ESTRO 2024 - Abstract Book

S5432

Radiobiology - Tumour biology

ESTRO 2024

Purpose/Objective:

Prostate cancer (PCa) exhibits a strong affinity for spreading to bone tissue, giving rise to bone metastases, which are resistant to traditional treatments like radiotherapy and systemic approaches [1]. This resistance is often attributed to the presence of cancer stem cells (CSCs), with aldehyde dehydrogenase (ALDH) activity identified as a marker for PCa stem cells [2, 3]. The primary objective of our study is to investigate the role of ALDH proteins in the cellular processes and molecular mechanisms responsible for sustaining the persistence of PCa metastasis-initiating cells and their resistance to radiotherapy.

Material/Methods:

We carried out our study by employing in vitro radiobiological clonogenic and spherogenicity assays, RNA and protein analysis. We also validated ALDH1A1 and ALDH1A3 as potential biomarkers of clinical outcomes in the independent cohorts of patients with PCa. We conducted our study by employing in vivo syngeneic mouse model, zebrafish larval and mouse xenograft tumor models.

Results:

We found that ALDH1A1 was highly upregulated, whereas ALDH1A3 was significantly downregulated in radiation resistant (RR) cells. Radiobiological clonogenic analyses after siRNA-mediated knockdown, as well as western blot analysis of key DNA damage response proteins, demonstrated that both ALDH1A1 and ALDH1A3 contribute to the regulation of PCa cell radiosensitivity. Gene Set Enrichment Analysis (GSEA) analysis confirmed that genes deregulated by ALDH1A1 knockdown are associated with CHEK2 signalling network and DNA double-strand break repair. Furthermore, the knockdown of ALDH1A1 upregulates AR, a transcriptional regulator of DNA repair genes in PCa [4], suggesting a balancing feedback mechanism. To investigate the predictive value of ALDH1A1 and ALDH1A3 gene expression, we analyzed biochemical recurrence-free survival (BRFS) of The Cancer Genome Atlas Prostate Adenocarcinoma (TCGA-PRAD) patients’ cohort. Analysis demonstrated that ALDH1A1 and ALDH1A3 gene expression levels oppositely correlate with clinical outcomes. We next validated our findings by investigating a possible association of ALDH1A1 protein expression with BRFS in a retrospective, monocentric cohort including 177 patients diagnosed with PCa (Lübeck sub-cohort with sufficient follow-up data). We found that patients with high ALDH1A1 expression in primary tumors exhibited worse BRFS compared to the negative/single-cell positive subgroup. Analysis of the gene expression dataset from an independent cohort of patients with primary intermediate or high-risk PCa (Oslo cohort [5], n = 95) validated a negative correlation between ALDH1A1 and ALDH1A3 genes. To validate whether the expression of ALDH1A1 and ALDH1A3 genes also correlates with tumor radioresistance in PCa patients, we analyzed the expression of these genes in tumor tissues of patients with intermediate or high-risk localized PCa treated with radiotherapy (n = 67, Dresden cohort [6]). We found a significant association of high ALDH1A1 expression with lower relapse-free rates, whereas high expression of ALDH1A3 is significantly associated with higher rates of freedom from PSA relapse. ALDH1A1 and ALDH1A3 synthesize retinoic acid (RA) from retinol, potentially influencing retinoid receptor-mediated transcription. In turn, retinoid receptors interplay with androgen receptor (AR) to regulate common target genes [7]. To understand the mechanisms explaining the differential roles of ALDH1A1 and ALDH1A3, we performed RNA sequencing (RNAseq) upon siRNA-mediated knockdown of target genes or treatment with all trans retinoic acid (ATRA). We revealed that genes deregulated after ALDH1A1 knockdown are similarly regulated in response to the knockdown of each individual retinoid receptors and AR knockdown. We next focused on one of the druggable targets, Polo-like kinase 3 (PLK3), a nucleolus protein involved in the cell cycle and DNA repair regulation. Depletion of ALDH1A1 led to a decrease in the PLK3 gene and protein expression, while downregulation of the