CAFs-CM promotes PCa progression under hypoxic condition
In previous studies, a characteristic hypoxic microenvironment has been identified in PCa tissues, potentially contributing to tumor development via multiple signaling pathways [15, 16]. Consequently, we detected the expression of HIF-1α, a member of the hypoxia-inducible factor, in 15 BPH, 15 organ-confined PCa, and 15 primary tumor tissues of mPCa. IHC analysis revealed minimal HIF-1α expression in BPH tissues, while significantly increased expression with PCa progression (Fig. 1A).
To explore the influence of stromal cells on PCa progression under hypoxic condition, we collected the supernatants from cultured WPMY-1 exposed to hypoxic and normoxic conditions. These supernatants were subsequently employed as conditioned media (CM) for treating PCa cells. Regrettably, treatment with WPMY-1 CM did not result in any significant alterations in the proliferation or metastatic potential of PCa cells (Supplementary Fig. 1A, B). Subsequently, primary CAFs and normal fibroblasts (NFs) were isolated from PCa tissues and adjacent normal tissues, respectively. Under white light microscopy, both CAFs and NFs displayed a spindle-shaped morphology, with CAFs exhibiting a more elongated appearance (Fig. 1B). Western blotting and immunofluorescence analysis revealed elevated levels of α-SMA expression within CAFs (Fig. 1C).
To investigate the effect of CAFs on PCa cells in hypoxic conditions, PC-3 and C4-2 cells were exposed to hypoxic and normoxic CM obtained from CAFs. PC-3 and C4-2 cells were slightly accelerated in their growth capability by hypoxic CM, but their apoptosis levels were not affected (Fig. 1E, Supplementary Fig. 2A). Additionally, scratch assays revealed an augmented migratory capacity of PC-3 cells following treatment with hypoxic CM derived from CAFs (Fig. 1F). In addition, Transwell invasion assays demonstrated that hypoxic CM promoted the invasive ability of PC-3 and C4-2 cells (Fig. 1G). Collectively, these findings indicated that hypoxia could promote PCa progression through the modulation of CAFs in TME.
CAFs-secreted exosomes under hypoxia promote PCa in vitro and in vivo.
Considering the significant regulatory influence of exosomes on tumor progression[8], we collected exosomes secreted by CAFs to investigate their impact on PCa cells under hypoxia and normoxia conditions. The exosomes released by CAFs were identified through the common exosomal markers (TSG101, Alix, CD63), nanoparticle tracking analysis (NTA) and transmission electron microscope (TEM). The results showed that exosomal markers were highly expressed in CAFs exosomes while being nearly absent in CAF cells (Fig. 2A). Furthermore, NTA revealed that these vesicles possessed a diameter of approximately 100nm, with TEM revealing their characteristic cup-shaped morphology (Fig. 2B, C). These results indicated that the isolated vesicles were exosomes.
To investigate whether PCa cells could internalize CAFs-derived exosomes, PC-3 and C4-2 cells were treated with exosomes labeled by PKH67 for 16 hours. Confocal microscope showed the successful internalization of exosomes by both PC-3 and C4-2 cells (Fig. 2D). Subsequently, we assessed the impact of CAFs-secreted exosomes on PCa cells. As anticipated, CAFs hypoxic exosomes significantly enhanced the migration and invasion capacities of PC-3 and C4-2 cells (Fig. 2E, F). However, only minor changes were observed in the proliferation and apoptosis of both PC-3 and C4-2 cells following treatment with CAFs hypoxic exosomes (Fig. 2G, Supply Fig. 2B). Notably, the promoting effect exerted by CAFs hypoxic exosomes on PCa cells could be counteracted by the exosome inhibitor GW4869 (Supplementary Fig. 3A, B). To assess the influence of CAFs hypoxic exosomes on PCa metastasis in vivo, we established a lung metastasis model by injecting PC-3 cells via the tail vein. As illustrated in the schematic (Fig. 2.H), PC-3 cells were treated with CAFs-derived exosomes for 48 hours prior to injection. Subsequently, exosomes (40µg) were administered via tail vein injection every third day for two weeks after tumor cells injection. Tumor metastases were evaluated after eight weeks. We observed a steady increase in body weight in nude mice during the first six weeks after tumor injection, followed by a decline from the seventh week (Supplementary Fig. 4). Histological examination confirmed the presence of tumor metastases, with a higher incidence of lung metastases observed in the group of mice injected with CAFs hypoxic exosomes (Fig. 2I). Additionally, P504S staining confirmed the histological origin of PCa (Fig. 2J). Collectively, these results underscored the role of CAFs-derived exosomes in accelerating PCa metastasis under the hypoxic microenvironment.
