The Role of Brm in Non-­‐Melanoma Skin Cancer Progression

Abstract

Background: Australia has the highest incidence of non-melanoma skin cancer (NMSC) in the world. These cancers are predominantly caused by exposure to ultraviolet radiation (UVR) in sunlight. Brm is an ATPase subunit that drives chromatin remodelling via the SWI/SNF complex. Thus, Brm is able to regulate access to transcription factors and repair enzymes to DNA, in addition to cell division. The loss of Brm has been observed in several cancer types, where its loss correlates with decreased patient survival. The loss of Brm has been identified in NMSC, but not pre-malignant lesions, suggesting Brm plays a role in the progression of a benign skin lesion into a malignant skin cancer. Brm-deficient mice also harbour excessive levels of epidermal hyperplasia as well as heightened tumour incidence following UVR as compared to mice with wildtype Brm. Moreover, a novel hotspot mutation of Brm (Q203K) has also been discovered in 17% of NMSC studied, which was the first mutation of Brm discovered in any type of cancer. Aims: This thesis aims to study the function of the Q203K mutation as compared to the loss of Brm in several key aspects of keratinocyte responses to UV-induced DNA damage: proliferation, cell cycle regulation, UVR-induced cell death and DNA repair. Methods: The loss of Brm was studied with keratinocytes grown ex vivo from neonatal Brm-/- mice and compared to keratinocytes grown from Brm+/+ mice. Further, a human keratinocyte cell line, HaCaT was also studied, in combination with Brm miRNAs to knockdown Brm expression in these cells. The overexpression of either wildtype or Q203K Brm was also studied in HaCaTs. These cells were treated with UV, and their responses were observed using assays for cell growth, cell death, cell cycle regulation and DNA repair. Results: In neonatal mouse keratinocytes, as well as HaCaT cells, the loss of Brm expression led to an altered cellular response to UVR. Cells lacking Brm exhibited increased proliferative potential following UVR due to a reduced time spent in G1 cell cycle arrest. Further, Brm-deficient cells displayed increased formation of UVR-induced cyclobutane pyrimidine dimers (CPDs) in the dark. Interestingly, when Brm was knocked down in the immortalised human keratinocyte cell line HaCaT, these cells were not as sensitive to the loss of Brm in combination with UVR, and showed less prominent effects on both cell cycle regulation and CPD formation. It was hypothesised that primary neonatal keratinocytes were more sensitive to UVR due to these cells being naïve, whereas HaCaT cells already exhibit several UVR-induced mutations, such as that of p53. Therefore, the loss of Brm in mouse neonatal keratinocytes was more catastrophic to the cell. Lastly, the mutation of Q203K did not lead to increased cellular sensitivity to UVR, suggesting the presence of this mutation in NMSC was most likely non-functional. However, increasing levels of Brm in HaCaT cells did lead to enhanced levels of cell viability at high density, which the Q203K mutation was able to reverse, suggesting Q203K does display some functionality, and thus may play a role in other functions not studied in this thesis. Conclusions: The loss of Brm but not the Q203K mutant in combination with UVR is enough to cause increased cellular sensitivity in the cell, leading to the proliferation of cells whilst harbouring DNA damage via defective cell cycle checkpoints. This would likely lead to heightened levels of mutations in the cell, and increased carcinogenesis downstream, as previously witnessed in Brm-deficient mice. The Q203K mutation was not comparable to the loss of Brm, and this thesis suggests that the Q203K mutation in NMSC is not likely to contribute to increased carcinogenesis, however further testing would have to be conducted to fully determine this.