Brown, Thomas J.
(2024)
Investigating extracellular vesicle communication in the endocrine cancers of the prostate and breast.
PhD thesis, University of Nottingham.
Abstract
Introduction
Across different cancer types, extracellular vesicles (EVs) have been shown to play a direct role in promoting the progression and metastasis of cancer cells. Furthermore, cancer derived EVs have been shown to enable crosstalk between cancer cells and other non-tumour cell types, aiding cancer progression. We hypothesise that EVs produced by endocrine-related cancers of the prostate and breast, mediate communication with stromal cells of distant tissues and the localised microenvironment to aid in cancer progression and metastasis. This hypothesis is explored through the three aims: 1) analyse the RNA cargos of EVs produced by bone metastatic prostate cancer cells to predict their potential effects; 2) determine the molecular effects of EVs produced by bone metastatic prostate cancer cell line PC3MLuc2a in vivo; and 3) investigate the effects of EVs produced by triple negative breast cancer cells on stromal mesenchymal cells to determine changes in chemoresistance and tumour cell growth.
Methods
EV cargos were investigated by extracting both proteins and RNA from EVs derived from non-cancer prostate epithelial PNT1A and bone-metastatic prostate cancer cell line PC3 for RNAseq and LS-MS/MS mass spectrometry with subsequent qPCR validation. The resulting data was compared to previously published data to determine small regulatory RNA of interest and their protein binding partners/complexes. Further RNAseq data was collected from osteoblasts treated with PC3 derived EVs for 24 hours to determine the downstream effects of identified RNAs and proteins involved in splicing.
To assess the molecular effects, mediated by EVs produced by bone-metastatic prostate cancer cells in vivo, CD1 nude male adult mice were injected with fluorescent labelled EVs isolated from PC3MLuc2a cells. Subsequent tissue RNA extraction from hind leg knee joints was conducted and subjected to RNAseq, the resulting data was used to determine molecular changes via integrated pathway analysis.
Finally, to determine how EVs produced by triple negative breast cancer cells influence stromal cells to alter the niche to support tumour growth and chemoresistance, a co-culture system of MDA-MB-231 TNBC cells with mesenchymal cells was developed. Within which mesenchymal cells were educated with MDA-MB-231 EVs prior to co-culture, enabling us to determine subsequent effects of the EV exposure on chemoresistance to paclitaxel and cisplatin. As well as, determining tumour cell growth. Cell metabolic activity or survival after treatment was determined by total cell count, or RealTime-Glo MT Cell Viability Assay were used.
Results
From RNA-seq datsets showing differential levels of 7877 RNAs, 23 target were chosen from P value and fold change in EVs derived from bone metastatic prostate cancer cell lines compared to normal prostate epithial cell lines. Target analysis was successful with 19 RNA targets, 8 of which showed significantly different expression levels within prostate cancer derived EVs, with CTD and RNU2 being significantly increased in bone metastatic lines p=0.05. Proteomic analysis showed 9 out of 10 of the known associated proteins of RNU2. In vitro analysis of osteoblasts treated with PC3 EVs demonstrated a splicing effect mediated by PC3 EVs that was not replicated with EVs isolated from non-cancer prostate epithelial PNT1A cells.
In vivo analysis of fluorescently labelled EVs produced by bone metastatic PC3MLuc2a cells resulted in evidence of EV localisation in limb joints 27 days after treatment. Analysis of RNA extracted from the joints of those bones showed increases to expression in multiple transcription and bone specific pathways.
Breast cancer co-culture showed that treatment of mesenchymal cells with triple negative breast cancer (TNBC) derived EVs had no effect on the chemoresistance to either cisplatin or paclitaxel. However, TNBC EV exposure to mesenchymal cells before co culture revealed a significant increase to total cell count of TNBC cells p=0.001.
Conclusions
Our data suggest that EVs derived from prostate and breast cancer may assist in cancer progression by influencing osteoblasts and mesenchymal cells respectively. In prostate cancer we have elucidated several internal cargo elements which potential underpin the mechanisms used by EVs to influence cancer progression, leading to specific alterations to gene expression. In breast cancer, although not contributing to chemoresistance, prior treatment of non-cancerous stromal cell types with EVs increased the total number of breast cancer cells, suggesting a role in supporting cancer cell growth/survival. Together, this data adds to the foundation of knowledge of EVs roles and mechanisms in progression of endocrine cancers of the prostate and breast.
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