Role of caspase-3 in supporting normal and malignant human mammary cells

Mechanisms that regulate cell survival and proliferation are important for the both the development and homeostasis of normal tissue. Elements of the pathways that mediate these roles are also variably deregulated both during the establishment and perpetuation of malignant states. CASPASE-3 (CASP3) is well known for its key function in apoptosis, but an involvement in other cellular processes has also been documented. The investigations carried out in this thesis focused on understanding if and how CASP3 might affect the proliferation, survival, and cell cycle progression of normal and malignant human mammary cells. A series of lentiviral-mediated CASP3 knockdown (KD) experiments ± vectors encoding the wild-type CASP3 cDNA or a cDNA lacking the catalytic domain required for its apoptotic function showed that loss of CASP3 diminished both the growth and survival of normal and malignant human mammary cells in vitro and the growth of malignant human mammary cells in vivo. Moreover, the “rescue” experiments showed these activities of CASP3 did not require its known catalytic function. Initial mass spectrometry-based proteomic comparisons of control and CASP3 KD cells were then used to identify molecular targets and pathways dependent on CASP3. This revealed a number of candidates with known roles in controlling the clearance of protein aggregates by the ubiquitin–proteasome system (UPS) and components of the autophagy process. Additional intracellular flow cytometric analyses of these candidate molecular targets in both normal and malignant human mammary cells showed that the levels of protein aggregates were consistently highest in the CASP3 KD cells and, conversely, CASP3 overexpressing cells harboured reduced levels of protein aggregates further implicating an important role of CASP3 in the clearance of protein aggregates. These observations suggest that the toxic consequences of an accumulation of protein aggregates might be a driving factor in causing the impaired survival and proliferation of CASP3 KD human mammary cells. The results of functional assays of the effect of inhibiting these targets added strength to the idea of their involvement in the dependency function of CASP3 in normal mammary cells that may be exploited to enhance the treatment of highly aggressive types of human breast cancer cells.Medicine, Faculty ofMedical Genetics, Department ofGraduat