Fig 2: RARA- and AR- interplay drives TGFB1 transcription. A qPCR analysis of the relative TGFB1 expression in LNCaP cells upon knockdown of AR, RARA, and RXRA expression alone or in combination. Cells transfected with scrambled siRNA (siScr) were used as a control. n ≥ 3; Error bars = SD; *p < 0.05; **p < 0.01; ***p < 0.001. B Chromatin immunoprecipitation (ChIP)-qPCR analysis in LNCaP cells for assessment of the direct binding of RARA and AR proteins to the putative binding regions in the promoter of the TGFB1 gene. RARA and AR binding sites were taken from the JASPAR CORE database [64]. n ≥ 3; Error bars = SD; *p < 0.05; **p < 0.001; ***p < 0.001

Fig 3: TGFβ1 regulates the members of the MMP family. A The ROC curve analysis of the potential association of the expression levels of 20 MMP genes with biochemical recurrence-free survival (BRFS) in the TCGA PRAD gene expression dataset (n = 493). ROC analysis was conducted using easyROC web-tool [57]. B Correlation of MMP11 and MMP26 gene expression with TGFB1 mRNA levels in the PCa gene expression datasets TCGA PRAD (n = 493), MSKCC primary (n = 131), Broad/Cornell (n = 31), DKFZ (n = 118), and SMMU (n = 65) and non-cancerous tissues (MSKCC cohort, n = 29); *p < 0.05; n.s.- non significant. C PC3 and DU145 cells were treated with 5 ng/ml of TGFβ1 for 48 h, and MMP11 levels were analysed by qPCR. n ≥ 3; Error bars = SD; *p < 0.05; **p < 0.01. D Analysis of the previously published dataset [68] indicated that TGF-β signaling in stroma cells induces MMP expression in LNCaP cells. LNCaP cells overexpressing TGFB1 and co-cultured with stroma cells were compared to the control LNCaP co-cultured with stroma. *p < 0.05. E qPCR analysis of the relative MMP11 expression in LNCaP and PC3 cells upon knockdown of ALDH1A1 or ALDH1A3. Cells transfected with scrambled siRNA (siScr) were used as controls. n = 3; Error bars = SD; *p < 0.05; **p < 0.01; n.s.- non significant. F qPCR analysis of the relative MMP11, ALDH1A1 and ALDH1A3 expression in LNCaP and PC3 cells upon MMP11 knockdown. Cells transfected with scrambled siRNA (siScr) were used as controls. n ≥ 3; Error bars = SD; *p < 0.05; **p < 0.01; ***p < 0.001. G Relative cell radiosensitivity was analysed by 2D radiobiological colony forming assay after siRNA-mediated knockdown of MMP11 in LNCaP and PC3 cells. Cells transfected with scrambled (Scr) siRNA were used as controls. n = 3; Error bars = SD; **p < 0.01. H PC3 cells were cultured either under 2D conventional adherent conditions, in the polymer-based microcapsules mimicking mechanical stress within tumors [42], or in 3D anchorage-free cultures as a model of the intermediate stage of metastasis, circulating tumor cells (CTC). In all conditions, cells were kept in the same culture media. At day 3, the cells were collected, and relative gene expression of TGFB1, MMP11, ALDHA1 and ALDHA3 was analysed by qPCR. n ≥ 3; Error bars = SD; *p < 0.05; ***p < 0.001. I ALDH1A1 regulates TGFB1 expression in androgen-sensitive cells through AR- and RA-dependent mechanisms. In contrast, the interplay between TGF-β1 and MMP11 is present in the androgen-sensitive and castration-resistant models of PCa (like in LNCaP and PC3 cells, respectively)

Fig 4: ALDH1A1 and ALDH1A3 differentially regulate migration depending on the AR status. A Analysis of the relative cell migration of LNCaP, C4-2B, and PC3 cells upon knockdown of ALDH1A1 or ALDH1A3 expression using Oris migration assay. Cells transfected with scrambled siRNA (siScr) were used as a control. Cell migration was analysed 24 h after cell plating. AR + : androgen receptor positive; AR-: androgen receptor negative; AD: androgen dependent; AI: androgen independent; n ≥ 3; Error bars = SD; *p < 0.05. B The Kaplan–Meier analyses of the biochemical recurrence-free survival (BRFS) and metastasis-free survival (MFS) for patients with high-risk and locally advanced PCa treated with ADT and then concurrently with one of three radiotherapy regimens as described in Table 3. The patients were stratified by the most significant cut-off for ALDH1A1 expression levels. C Correlation of mRNA expression levels of ALDH1A1 and ALDH1A3 genes and gene sets related to cell motility, tumor metastasis, EMT and androgen signaling in the TCGA PRAD patient cohort (n = 490). The gene lists are provided in Table S2. D Correlation of mRNA expression levels of ALDH1A1 and ALDH1A3 genes and metastasis-related genes in the TCGA PRAD patient cohort (n = 490). The arrow indicates a correlation with TGFB1. E Levels of mRNA expression of the metastasis-related genes which highly correlate with ALDH1A1 expression (as in panel D) in PC3 and LNCaP cell lines. The arrow indicates the level of TGFB1 expression. F Correlation of mRNA expression levels of ALDH1A1 and ALDH1A3 genes and gene sets related to the TGFβ1 signaling targets and TGFβ1 / BMP signaling targets in the TCGA PRAD patient cohort (n = 490). The gene lists are provided in Table S2