Abstract
Pathogenic variants in three genes associated with DNA replication licensing: ORC1, CDC6 and CDT1 are associated with the rare autosomal disorder, Meier-Gorlin Syndrome (MGORS). Those with MGORS present with a global reduction in body size alongside other characteristics such as mammary hypoplasia. Intriguingly, these same three genes are also overexpressed in breast cancer, elucidating a direct link between these pre-replication complex (PRE-RC) genes and breast tissue. Post-developmentally, the somatic tissue of those with MGORS is unaffected. This suggests that a reduced functioning of the PRE-RC is tolerable and not lethal. Subsequently, these pathogenic variants provide a potential guide for a novel therapeutic treatment without the cytotoxic effects that conventional anti-cancer treatments offer for breast cancer patients.
The R105Q (p.Arg105Gln) and R462Q (p.Arg462Gln) amino acid substitutions are the most common MGORS variants affecting ORC1 and CDT1, respectively. Functionally, they partially hamper the ability of the proteins to form a pre-replication complex and appropriately license replication origins on DNA. The molecular pathophysiology of MGORS suggests that this causes a reduction in cellular proliferation and overall body size. Transient transfection of mammary epithelial MCF10A cells with plasmid constructs harbouring wild type ORC1, CDC6 and CDT1 increased their proliferation rate 3.84-fold, 1.96-fold and 4.39-fold respectively over 3 days compared to an empty vector control plasmid. Furthermore, transient transfection of ORC1 and CDT1 pathogenic variants into MCF10A cells resulted in proliferation rates the same as that of the empty vector control. This indicates that the effects we see for ORC1, CDT1 and CDT1 on proliferation is a direct result of the open reading frames, an effect that is completely lost with ORC1 and CDT1 pathogenic variants associated with MGORS. These results prove a functional relationship between MGORS variants R105Q and R462Q and proliferation in the MCF10A cell line.
MCF10A expresses low RNA transcripts of ORC1, CDC6 and CDT1. Intriguingly, protein analysis by western blot identified higher than expected expression levels of these proteins in MCF10A cells. We established stable isogenic derivatives of MCF10A cells overexpressing ORC1, CDC6 and CDT1, and confirmed stable expression by western blot (CDC6 and CDT1) and qRT-PCR (ORC1). An initial increase in CDC6 protein expression was followed by a decrease in CDC6 expression as cell passaging continued. This suggested that MCF10A cells expressing high levels of the CDC6 transgene may be being outcompeted in the polyclonal cell population, and that the baseline level of expression that these MCF10A cells show, could be at their maximum tolerated limit. Thus, exceeding this limit with overexpression could incur DNA damaging effects. Potential evidence of MGORS transgenes causing genomic instability was observed by an increase in DNA damage foci in MCF10A cells transiently transfected with ORC1 or CDC6 compared to empty vector control, but further repetitions of this experiment are required.
Overall, this project aimed to prove a functionality between DNA replication licensing genes, their MGORS-associated variants, and mammary epithelial cell proliferation to guide a novel approach for breast cancer treatment.