Cancer Research @ UCC - Doctoral Theses
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Item Evaluation of the role of MxA in oesophageal cancer(University College Cork, 2024) Hayes, Robert Michael; McKenna, Sharon L.; O'Donovan, Tracey; Breakthrough Cancer Research; Health Research BoardOesophageal cancer is the seventh most common cancer worldwide and the sixth leading cause of cancer death. The incidence of oesophageal cancer is predicted to increase by 30% by the year 2040, however five-year survival remains at 20% in Ireland. There are two main subtypes, oesophageal squamous cell carcinoma and oesophageal adenocarcinoma. Both are aggressive cancers which frequently develop resistance to chemotherapeutics. Thus, novel therapeutic options are needed. Myxovirus resistance A (MxA) is an interferon inducible antiviral protein that is frequently upregulated in oesophageal cancer. It is currently unknown whether MxA plays a role in the development and progression of oesophageal cancer. We investigated the relationship between MxA expression and the response to chemotherapeutics (5-fluorouracil and oxaliplatin) in oesophageal cancer cell lines. MxA was differentially expressed in five oesophageal cancer cell lines. KYSE450 and KYSE140 cells did not express MxA and were apoptosis incompetent. FLO-1, KYSE270 and OE21 cells expressed MxA and were apoptosis competent. MxA was artificially overexpressed in cell lines without endogenous expression. This increased the resistance of KYSE450 but not KYSE140 cells. Both cell lines remained apoptosis incompetent. MxA expression was depleted in FLO-1 cells using siRNA, and in OE21 cells using CRISPR knockout. Knockdown of MxA significantly increased drug sensitivity and caspase-3 activation in FLO-1 cells. OE21-MX1KO cells were also more drug-sensitive, but caspase-3 activation was reduced. Therefore, these data indicate MxA can influence drug sensitivity, but this is not always related to apoptosis. We examined the impact of MxA on autophagy. Loss of MxA led to significant elevation of basal and drug-induced autophagy in all cell lines. Limited colocalisation was observed between MxA and the autophagosome marker LC3, suggesting MxA’s negative regulatory effect on autophagy is unlikely to involve direct interaction with autophagosomes. MxA was distributed in variably sized, discrete structures which were more numerous and peripherally located following treatment with a proteasome inhibitor or oxaliplatin. We also found that MxA is a significant component of the secretome of oesophageal cancer cells. This secretome included the mitochondrial protein TOM20. MxA knockout diminished the secretion of TOM20 from cells, in conjunction with the autophagy marker LC3. Therefore, we have established that MxA plays a role in the secretion of mitochondria and may act at the intersection between autophagic degradation and export of cellular material in oesophageal cancer cells. We evaluated the effects of MxA knockout on gene expression by RNAseq analysis. 868 genes were differentially expressed, including interconnected networks of genes involved in cell motility, immune/inflammatory signalling, metabolism, and cell differentiation. 59 differentially expressed genes overlapped with genes affected by inhibition of HDAC6, a central mediator of cancer associated pathways. We examined a potential interaction between MxA and HDAC6. Immunofluorescence analysis of OE21 and FLO-1 cells identified colocalisation between MxA and HDAC6 in particulate structures resembling stress granules. Colocalisation was increased in response to treatment with a chemotherapeutic agent or proteasomal inhibitor. Co-immunoprecipitation analysis indicated a specific interaction between MxA and HDAC6. We then compared migration in OE21 and OE21-MX1KO cells. A wound-healing assay demonstrated that migration was significantly reduced in MxA KO cells. Therefore, our data indicates a diverse role for MxA in oesophageal cancer biology. Depletion of MxA expression increased sensitivity to chemotherapeutics. MxA knockout resulted in differential expression of interconnected networks of genes which are associated with cancer progression. MxA negatively regulated autophagy and promoted secretion. A novel interaction between MxA and HDAC6 was identified, which may have significant consequences for the activity of both proteins. Importantly, as depletion of MxA can increase drug sensitivity and reduce migration, targeting this protein may be beneficial for the treatment of oesophageal cancer in future.Item The perioperative setting as a platform for breast cancer biomarker development(University College Cork, 2024) Hennessy, Maeve; Connolly, Roisin; Dahly, Darren; O'Reilly, Seamus; Breast Cancer Research Foundation; American Society of Clinical Oncology; Conquer Cancer FoundationBreast cancer is the most common cancer in women, with 2.3 million new cases globally in 2022 (1). ‘Neoadjuvant’ systemic therapy prior to surgery is the standard of care for stage II-III human epidermal growth factor receptor-2 (HER2) positive breast cancer. The identification of predictive biomarkers of response to systemic therapy is of great research interest, as this may allow treatment to be tailored to the individual. Pathologic complete response (pCR), the absence of residual invasive carcinoma in the breast or axillary lymph nodes following completion of neoadjuvant systemic therapy, is an accepted surrogate marker for survival outcomes in clinical trials (2). An advantage of the neoadjuvant setting is that tumour response to treatment can be assessed through tissue-based, biochemical, or radiologic changes throughout administration of therapy; allowing valuable information to be obtained in a short time period. Ideally, these potential biomarkers would be used early in the treatment paradigm allowing ‘individualisation’ of treatment decision-making. Certain patients could avoid receiving futile therapies, while others could be directed towards a more intensive approach to maximise their outcome. The studies in this Thesis were undertaken to evaluate imaging and tissue-based biomarkers for oestrogen receptor (ER)-negative, HER2-positive early-stage breast cancer in the Translational Breast Cancer Research Consortium (TBCRC) 026 trial (NCT01937117). TBCRC026 was a phase II, multicentre clinical trial incorporating serial positron emission tomography–computed tomography (PET-CT) imaging [baseline and 15 days after trastuzumab/pertuzumab (HP) initiation (C1D15)] and tumour biopsy collection (3). The primary hypothesis was that early measurements of tumour maximum standardised uptake value corrected for lean body mass (SULmax) on PET/CT would predict pCR to HP. Results found that participants not obtaining a 40% reduction in SULmax by C1D15 were unlikely to obtain pCR (3). Thus, the use of PET/CT as an imaging biomarker may provide a more individualised approach to treatment in early-stage HER2-positive breast cancer. This Thesis presents secondary analyses of the TBCRC026 clinical trial. Chapter 2 builds on the primary study analysis and evaluates the correlation between early metabolic changes on PET/CT and survival outcomes in this cohort. A potential association between SULmax parameters on PET/CT and recurrence free survival (RFS) and overall survival (OS) outcomes was identified. This imaging-based biomarker has not previously been assessed in patients receiving anti-HER2 therapy alone and therefore is novel and potentially practice changing if validated in future trials. Chapter 3 explores the relationship between HER2 and immune tissue-based biomarkers and pCR in this dataset, through analysis of baseline archived tumour samples. NanoString codesets BC360 and IO360 were used to compare gene expression in baseline tumours that underwent pCR versus no pCR. NanoString GeoMx digital spatial profiling (DSP) assessed immune protein abundance in intraepithelial and stromal segments. There was a significant association between intraepithelial HER2 protein abundance and pCR. Overall, the results demonstrated the unique molecular and immunological features of ER-negative, HER2-positive breast cancer, which warrant further exploration as potential predictive biomarkers. Chapter 4 investigates improving on the prediction power of the PET/CT imaging biomarker by combining it with a HER2-tissue based biomarker. Promising HER2-tissue biomarkers available from our analysis included high HER2 expression by immunohistochemistry (IHC), HER2-enriched (HER2-E) intrinsic subtype and high levels of HER2 protein abundance. While the composite HER2/PET biomarker comprising D15 SULmax, % reduction in SULmax and tumour HER2 protein abundance had high prediction power for pCR, it was not significantly superior to the prediction power of PET/CT parameters alone. The combined data from the above analyses suggest clinical trials informed by early PET/CT biomarkers and promising HER2-tissue based biomarkers should be further evaluated to personalise breast cancer care. If these potential biomarkers can be validated, results will be used to design clinical utility studies, facilitating early individualisation of therapy for patients.Item Characterisation of the effect of electroporation and electrochemotherapy on cancer cells and immune cells in the tumour microenvironment(University College Cork, 2022-10) Bendix, Maura; Brint, Elizabeth K.; Houston, Aileen M.; Amu, Sylvie; Forde, Patrick; Health Research Board; Breakthrough Cancer ResearchLung cancer is the leading cause of cancer-related death worldwide, with the lung cancer incidence rate expected to rise further. Despite recently developed novel therapy options, 5-year survival rates for lung cancer patients remains below 20% generally and below 5% for late-stage diagnosis, thus additional therapy options are still needed. Electrochemotherapy (ECT), the application of an electric pulse to deliver chemotherapy drugs into cells, could be a new treatment option for lung cancer patients. ECT is a locally very effective treatment, with local tumour reduction of up to 85%, while the systemic effects are more varied. For clinical application ECT treatment modalities have been standardized since 2006, after the ESOPE study, which optimized ECT parameters to 8 pulses at 1000V/cm with 100µs pulse length at 1Hz frequency and either bleomycin or cisplatin as the drug of choice. To evaluate whether ECT could be a potential treatment option for lung cancer patients’ ECT parameters, the needed electric field strength and the needed drug and drug concentration, were optimized for in vitro lung cancer research. In our study, we initially developed a standard operating protocol (SOP) to determine the optimal electric field strength for a given cancer cell line in vitro, while keeping the other ESOPE parameters constant. The developed SOP combined short-, medium-, and long-term assays to fully visualize the impact treatment, at a given field strength, has on the tested cancer cell line. This evaluation showed that human lung cancer cell lines (A549, H460 and SK-MES 1) and the human pancreatic Pan02 cell line have an optimal electric field strength of 800V/cm, the melanoma A375 (human) and B16F10 (murine) cell lines as well as the murine pancreatic Mia-PACA2 cell line have an optimal electric field strength of 700V/cm, while the murine Lewis Lung carcinoma (LLC) cell line has an optimal electric field strength of 1300V/cm. In addition, our study findings demonstrate that cisplatin at 11µM would be the drug of choice when using ECT for lung cancer treatments. In recent years while the importance of the immune system in lung cancer development and treatment results has become increasingly clear, little is known about how ECT treatments impact immune cells. Therefore, the impact of ECT on murine T cells, dendritic cells (DCs) and macrophages was evaluated in vitro. Our data indicates that while T cells are able to tolerate electric field strengths of up to 1400V/cm, DCs and macrophages are significantly negatively impacted by electric field strengths exceeding 800V/cm. Further investigation on the impact of ECT on dendritic cells demonstrated that DCs die via necrosis following ECT treatment, while ECT at electric field strength exceeding 1000V/cm leads to DC maturation and activation of the surviving cells. In addition, DCs remain partially functional following ECT treatment in a stimuli and treatment dependent manner with distinctively different sets of genes upregulated 4-hours post treatment at 800V/cm compared to treatment at 1000V/cm and 1300V/cm. Taken together, our data indicates that it is worthwhile to further investigate ECT as a potential therapy option for lung cancer patients, while more attention needs to be paid to the impact ECT has on immune cells in order to maximize treatment results.Item Investigating the utility of blood borne oncological biomarkers in solid tumours: glioma and melanoma(University College Cork, 2021-07-15) Ita, Michael Itak; Lim, Chris; Wang, Jianghuai; Redmond, Henry Paul; University College CorkBloodborne molecular biomarkers are increasingly emerging as significant non-invasive adjuncts to current methods of disease status evaluation in cancer patients. Investigations into the potential utility of these circulating biomarkers as analytic test measures complementing radiological imaging have been occasioned by the invasive nature of malignant tumour tissue biopsy, the need for serial evaluation of tumour burden during therapy, and the need for prognostication. In a series of five studies (four clinical studies and one pre-clinical study), this research work explored the potential utility of plasma cell-free DNA, circulating tumour DNA, cell-free messenger RNA, and bloodborne tumour related proteins as disease biomarkers in patients with glioma and metastatic melanoma. The in vivo research work employed an animal model to study micro RNA mediated epigenetic regulatory mechanisms implicated in therapeutic resistance which is prevalent in melanoma brain metastasis. Specifically, this research work sought to determine whether somatic mutations identified in the plasma samples of patients with glioma were identical or representative of the somatic mutations in synchronously obtained glioma tumour tissue samples. It further sought to determine whether significant differences exist in the plasma transcriptomic profile of glioma patients relative to differences in their tumour characteristics, and also whether any observed differences were representative of synchronously obtained glioma samples and the human cancer genome atlas (TCGA) glioma derived RNA profile. Moreover, this research work explored the relationship between plasma cell-free DNA (cfDNA), serum lactate dehydrogenase (LDH), plasma