This proposal will address the hypothesis that AKT1, the cellular homolog of the protein-serine/threonine kinase oncogene v-akt, plays a key role in the proliferation, survival and etiology of breast cancer. This will be addressed by the following Specific Aims: 1) Determine the role of AKT1, and possibly other AKT isoforms, in estrogen- and growth factor-dependent proliferation of human breast cancer and breast epithelial cells. AKT1 activity will be quantified by immunocomplex kinase assays and levels will be determined by RNase protection assay and western blotting. AKT1 activity will be correlated to proliferation in response to estradiol (in ER+ cells) and growth factors (eg. IGF-I). The role of different AKT isoforms will also be assessed with dominant-negative AKT retroviruses and isoform-specific AKT antisense oligonucleotides (AONs). 2) Determine the role of AKT1 in preventing apoptosis in breast cancer and breast epithelial cells. The role of AKT1 in apoptosis will be evaluated with dominant-negative forms of AKT1 or isoform-specific AKT AONs. Apoptosis will be characterized by in situ end-labeling of DNA breaks, and caspase-3 activation. Changes in gene expression resulting from inhibition of AKT1 will be assessed by GeneChip Oligonucleotide microarray analysis and subtractive hybridization. The post-translational effects of AKT1 in preventing apoptosis will be evaluated by determining the phosphorylation of bcl-2 family members in breast cancer cells transduced with a dominant-negative AKT1 and AKT AONs. 3) Determine if mammary gland-directed expression of the AKT1 and Gag-akt1 transgenes results in hyperplasia and transformation. Transgenic mice will be generated that express AKT1 or the constitutively active Gag-akt1 oncogene in the mammary gland under the control of the MMTV LTR. Gene expression in mammary tissue from wild-type and transgenic mice will be assessed by GeneChip and subtractive hybridization analyses. Primary cultures of mammary epithelium and tumors arising from transgenic animals will be characterized for growth factor-dependent proliferation, susceptibility to apoptosis, anchorage-independent growth in soft agar and tumorigenicity in nude mice.
