Uncovering the molecular circuitries of deleted in oral cancer 1 (DOC1) in oral squamous cell carcinomas (OSCC)

Abstract

Oral squamous cell carcinomas (OSCCs) are the most common type of malignancy affecting the oral cavity, characterised by formation and invasion of squamous cell islands deep to the surface epithelium. Tumour resection is the first line of treatment for early stages of OSCC. However, OSCC is often detected in later stages at which point adjuvant radiotherapy and/or chemotherapy are required. Currently, there are no targeted therapies for OSCC. As such, 5-year survival rate following oral cancer diagnosis is 60% in the UK. Indeed, there is a need to develop new treatment strategies for OSCC.

Deleted in Oral Cancer 1 (DOC1) plays diverse roles in epigenetic reprogramming as part of the Nucleosome Remodeling and Deacetylase (NuRD) complex, as well as in cell cycle regulation through its reported interactions with the cyclin-dependent kinase, CDK2. Crucially, DOC1 protein is not expressed in the majority of cases of OSCCs, and this loss of expression is corelated to poorer prognosis in patients. Further, the lab previously demonstrated a transition from highly metastatic mesenchymal phenotype to a less tumourigenic epithelial phenotype in OSCC cell line SCC9 following lentiviral overexpression of DOC1. This points to a prominent role for DOC1 in OSCC, one that could be exploited for targeted OSCC therapy. Yet, our knowledge of the molecular circuitries of DOC1 in OSCC is lacking.

To better understand the functions of DOC1, its interactome needed to be elucidated. Through unbiased proteomics, we confirmed DOC1’s association with subunits of the chromatin remodeler NuRD. Ubiquitous and strongest DOC1 associations were reported for subunits CHD4 and GATAD2A/B based on peptide intensity values from pull-down (PD) assays on HaCaT and OSCC cell lines SCC4 and SCC9 cells, and immunoprecipitation (IP) assays using antibodies against DOC1 on HeLa and HaCaT cells. Moreover, DOC1 interactions with cell cycle regulators and proliferative markers including CDK1 and PCNA were identified. These associations were cell-type-dependent and highlighted the heterogenous interactome of DOC1. These findings directly link DOC1 to molecular functions such as epigenetic reprogramming and cell cycle regulation.

To further investigate DOC1 biology in OSCCs, a structure-function analysis was performed through lentiviral overexpression of wild-type (WT) DOC1 or its amino acid substitution mutants in SCC9. Overexpression of WT DOC1 did not result in discernible changes in assessed phenotypes. Strikingly, several mutants induced attenuation in proliferations rates, increased DNA damage, and alterations in histone H3K27 marks, suggesting de novo functions of DOC1 mutants. These findings elucidate the mechanistic roles played by DOC1’s amino acid residues towards DOC1 functions.

While DOC1 expression is absent at the protein level, doc1 gene is intact in OSCC. It was therefore hypothesised that doc1 gene is epigenetically silenced. To interrogate the mechanism by which doc1 is repressed, inhibitors of enzyme DNA methyltransferase (DNMT) such as decitabine and 5-azacytidine were utilised. Our findings revealed the rescue of DOC1 protein expression following DNMT inhibitor treatment. Further, we demonstrated that a locus within the doc1 gene promoter is hypermethylated in SCC9 cells and doc1 de-repression following treatment with decitabine is a direct result of its hypomethylation.

The epigenetic landscape in the presence or absence of DOC1 in SCC9 cells was also investigated using single cell Assay for Transposase-Accessible Chromatin followed by sequencing (scATAC-seq). Variations in chromatin accessibility were observed following treatment with decitabine or lentiviral overexpression of wild-type DOC1 or its mutant Serine-24-Alanine. scATAC-seq data generated in this work could be integrated into future multi-omics approaches. Finally, we demonstrated alterations in enrichment of histone marks H3K27me3 and H3K27ac as well as DOC1 binding to selected gene promoters using chromatin immunoprecipitation followed by quantitative polymerase chain reaction (ChIP-qPCR).

Findings in this thesis support the crucial role for epigenetic regulation in OSCCs. These represent the quintessential features of chromatin remodelers and epigenetic modifiers to adapt their identity to an ever-changing environment in the journey from genomic instability to cancer. Our studies highlight the dynamic equilibrium between epigenetic regulation and cell cycle pathways in OSCC. Such an intertwined system of control opens novel opportunities for the development of targeted therapeutic strategies for OSCCs.

Thesis is embargoed until 31 December 2024.

Date of Award	Dec 2023
Original language	English
Awarding Institution	Queen's University Belfast
Sponsors	Northern Ireland Department for the Economy
Supervisor	Adone Tielenius Kruythoff-Mohd Sarip (Supervisor) & Paul Mullan (Supervisor)