The investigation of novel drug targets for triple negative breast cancer

Abstract

Histone Deacetylase 6 (HDAC6) is a unique member of the Histone Deacetylase protein family. Unlike other HDAC proteins, it consists of two tandem catalytic domains and is localised in the cytoplasm. HDAC6 is involved in various cellular processes including cancer transformation, cell spreading, cell motility, and cell apoptosis. The selective HDAC6 inhibitors continue to be assessed under clinical trials, although their role in high grade serous ovarian cancer (HGSOC) and breast cancer are not clear.This study investigated the influence of pharmacological inhibition with ACY-1215 and protein knockdown of HDAC6 with siRNA and a HDAC6 targeting PROTAC in triple negative breast cancer (TNBC) cells. The stable overexpression of wildtype and a catalytically dead HDAC6 mutant in TNBC cells was also investigated. The enzymatic inhibition of HDAC6 with ACY-1215 was insufficient to inhibit TNBC cell viability, while elimination of HDAC6 protein expression with PROTAC led to significant growth suppression. Here, we hypothesise that HDAC6 might have enzymatic independent functions, and ACY-1215 prevents catalytic dependent functions but has no effect on catalytic domain independent functions of HDAC6. Cell viability is decreased with ACY-1215 at high concentrations, however, this is mediated by inhibition of other HDACs, whereas the PROTAC produced a stronger inhibition of cell viability following complete protein degradation. Interestingly, the most pronounced inhibition of HDAC6 with the PROTAC was strongest when the molecule produced a ‘hook effect' complete inhibition of the activity of both HDAC6 and E3 ligases. SiRNA knockdown of HDAC6 with four different siRNAs produced conflicting responses on TNBC cell proliferation with commercially validated HDAC6 targeting siRNA oligos. This demonstrates the complex nature of HDAC6 knockdown and why discrepancies in HDAC6 mediated functions have been reported in the literature. Finally, the overexpression of both full length HDAC6 and the double dead HDAC6 had similar effects on the growth rate of TNBC cells and demonstrated no significant difference compared with the empty vector control cells. In conclusion, degradation of the HDAC6 protein and not HDAC6 transcripts produced more efficacious inhibition of TNBC cell viability. However, the data suggests that HDAC6 inhibition alone is an insufficient therapy strategy for TNBC cells.

In the last experimental chapter, we validated the novel long read sequencing technology, Oxford Nanopore Technology (ONT), in detecting RNA methylation in TNBC cell MDA-MB-231. The levels of RNA methylation on m6A is modified in MDA-MB-231 cells by manipulating the expression of the m6A writer protein, METTL3, with siRNA. Changes in the levels of m6A were detected in scramble control versus siRNA treated samples. In addition, differential gene transcription by direct RNA sequencing (DRS) was evaluated in which we confirmed the knockdown of METTL3 in the siRNA treated samples. Furthermore, m6A sites were annotated by this method in which the HSP90 pathway was identified with a strong association with m6A methylation. In conclusion, we successfully detected methylation sites in MDA-MB-231 cells at nucleotide resolution and set up a validated protocol for future experiments to measure RNA methylation by DRS.

Thesis is embargoed until 31 July 2026.

Date of Award	Jul 2024
Original language	English
Awarding Institution	Queen's University Belfast
Supervisor	Fiona Furlong (Supervisor) & Niamh Buckley (Supervisor)