Interplay of nuclear signalling kinase PKC theta and the gene regulatory machinery in T-cells and tumour cells

Abstract

The implications of targeting a novel class of epigenetic enzymes, nuclear signalling kinases, are beginning to be appreciated in clinical oncology and T-cell biology. In this thesis, I will cover the regulatory role of one of these nuclear signalling kinases, PKC-θ, its mechanism of nuclear localisation and the interplay with components of the transcriptional regulatory machinery such as transcription factors, splicing factors and epigenetic enzymes in the context of the immune and cancer setting.
This thesis examines the nuclear translocation and expression of PKC-θ. Showing for the first time that PKC-θ is expressed within the nucleus of Jurkat-T-cells and MCF-7 cancer cells and has two separate NLS’s, a canonical NLS and a novel SPT NLS motif activity that only functions within the T-cell line. This thesis also addresses the importance of PKC-θ in cancer cell mesenchymal signatures showing for the first time that overexpression of nuclear PKC-θ can drive a mesenchymal, stem-like cancer phenotype in cancer cell lines (Chapter 3). To address the functions of nuclear PKC-θ in regulation this thesis investigates the indispensable role of PKC-θ in splicing and transcriptional machinery. Briefly, this thesis demonstrates that PKC-θ is responsible for the phosphorylation of histone marks H2Bs32 and H2Bs36 as well as the nuclear expression and phosphorylation of the transcription factor p65 and the splicing factor SC35 in T-Cells and regulation of H2Bs32 and p65 in mesenchymal breast cancer cells (Chapter 4).
Lastly this thesis examines the implications of PKC-θ and LSD1 in variety of cancer models including cell-lines, mouse cancer models and patient derived circulating tumour cells (CTCs). This chapter shows for the first time that LSD1 and PKC-θ have a role as onco-epi-enzymes and may be implicated in cancer progression, metastasis, and the ability of cancer to evade therapy and express stem-like signatures, regulating mediators of mesenchymal signatures such as FOXQ1. This chapter also discusses the implications of onco-epi-enymes as prognostic and predictive biomarkers and drug targets to assist in patient stratification and therapeutic intervention. Further to this work I will discuss the development of novel liquid biopsy-based biomarkers in this context and discuss the potential implications for the utility of epi-therapy in combination with immunotherapy and chemotherapy.
In summary, this thesis has demonstrated the importance of nuclear PKC-θ in regulating the nuclear dynamics of the splicing factor SC35 and the transcription factor P65, influencing both protein expression and transcriptional programs in T-Cells. Hence demonstrating an elegant epigenetic enzyme that has implications in the transcriptional memory model of T-Cells through specific phosphorylation of precise histone marks, in particular H2Bs32p and H2Bs36p. PKC-θ’s critical role is not restricted to that of T-Cells as this thesis also demonstrates the importance of nuclear PKC-θ expression in cancer cells, particularly in mesenchymal, breast cancer cells and chemo-resistant cancer cells in mouse xenograft models as well as PKC- θ’s role in regulation FOXQ1 a mesenchymal promoting transcription factor (Chapter 5).
This thesis has also demonstrated an approach to isolate circulating tumour cells (CTCs) from liquid biopsies to examine protein expression and subsequently demonstrated the high expression of PKC- θ in CTCs derived from patients with high disease burden and its potential as a biomarker of disease burden and tumour response (Chapter 5).
In summary, this Thesis demonstrates that nuclear PKC-θ has a critical role in normal cell responses regulating T-Cell biology, transcription, splicing and protein expression and is critical for normal immune function, however dysregulation of PKC-θ expression is intimately tied to a progressive cancer disease burden, metastatic events and cancer therapeutic evasion as well as regulating critical factors such as LSD1 and FOXQ1 implicated in mesenchymal cancer. PKC-θ is therefore a important target for both therapeutic intervention and a biomarker for tracking disease burden and immune responses.

Date of Award	2019
Original language	English
Supervisor	Reena GHILDYAL (Supervisor), Sudha Rao (Supervisor) & Chloe Lim (Supervisor)