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Aldehyde dehydrogenase (ALDH) high activity is a marker of PCa stem cells with increased therapy resistance, enhanced DNA double-strand break repair, and activation of epithelial to mesenchymal transition (EMT). We investigated the role of two ALDH genes, ALDH1A1 and ALDH1A3, in regulating prostate cancer radiation resistance, cancer stem cell (CSC) phenotype and metastases formation.

We conducted our study by employing in vitro radiobiological clonogenic and spherogenic assays and in vivo syngeneic mouse model, zebrafish larval and mouse xenograft tumor models. We also validated our findings on the patient’s tumor samples by immunohistochemical staining of ALDH1A1 and ALDH1A3 protein expression (N=613). 

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. We analyzed the association of the functional gene signatures with ALDH1A1 and ALDH1A3 gene expression in the TCGA gene expression dataset and found a high positive correlation of the ALDH1A1 expression with WNT/β-Catenin, extracellular matrix and adhesion, EMT, angio- and osteogenesis gene sets. Contrary, the ALDH1A3 showed a negative correlation with those gene sets but a high correlation with androgen receptor (AR) signalling. We next validated our findings in knockdown experiments, as well as by using inhibitors against AR and CTNNB1 (β-Catenin) genes. We also compared the initial prostate-specific antigen (iPSA) serum level preoperatively between patients with tumors overexpressing ALDH1A1 and ALDH1A3 and observed a significantly increased iPSA level in patients with ALDH1A3 overexpressing tumors. Mechanistically, ALDH1A1 and ALDH1A3 differentially influence the TGFB1 mRNA gene expression, a potent inducer of EMT. Our in vivo zebrafish larval model showed that PCa cells lacking ALDH1A3 exhibit higher extravasation potential than those with suppressed ALDH1A1. The syngeneic mouse model demonstrated that Aldh1a1 depleted cells had reduced bone homing potential while Aldh1a3 suppressed cells formed a higher number of tumor nodules in the bone marrow. The analysis of the patient’s cohort showed the overexpression of ALDH1A1 (N=613) in lymph node and distant metastasis, whereas the ALDH1A3 protein (N=325) was highly expressed in primary prostate tumors. Additionally, Kaplan-Meier survival analyses of PCa patients with clinical follow-up information showed that high ALDH1A1 expression predicts disease recurrence.

Overall, ALDH1A1 and ALDH1A3 have opposite effects on PCa metastatic dissemination, with ALDH1A1 associated with increased metastatic burden and biochemical failure in PCa patients.