To investigate the functional role of PLAC8 in a human primary breast cancer cell, Hs578T(P) and its invasive variant, Hs578T(i)8

Breast cancer is the most common cancer and the second leading cause of cancer-related death in women in Ireland. Due to the luck of a clinically relevant cell line model of breast cancer progression, our lab previously established an isogenic invasive variant, Hs578T(i)8 cell line from the original Hs578T cell line produced by Hackett et al 1977. Analysis of gene expression profiles in the parental Hs578T and Hs578T(i)8 cells using microarrays identified a gene called placenta specific 8 (PLAC8) as being the most upregulated gene in the Hs578T(i)8 cells. Gene expression data was confirmed at the mRNA level, but protein expression could not be confirmed due to the lack of availability of an antibody to PLAC8. In this current study the expression and localisation of PLAC8 protein was investigated in the parental cell line, Hs578T and the invasive variant Hs578T(i)8 cells. Using immune-histochemistry PLAC8 was shown to localise in the cytoplasm and nucleus. Western blot analysis showed a 14-fold increase in PLAC8 protein expression in the Hs578T(i)8 cells relative to the Hs578T cells. Analysis of the growth rate in batch culture showed a shorter doubling time of 26.5 hours in the Hs578T(i)8 in comparison to 32 hours in Hs578T cells. In addition, this study highlighted a potential role of PLAC8 overexpression in modulating apoptosis and autophagy following treatment with pharmacological inducers, doxorubicin and rapamycin. Preliminary results indicated that induction of apoptosis following treatment with doxorubicin showed an increase in PLAC8 expression in the Hs578T(i)8 cells relative to the nontreated control with reducing PLAC8 expression during treatment. On the other hand, there was a correlated decrease in PLAC8 relative expression determined from the rapamycin induced treatment in the Hs578T(i)8 cells. A study examining the effect of serum starvation was carried out to examine the role of PLAC8 in cell survival. Serum starvation demonstrated a negative correlation with reducing PLAC8 expression plus a reduction in apoptosis and autophagy through p62 expression. This thesis offers novel methodologies including live cell assessment of apoptosis using flow cytometry and a defined procedure for the measurement of autophagy using western blotting with LC3I/II turnover and p62. The results obtained from these experiments supports a role for PLAC8 in modulating both apoptosis and autophagy processes in the invasive cell line model of breast cancer.