Elucidating the clinical and molecular significance of retrograde trafficking in triple negative breast cancer

Abstract

Triple Negative Breast Cancer (TNBC) is an aggressive and heterogeneous subtype of breast cancer (BC) accounting for 15-20% of BC cases but a disproportionate 40% of deaths. Despite some progress in targeted therapies for specific TNBC subgroups, chemotherapy remains the backbone of treatment, however, response rates vary and reliable predictors of response to treatment remain elusive. Previous analysis by the Buckley group identified several genes linked to retrograde trafficking (RT), a process which describes the trafficking of cargo in a plasma membrane to endoplasmic reticulum direction, as potential biomarkers for patient outcomes. Given the known dysregulation of RT in cancer and other diseases, it was hypothesised that deregulated RT drives poor outcomes in TNBC and could serve as a novel biomarker and therapeutic target.

Bioinformatic analyses revealed that high expression of four RT genes, Rab6A, VPS35, Rab2A or COPZ1, correlates with significantly poorer outcomes in TNBC patients, while high expression of ANKFY1 and FAM21A is associated with improved survival. These results highlight the potential of these genes as novel biomarkers in TNBC, and subsequently two genes, Rab6A and VPS35, were further explored in vitro. Gene expression modulation in cell line models through siRNA-mediated knockdown or lentiviral overexpression demonstrated an observable impact on cell proliferation, underscoring their functional relevance.

Building on these insights, a novel RT gene signature was developed from all six RT genes that can significantly predict TNBC patient outcomes in the context of SoC chemotherapy and is superior to the prediction capabilities of each gene individually. A high RT score (as defined by median gene expression), indicative of poor prognosis, was linked to ferroptosis, a recently identified form of iron-dependent cell death, suggesting a new avenue for therapeutic intervention.

Finally, the therapeutic potential of targeting RT and ferroptosis in TNBC was investigated. Notably, TNBC cell lines with higher RT scores were more sensitive to ferroptosis inducers including RSL3. Furthermore, a functional link between RT and ferroptosis was uncovered, whereby RT inhibition reduces intracellular iron levels, protecting cells from ferroptosis. Additionally, the combination of the chemotherapy regime, FEM, with RSL3 synergistically induced cell death in TNBC cells, a process partially dependent on RT, as RT inhibition rescued cells from FEM/RSL3-induced death.

The development and validation of this RT gene signature not only provides a powerful tool for predicting patient outcomes in TNBC but also identifies RT-related pathways as promising targets for tailored treatment strategies. This approach has significant translational potential, offering new hope for more effective and personalised therapies for TNBC patients.

Thesis is embargoed until 31st December 2029.

Date of Award	Dec 2024
Original language	English
Awarding Institution	Queen's University Belfast
Sponsors	Northern Ireland Department for the Economy
Supervisor	Niamh Buckley (Supervisor) & Helen McCarthy (Supervisor)