The Nuclear Interactome of Chromatin-bound YAP in Cutaneous Squamous Cell Carcinoma

: (Alternative Format Thesis)

Abstract

Non-melanoma skin cancer, also called keratinocyte cancer, is the most frequently diagnosed cancer globally. Keratinocyte cancers are subcategorised into basal cell carcinoma (BCC) (73% of diagnosed cases) and cutaneous squamous cell carcinoma (cSCC) (23% of diagnosed cases) and are largely attributable to accumulated mutations caused by life-long sunlight exposure. BCC rarely metastasises, while cSCC has a higher incidence of recurrence and metastatic potential. Although only ~5% of cSCC patients progress to metastatic disease, the mortality rate is >70%. Current therapies for cSCC have limited success and exhibit significant side effects and therefore there is an urgent need to identify novel therapeutic approaches for cSCC. YAP and TAZ are transcriptional co-regulators and are key oncoproteins in the development of cSCC. The subcellular localisation of YAP/TAZ is predominantly governed by the Hippo signalling network. In their activated state, YAP/TAZ accumulate in the nucleus, where, by interacting with transcription factors and chromatin remodelling complexes, they transcriptionally regulate the balance between keratinocyte proliferation and terminal differentiation in the healthy epidermis during wound healing, and during cancer development. However, how YAP/TAZ execute their transcriptional roles in cSCC cells has not been studied. The main objective of this project was therefore to identify the protein interactome of chromatin-bound, transcriptionally engaged YAP in cSCC cells to identify potentially targetable YAP/TAZ-dependent vulnerabilities in cSCC. By using rapid immunoprecipitation mass spectrometry (RIME), and by performing co-immunoprecipitation experiments and in-situ proximity ligation assays (PLA), I have discovered that YAP interacts with a protein called RIF1 in cSCC cells. RIF1 regulates the replication timing programme and participates in DNA repair pathway choice for double-strand DNA break repair. RIME also identified previously known YAP interactors including the nucleosome remodelling deacetylase (NuRD) complex protein CHD4, and the SWI/SNF chromatin remodelling complex protein ARID1A. YAP was confirmed to interact with CHD4 and ARID1A in cSCC cells by co-immunoprecipitation and PLA. YAP/TAZ, RIF1, CHD4 and ARID1A target genes were identified using siRNA-mediated YAP/TAZ, RIF1, CHD4 or ARID1A knockdown followed by RNA-sequencing. Of note, knockdown of YAP/TAZ increased the expression of genes associated with terminal differentiation, highlighting YAP/TAZ as key suppressors of keratinocyte terminal differentiation. Bioinformatic interrogation of biological pathway enrichment within differentially regulated genes in response to knockdown of YAP/TAZ, RIF1, ARID1A, and CHD4 determined that loss of either RIF1, ARID1A, or CHD4 alone was not sufficient to elicit gene expression changes that mirrored those observed upon knockdown of YAP/TAZ. However, YAP was identified as a positive regulator of the gene expression of RIF1, acting through TEAD transcription factors. Knockdown of YAP or RIF1 caused decreased cell growth in in vitro cell growth assays. YAP knockdown increased expression of the cell cycle regulator p21, resulting in cell cycle arrest, while RIF1 knockdown caused accelerated S-phase progression and activated the replication stress pathway. Immunoblot analysis of cell cycle regulator proteins and high-content imaging-based determination of the numbers of cells in S-phase showed that ARID1A and CHD4 were both dispensable for cell cycle progression in cSCC cells. In response to replication stress and DNA damage induced by the chemotherapeutics hydroxyurea and cisplatin, YAP-RIF1 and YAP-γH2AX interactions increased when measured by using PLA. Together, these findings have identified RIF1 as an important and novel protein implicated in the proliferation of cSCC cells and establish novel transcriptional and non-transcriptional functions of YAP in cSCC. Summarised, the findings in this thesis inform on the molecular biology of YAP-addicted cancers for the purpose of developing molecular targeted therapeutics.

Date of Award	24 May 2023
Original language	English
Awarding Institution	University of Bath
Supervisor	Gernot Walko (Supervisor), David Tosh (Supervisor) & Keith Vance (Supervisor)