Functional characterization of MCPH1/BRIT1 effector genes in oncogenesis

The MCPH1 protein plays an essential role in many functions including DNA repair, cell cycle checkpoints and apoptosis. Premature chromosome condensation (PCC) is a hallmark of MCPH1 knockdown in which cells enter into mitosis prematurely before they complete DNA synthesis. Previously reduced levels of MCPH1 has been identified in 29% (93/319) of breast cancers (BC), associated with high tumour grade and reduced survival. Previously, a high throughput human protein kinase (hPK) siRNA sublibrary has identified a protein kinase (PKMYT1) as PCC modifier. PKMYT1 and WEE1 play a role in the regulation of mitosis through phosphorylation of CDK1. Double knockdown of PKMYT1 or WEE1, in combination with MCPH1, showed mitotic abnormalities and increased cell death compared to single siPKMYT1/NT or siWEE1/NT using time-lapse imaging. siMCPH1 knockdown cells showed WEE1 up-regulation at both the RNA and protein level suggesting that MCPH1 represses WEE1 expression. Furthermore, MCPH1 knockdown in interphase and PCC cells demonstrated dispersed PKMYT1 staining associated with dispersal of the Golgi apparatus which suggested a novel role of MCPH1 in Golgi regulation. ARF1 is a potential mediator of these processes, since ARF1 plays a role in both chromosome condensation and Golgi dispersal, suggesting a potential link between ARF1 and MCPH1 in Golgi regulation. Previously, a second hPK siRNA screen identified candidate synthetic lethal (SL) genes in MCPH1/BRIT1-deficient cells, TTK and CDK1. TTK, a spindle checkpoint gene and cyclin dependant kinase CDK1, an essential cell cycle regulator were subsequently validated as potential MCPH1 SL genes. The expression of TTK and CDK1 proteins was examined in BC tissue pre- and post-NACT samples using immunohistochemistry. TTK expression showed significant correlation with TNBC (p =0.020) and metastases (p =0.023) in post-NACT cohort. Reduced TTK expression correlated with longer overall survival (OS) pre-NACT (p < 0.000). TTK expression increased significantly in response to NACT in post-NACT samples (p=0.007). Increased CDK1 expression correlated with high tumour grade (p=0.023) in pre-NACT samples. However, no correlation between CDK1 expression and OS was found. Correlation analysis identified significant correlation between TTK and MCPH1 expression (p=0.036). These findings suggest that TTK and CDK1 are potential BC biomarkers and therapeutic targets in MCPH1-deficient cancer cells. In conclusion, the study of the function of genes whose inhibition promote cell death in MCPH1-deficient cells would identify potential therapeutic targets in tumours with compromised MCPH1 expression or function.