Abstract
Renal cell carcinoma (RCC), particularly its most common subtype clear cell RCC (ccRCC), remains a clinically challenging malignancy characterized by late-stage diagnosis, high recurrence rates, and resistance to conventional therapies. Paired box 2 (PAX2), a developmental transcription factor involved in renal organogenesis, is frequently overexpressed in ccRCC and has been proposed as a potential therapeutic target. However, several studies have reported a reduction in PAX2 expression in advanced-stage or high-grade ccRCC. The biological consequences of this decline, particularly how reduced PAX2 expression may influence tumour behaviour and metabolic characteristics, remain poorly defined.
The aim of this thesis was to investigate the expression patterns of PAX2 in ccRCC and to examine the transcriptional and functional changes associated with reduced PAX2 expression, with a particular focus on mitochondrial regulation. Analysis of public clinical and transcriptomic datasets demonstrated that lower PAX2 expression is associated with adverse clinicopathological features, including higher tumour grade, advanced stage, and metastatic disease. These findings suggest that PAX2 downregulation accompanies tumour progression in ccRCC. To explore downstream associations of reduced PAX2 expression, candidate-based analyses identified mitochondrial transcription factor A (TFAM) as a potential gene associated with PAX2 expression. Following siRNA-mediated PAX2 knockdown, TFAM expression was significantly reduced, and TFAM promoter–reporter assays revealed a pronounced decrease in promoter activity. Collectively, these data strongly suggest that TFAM is a direct downstream transcriptional target of PAX2, although the precise promoter regions involved in PAX2 binding remain to be defined. RNA sequencing further revealed that PAX2 knockdown is accompanied by coordinated changes in gene networks related to mitochondrial-associated processes, cellular stress responses, and pro-survival pathways, particularly in A498 ccRCC cells, with more limited transcriptomic responses observed in other cell lines. Functional analyses demonstrated that PAX2 knockdown is consistently associated with impaired mitochondrial function across multiple ccRCC cell lines. Reductions in mitochondrial mass, mitochondrial membrane potential, and mitochondrial ATP contribution were observed using fluorescence intensity assays and ATP measurements normalised to cell number. Although modest changes in glycolysis-related readouts were observed under specific conditions, the current evidence is insufficient to support a direct role for reduced PAX2 expression in driving glycolytic reprogramming.
Overall, this thesis provides new insight into the potential role of PAX2 downregulation in ccRCC, suggesting that reduced PAX2 expression may accompany tumour progression and be associated with alterations in mitochondrial function. Future studies should prioritise experimental validation of genes and signalling pathways identified by RNA sequencing, particularly those involved in cellular metabolism and mitochondrial regulation, and further investigate the mechanisms linking PAX2 downregulation to metabolic reprogramming.