vascular endothelial growth factor (VEGF), programmed death ligand-1 (PD-L1), interferon-gamma (IFN-γ), and tumour burden in advanced melanoma patients. Furthermore, it sought to examine whether important differences exist in the plasma transcriptomic profile of advanced melanoma patients with a high disease burden compared to patients with a low disease burden or therapeutic response and whether the plasma transcriptomic profile of advanced melanoma patients was representative of TCGA melanoma tumour tissue-derived RNA profile. The methods employed in this research work include; the purification and quantification of circulating cell-free DNA and total RNA from the plasma samples of glioma and metastatic melanoma patients, somatic mutation profiling using DNA derived from FFPE glioma tumour tissue curls and plasma circulating cell-free DNA by amplification refractory mutation system (ARMS®) PCR, pathway-focused gene expression analysis using complementary DNA synthesized from the plasma circulating cell-free messenger ribonucleic acid (ccfmRNA) samples of patients with glioma and advanced melanoma, the extraction and quantification of tumour-related proteins such as LDH, VEGF, PD-L1, and IFN-γ from patients with advanced melanoma by the enzyme-linked immunosorbent assay technique (ELISA), in vivo malignant brain tumour model development, bioluminescence imaging study, immunohistochemistry and microscopy, evaluation of protein expression by flow cytometry, and genomic profiling of total cellular micro RNA using RT- PCR. This research work was able to establish that the detection of plasma circulating tumour DNA originating from the glioma tumour tissues of affected patients is feasible, albeit with a low tumour to plasma mutation concordance. It identified significant differential expression of genes involved in cancer inflammation and immunity crosstalk among patients with different glioma grades, and a positive correlation between the transcriptomic profile of these genes in plasma and tumour samples, and with TCGA glioma derived RNA. Moreover, this research work identified that the incorporation of the quantitative measures of cfDNA, LDH, VEGF and PD-L1 in a suitable multiple regression analysis model was capable of predicting changes in tumour burden in patients with advanced melanoma. Furthermore, it identified and characterized the plasma transcriptomic profile associated with therapeutic response in advanced melanoma patients during immunotherapy. This research work also characterized in a limited way, the tissue and blood markers of therapeutic response and resistance in an in vivo model of melanoma brain metastasis.Item Malnutrition & altered body composition in oncology: prevalence, aetiology, consequences & potential therapies(University College Cork, 2020-08-10) Sullivan, Erin S.; Ryan, Aoife; Power, DerekMalnutrition is common across all cancer sites and stages and its aetiology is multifactorial and complex. It is associated with poorer quality of life, increased morbidity and mortality and is often considered an inevitable consequence of cancer and its treatments. However, we lack efficacious treatments for cancer-related malnutrition. The aim of this thesis was to describe the epidemiology of malnutrition in cancer, examine the causes and consequences of the condition and explore potential treatment strategies. This thesis begins by estimating that across Ireland and the UK, 34% of cancer patients (128,892) are affected by clinically significant weight loss annually and there are 133,707 annual cases of cancer-related sarcopenia (35% patients affected). This thesis shows using computed tomography scans (the gold standard in body composition analysis) that abnormalities of body composition, including loss of fat without loss of muscle, are predictive of poor survival in advanced cancer. Furthermore, cachexia (a syndrome of disease-related appetite loss and wasting) was shown to be more prevalent in those with inflammation and poor performance status and the obesity paradox in colorectal cancer was confirmed (obesity is a risk factor for the disease, but is associated with improved survival). The nutritional experience of patients with cancer is described, namely that nutrition is a high priority for patients, who experience many dietary issues throughout their journey, but that information available to patients is lacking and referral to dietitians is very inconsistent. Finally, a placebo controlled trial of 2 novel dairy-derived, ghrelinergic peptides showed that one of the peptides investigated increased protein intake in healthy males by 23 g per day. Prompt identification of patients with cancer-related malnutrition must be optimised and development of an effective, evidence-based treatment strategy is of the utmost importance as it stands to improve longevity and quality of life for cancer survivors.
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