Investigating the pathophysiological mechanisms of novel therapeutic targets in triple-negative breast cancer

Abstract

Triple-negative breast cancer (TNBC) patients have the worst prognosis and are the most aggressive metastatic subtype. The heterogenicity of the TNBC subtype is closely related to variable clinical outcomes. Therefore, developing new therapeutic targets for this poorly managed breast cancer subtype continues to be an urgent, unmet clinical need. This thesis investigated the therapeutic inhibition of the histone deacetylase 6 (HDAC6) protein in TNBC cells and measured the post-transcriptional chemical modifications on the transcriptome of a TNBC cell line.

HDAC6 protein is the largest class IIb histone deacetylase isoform and deacetylates α-tubulin, HSP90 and cortactin, mediating cellular responses such as cell migration, proliferation, and survival. The overexpression of HDAC6 has been identified in a wide variety of tumours. It is required for malignant transformation and tumorigenesis. Interestingly, high expression of HDAC6 is associated with the more aggressive and invasive breast cancer subtypes, such as TNBC. Recent research suggests that up to 30% of breast cancers could be sensitive to HDAC6 inhibition.

However, what makes a breast cancer tumour sensitive to HDAC6 inhibition is still unclear. Therefore, this study aimed to evaluate the underlying molecular characteristics of effective HDAC6 inhibition in TNBC cells. Our study demonstrated that TNBC cell proliferation cultured in 2D was unaffected by HDAC6 inhibition. In contrast, TNBC cells cultured as tumour spheroids in the presence of Matrigel® were sensitised to selective inhibition of HDAC6. Similarly, the tumour spheroid growth of the MDA-MB-231 TNBC cells stably overexpressing a catalytically dead HDAC6 mutant protein was reduced compared to control cells by the presence of Matrigel®. This study tested the hypothesis that the basement membrane proteins and growth factors in Matrigel® promoted oncogenic stress signalling in the tumour spheroid cultures, increasing the cells' reliance on stress responses mediated by HDAC6 for survival. To upregulate oncogenic stress in TNBC cells, 2D cultures were treated with a phosphatase inhibitor, LB100. LB100 produced increased phospho-activated protein kinase (AKT) in the 2D cells, similar to responses measured in the spheroid cultures, suggesting activation of increased oncogenic signalling. The presence of LB100 also sensitised TNBC cells to HDAC6 inhibition. Western blot analysis revealed the presence of increased y-H2AX, acetylated Histone 3 and phosphorylation of AMP-protein kinase (AMPK) levels in LB100 treated cells, which was further increased by HDAC6 inhibition. This suggested that increased DNA damage, cell cycle and metabolic stress responses due to increased oncogenic signalling produced in the presence of LB100 became toxic to the TNBC cells when HDAC6 was inhibited. In silico analysis revealed that HDAC6 was co-enriched with the proliferative marker Ki67 in TNBC patients. Moreover, patient data showed a significant positive correlation between HDAC6 and Ki67 among TNBC subtypes, suggesting that TNBCs expressing high Ki67 levels may be predictive of a hyperactive oncogenic profile that could be targeted with selective HDAC6
inhibitory therapy.

To investigate additional therapeutic targets for TNBC, we measured the RNA methylation profile of the MDA-MB-231 cell line. RNA modifications in breast cancer were reported to be significantly altered. Public databases and clinical studies also showed that the regulators of RNA modifications have prognostic value in TNBC. The methylation of the MDA-MB-231 cell transcriptome was measured by Oxford Nanopore Technology (ONT) direct RNA sequencing (DRS). To investigate the levels of 1-methyladenosine (m1A) and 5-methylcytosine (m5C) RNA methylation, the MDA-MB-231 cell lines were enriched for RNA methylation on m1A and m5C by reducing their respective RNA writer and eraser enzymes with siRNA. Interestingly, the m1A and m5C RNA modifications were highly enriched in gene-mediated protein synthesis and cellular metabolism. These cellular pathways are significantly altered and associated with TNBC pathogenesis. Thus, studying the post-transcriptional chemical modification of RNA by DRS is a novel emerging technology that could reveal hidden biology in the pathogenesis of TNBC.

Thesis is embargoed until 31 December 2028.

Date of Award	Dec 2024
Original language	English
Awarding Institution	Queen's University Belfast
Sponsors	University of Petra
Supervisor	Fiona Furlong (Supervisor) & James Barrow (Supervisor)