Our study discussed the role of ferroptosis inducer erastin in two PCa cells, LNCaP and PC3. Unsurprisingly, we found that erastin exposure caused an impact on LNCaP and PC3 cells in terms of ferroptosis marker, cell survivability, and the concentration of MDA, Fe2+, GSH, and GSSG. In addition, we extracted DEGs of both two PCa cells at the transcriptional level. Subsequently, we identified various pathways, TFs and modules in different PCa cells. Moreover, we identified and validated several erastin-affected FRGs which were potentially meaningful in PCa treatment through a series of bioinformatics analyses and experiments.
First, we focused on the identification of FRGs in PCa cells after erastin exposure. From RNA-Seq results, 10 FRGs were identified as the hub genes via overlapping 295 DEGs. TMEFF2, a type I transmembrane protein with an EGF-like and two follistatin motifs 2 proteins, was selectively expressed in the brain and prostate [20]. Based on our analysis, TMEFF2 was a co-expressed gene and correlated with GPX4 and SLC7A11, as determined by GeneMANIA and GEPIA. Despite the absence of direct reports on the connection between TMEFF2 and ferroptosis, a study reported that oxidative stress downregulate the expression of TMEFF2 in PCa [21]. Moreover, TMEFF2 can influence the proliferative activity of PCa cells [22], which was consistent with our results. In addition, similar to our results, several researchers reported that the alternative expression levels of TMEFF2 changed the disease stage in PCa tissue of patients and mouse model [23–25]. Therefore, the above reports supported our view that the TMEFF2 is related to ferroptosis and can be the FRG of PCa. It was worth mentioning that the ferroptotic level was significantly affected by knockdown of TMEFF2 in LNCaP cells, but not in PC3 cells. Considering that LNCaP is an androgen-dependent cell and PC3 is an androgen-independent cell[11]. Furthermore, TMEFF2 has been identified as an androgen-related gene [22]. And there was a report claimed that knock-down of TMEFF2 could suppressed the androgen-response of LNCaP cells [26]. Thus, we speculated that TMEFF2 promotes ferroptosis via androgen, which means TMEFF2 only bring an influence on ferroptosis of androgen-sensitive LNCaP cells. Then if TMEFF2 were used as a ferroptotic treatment target in the future, it would be more suitable for PCa patients than CRPC patients.
Our results also indicated FRGs such as CLU, NRXN3, and UNC5B showed different expression levels between PCa and normal tissue, and their expression patterns were positively related to GPX4 and SLC7A11. Meanwhile, CLU caused ATP degradation which resulted in peroxidation in cells [27], while the upregulation of CLU inhibited PCa cell death [28]. Furthermore, NRXN3 is a gene associated with obesity [29], and we speculated that NRXN3 takes part in the metabolism process of lipids. Moreover, UNC5B is a crucial regulator of ferroptosis in osteosarcoma cells [30]. Thus, these four hub DEGs identified in LNCaP and PC3 cells under erastin exposure exhibit to play an essential role in ferroptotic process of PCa cells, and maybe the potential treatment targets of PCa.
Subsequently, we obtained a TFs network for the 10 DEGs in PCa cells after erastin treatment. In the top TFs, STAT3 and E2F1 were differently expressed in erastin-induced PCa cells, and accumulating evidence indicated that they were related to ferroptosis. STAT3, signal transduction and activators of transcription 3, the ferroptosis was stimulated in PCa cells with knockdown of STAT3 [31]. Mechanistically, STAT3 binds to GPX4 and regulates its expression in pancreatic cancer cells [32]. E2F1, E2F transcription factor 1, can be one of the ferroptosis-related gene prognostic indexes (FRGPI) to predict disease-free survival (DFS) for PCa patients’ radical prostatectomy, PCa patients with high FRGPI had worse DFS than patients with low FRGPI [33]. Hence, we speculated that these TFs associated with ferroptosis also participate to the regulation of hub genes’ expression to affect the ferroptosis in PCa cells.