miR-500a-3p is elevated in CAFs hypoxic exosomes.
Exosomes are known to contain various non-coding RNAs (ncRNAs), including miRNAs, lncRNAs, and circRNAs. MiRNAs derived from exosomes had been shown to form a new mode among cells communications in diseases[17]. To identify the differentially expressed miRNAs in normoxic and hypoxic exosomes secreted by CAFs, we conducted a miRNA sequencing analysis. The results revealed 14 upregulated and 19 downregulated miRNAs in CAFs hypoxic exosomes compared to their normoxic counterparts (Fig. 3A, B). Results of the target genes of different expressed miRNAs (DE-miRNAs) by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were shown in Fig. 3C, D, respectively. Subsequently, we further validated the expression levels of the top-ranked 13 miRNAs from the sequencing date in CAFs hypoxic and normoxic exosomes using RT-qPCR. The results confirmed that miR-500a-3p and miR-2682-3p were significantly upregulated, while miR-184 exhibited downregulation in CAF hypoxic exosomes, consistent with RNA sequencing assay findings (Fig. 3E). However, following treatment with CAFs hypoxic exosomes, only miR-500a-3p displayed the anticipated upregulation in PCa cells (Fig. 3F). Therefore, we embarked on exploring whether miR-500a-3p could affect the progression of PCa.
miR-500a-3p in CAFs exosomes promote PCa cells migration and invasion.
Firstly, we investigated whether miR-500a-3p derived from CAF exosomes could be taken by PCa cells. Cy3-labelled miR-500a-3p mimics were transfected into CAFs using siRNA-transfectmate (Genepharma). Subsequently, exosomes were isolated from the transfected CAFs and introduced into PC-3 and C4-2 cells. Under a fluorescence microscope, abundant cy3-miR-500a-3p could be observed within PC-3 and C4-2 cells (Fig. 4A). To visualize the spatial distribution of miR-500a-3p within CAFs- derived exosomes following transfection with cy3-miR-500a-3p, we co-labeled exosomes with PKH67. The results showed that the majority of labeled miR-500a-3p was effectively packaged within CAFs exosomes, which were subsequently taken by PCa cells (Fig. 4B).
To investigate the impact of miR-500a-3p on prostate cancer, miR-500a-3p mimics and inhibitor were transfected into PC-3 and C4-2 cells. The results demonstrated that miR-500a-3p mimics promoted the migration and invasion capabilities of PC-3 and C4-2 cells, while the miR-500a-3p inhibitor exhibited the opposite effect (Fig. 4C, D). However, miR-500a-3p did not influence the proliferation of PCa cells (Supplementary Fig. 5). Additionally, we found elevated miR-500a-3p expression in the serum exosomes of PCa patients with metastasis (Fig. 4E). Furthermore, an analysis of the TCGA database revealed a significant increase in miR-500a-3p expression in PCa tissues compared with normal tissues (Fig. 4F). Similarly, data from the GEO database indicated elevated miR-500a-3p levels in the serum of PCa patients (Fig. 4G). Collectively, these findings indicated that miR-500a-3p promoted the migration and invasion of PCa cells and might serve as a potential marker for mPCa diagnosis and prognosis.
miR-500a-3p promoted PCa progression through targeting FBXW7.
