the mouse mammary sublines primarily a the transcriptional levels or also by altering the stability of the proteins. Completion of these studies in a well-defined mouse metastasis a model system will provide valuable information on the mechanisms mediating transcriptions regulation of the Ha-ras gene in mammary tumor cells. Once characterized, trans-acting factor(s) specific to metastatic mammary cells will be of use in functional assays to more completely define the role of ras in metastasis.