In addition to hub DEGs and their TFs, several pathways play an essential role in ferroptotic cells. Based on the GO functional analysis, the DEGs were primarily enriched in DNA replication, Chromosome segregation, and DNA helicase activity pathway. These three pathways were closely related to DNA replication, meanwhile, DNA replication is the crucial link to cell growth. Our findings indicated that erastin remarkably reduced the proliferative activity of PCa cells. In addition, Fe2+ is one of the essential factors of DNA replication, which is vital for cell survivability [34]. Consequently, we believed that erastin primarily transforms PCa cell proliferation by altering DNA replication. Except for DNA replication, KEGG analysis revealed that these DEGs were also associated with steroid hormone biosynthesis, MARK signal pathway, and P53 signal pathway. Steroid hormone biosynthesis is in charge of ferroptosis induction in adrenocortical carcinoma cells [35]. Considering our TFs network, it can be speculated that erastin affects MAPK/ERK to regulate STAT3, then STAT3 is a TF to change the expression patterns of hub genes CLU in PCa cells. Furthermore, STAT3 participate into P53/SLC7A11 pathway in osteosarcoma cells [36]. In our results, SLC7A11 was also downregulated by erastin exposure. We speculated that STAT3 not only affects the expression level of CLU but also changes the P53 signal pathway to alter the expression of SLC7A11. Meanwhile, REACTOME enrichment analysis suggested that DEGs were enriched in cell cycle checkpoints and activation of E2F1 target genes at the G1/S pathway. Experimental evidence showed that erastin caused a change in the proportion of PCa cells in certain phases [10]. Based on our analysis, E2F1 may be the TFs of hub genes TMEFF2, thus, E2F1 could affect the transcript level of TMEFF2, which may involve the cell cycle of PCa cells. Therefore, erastin may affect the expression patterns of hub genes and their TFs through regulate various signal pathways, including DNA replication, steroid hormone biosynthesis, and cell cycle, to transform the cell growth and cell cycle of PCa cells.
Interestingly, we found the erastin-induced PC3 group owned more DEGs than erastin-induced LNCaP groups. In our module analysis, the top 1 module of the PC3 group had more direct ferroptosis-related pathways, such as positive regulation of bone mineralization and regulation of steroid metabolic process, than the LNCaP groups. Therefore, as to these two PCa cell lines, we suggested that PC3 cells are much more sensitive to ferroptosis inducer erastin than LNCaP cells. Subsequently, we wanted to know why CRPC cell lines PC3 exhibited more susceptibility to erastin. GSEA analysis was applied to study various cellular signal pathways, which other functions enrichment did not identify. As expected, several cell death-related pathways were enriched, and the PC3 group displayed more and several unique signaling pathways in GSEA, such as negative regulation of ERBB signaling pathway, negative regulation of Ras protein signal transduction, and regulation of JAK-STAT cascade. These signaling pathways are all associated with ferroptosis in various cells. A complex suppressed the ERBB signal pathway to inhibit ferroptosis in vivo and in vitro [37]. Moreover, the RAS/MAPK pathway may regulated oxidative stress and the related ferroptosis pathways in glioblastoma [38]. Furthermore, IFNγ enhanced the JAK-STAT signal pathway to activate ferroptosis in hepatocellular carcinoma cells [39]. Unfortunately, there were lack of direct reports about the relationship between these signal pathways and ferroptosis of PCa. Hence, erastin transformed more crucial signal pathways, which led to PC3 being more sensitive to erastin. Moreover, considering that PC3 is an androgen-independent cell and LNCaP is an androgen-sensitive cell [11], we speculated that CRPC patients may be more susceptible to targeting gene therapy related to ferroptosis than PCa patients.
This current study has some limitations. Firstly, two PCa cell lines, LNCaP and PC3, were selected to carry out the experiment. If we included more PCa cells, the results will be more comprehensive. Secondly, although we verified the FRGs in cell experiments and in public databases, our further study will be confirmed in animal experiments and clinical practices.