To investigate the mechanisms underlying the regulation of PCa progression by miR-500a-3p, we utilized several miRNA databases, including miRDB, TargetScan, miRWalk, mirDIP, and RNA22, to identify potential target mRNAs of miR-500a-3p. The analysis revealed 63 potential target mRNAs of miR-500a-3p (Fig. 5A). Among these candidates, we focused on 11 mRNAs with target scores exceeding 90 in the miRDB database (Supplementary Fig. 6). One among these mRNAs, FBXW7, is a recognized tumor suppressor gene validated by previous studies[18, 19]. Moreover, TCGA data indicated a decreased FBXW7 expression levels in PCa tissues (Fig. 5B), suggesting that FBXW7 could probably serve as a potential marker for PCa. Subsequently, we assessed the expression of FBXW7 in PCa cells upon treatment with CAFs-derived exosomes. Both RT-qPCR and western blot analysis demonstrated a significant decrease in FBXW7 expression following CAFs hypoxic exosomes treatment (Fig. 5C, D). Additionally, the transfection of miR-500a-3p mimics into PC-3 and C4-2 cells resulted in a significant reduction of FBXW7 mRNA and protein expression (Fig. 5E, F). To further verify the direct interaction between miR-500a-3p and the FBXW7 mRNA, we constructed two FBXW7 3’ untranslated regions (3’UTR) luciferase reporter constructs – one containing the conserved seed sequence and the other carrying a mutated (MUT) version of the seed sequence with nine nucleotides exchanged (Fig. 5G). When miR-500a-3p mimics and the wild-type (WT) FBXW7 3’UTR reporters were transfected into 293T cells, there was a noticeable decrease in luminescence, while not observed in case of the mutant reporters (Fig. 5H). These findings indicated the inhibitory role of exosomal miR-500a-3p on FBXW7 expression in PCa cells.
FBXW7 inhibit the progression of tumor by regulating the HSF1 expression in PCa cells
To explore the relationship between FBXW7 and PCa, we conducted IHC to examine the FBXW7 expression in BPH, organ-confined PCa, and primary lesions of mPCa. FBXW7 was distributed in both the nucleus and cytoplasm, with notably higher expression in the cytoplasm. Notably, as PCa progression, FBXW7 expression was found gradually decreased (Fig. 6A), indicating a negative correlation between FBXW7 levels and PCa progression.
To investigate the functional role of FBXW7 in PCa, we overexpressed FBXW7 in PC-3 and C4-2 cells using a pcDNA3.1(+) plasmid. RT-qPCR and western blotting confirmed a significant increase in FBXW7 expression after plasmid transfection (Supplementary Fig. 7A, B). The overexpression of FBXW7 triggered increased cell death in PC-3 cells and inhibited the growth of C4-2 cells (Supplementary Fig. 7C). Additionally, FBXW7 overexpression significantly suppressed PCa cells proliferation, migration, and invasion (Fig. 5B, C). Furthermore, the overexpression of FBXW7 effectively counteracted the stimulatory effects of miR-500a-3p on PCa cells (Supplementary Fig. 7D).
Previous studies have revealed that FBXW7 exerts its inhibitory effects on cancer progression by ubiquitinating multiple oncogenes, including c-Myc, cyclin E and Mcl-1[20]. Among these oncogenes, HSF1 and MAP7D1 expression were negatively correlated with FBXW7 in PCa tissues (Supplementary Fig. 7E). Furthermore, it has been demonstrated that abundance of HSF1 expressed in PCa cells nuclear and HSF1 inhibitor robustly inhibited cancer progression[21]. Therefore, we investigated whether FBXW7 inhibited PCa progression through the HSF1 pathway. IHC analysis demonstrated that HSF1 mainly localized in the nucleus and limited expression in the cytoplasm, and the HSF1 expression escalates with PCa progression, indicating a negative correlation between FBXW7 and HSF1 (Fig. 6D). Furthermore, FBXW7 overexpression resulted in significantly decreased HSF1 expression (Fig. 6E), indicating that FBXW7 negatively regulate the expression of HSF1 in PCa cells. In conclusion, our findings suggest that exosomes derived from CAFs under hypoxic conditions promote PCa progression through the miR-500a-3p/FBXW7/HSF1 signaling axis. FBXW7 acts as a tumor suppressor through repressing HSF1 expression, and its downregulation mediated by miR-500a-3p contributes to the progression of PCa.