Despite considerable progress in early diagnosis and treatment of breast cancer, this disease remains a most significant cause of morbidity and mortality. A variety of oncogenes implicated in human breast cancer, induce breast tumor formation when overexpressed in the breast of transgenic mice including activating mutations of membrane receptors (ErbB2 (Neu)), non receptor tyrosine kinase proteins (pp60src) and cytoplasmic (p21ras) and nuclear (c-Myc) oncogenes. Understanding the molecular mechanisms governing cellular proliferation in breast cancer will provide important insights into alternate strategies for treatment of breast cancer. The cyclin D1 gene encodes a regulatory subunit of a holoenzyme complex (Cyclin D1-kinase, CD1K) capable of phosphorylating and inactivating the tumor suppressor retinoblastoma protein (pRB). Activity of the cyclin D1 complex is inhibited by kinase inhibitor proteins including p16Knk4a and p27Kip1. Cyclin D1 levels are increased in 70-100 percent of breast tumor cell lines and the majority of human breast cancers, and p16Ink4a and p27Kip1 levels are reduced in a proportion of breast cancers. Hormones known to be risk factors for human breast cancer, estrogen and progesterone, stimulate cyclin D1 levels. Recent studies by the laboratory and others have shown that several oncogenes capable of inducing breast cancer formation induce cyclin D1 expression. Cyclin D1 abundance is required for the earlyproliferative step induced by serum in fibroblasts. The goal of these studies is to examine the role of the cyclin D1/p16Ink4a/pRB/p27Kip1 complex in breast cellular proliferation and transformation. The abundance and activity of this complex will be assessed in tumors from several different transgenic models of breast tumorigenesis (ErbB2(Neu),pp60src,p21ras,c-Myc) and in cell lines derived from these tumors. The role of cyclin D1/p16Ink4a in breast tumor formation will be examined in transgenic animals. Transgenic mice will be established in which anti-sense or dominant negative cyclin Dq cDNA are regulated by ecdysone in a breast tissue-specific manner. These mice will be back-crossed with the transgenic breast oncogene expressing mice. These studies aim to determine whether a reduction in cyclin D1 levels or overexpression of a dominant negative cyclin D1 mutant, is sufficient to reduce the prevalence of breast tumor formation in the offspring of the back-crossed mice. In complementary studies, p16Ink4a will be assayed for tumor suppressor function in breast cancer cell lines. The role of p16Ink4a as a tumor suppressor will be examined through mating the oncogene transgenic mice with mice homozygously deleted of the p16 locus to determine whether there is an increased proclivity to breast tumor formation. Establishing whether cyclin D1/16Ink4a encodes a rate-limiting step in breast oncogenesis will pave the way for alternate molecular strategies for breast tumor therapy.
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