In the present study, we successfully established LNCaP-AI cells by following the methods of a previous report. The characteristics of the ideal model for PCa cells are androgen dependence and wild-type AR expression. Moreover, these cells should also have the ability to convert into androgen-independent cells in vitro. Currently, common PCa cell lines include LNCaP, PC-3, DU-145, and C4–2 derived from different tissues. PC-3, DU-145, and C4–2 are all androgen-independent cell lines, and the markers of the above cell lines also differ [18,19,20]. The evolution of these androgen-independent cell lines is discontinuous, so it is difficult to draw a reliable conclusion from the comparison of these androgen-independent cell lines. Therefore, these cell lines are not the most ideal cell lines for castration-resistant growth research. LNCaP is an androgen-dependent cell line that is widely used in various experiments. LNCaP cells show the differentiation characteristics of primary PCa, including androgen-dependent growth and the ability to secrete PSA in vitro. In this study, charcoal/dextran  was used to remove most androgen from the culture medium, which mimicked the clinical process of androgen deficiency treatment. Therefore, compared with PC-3, DU145, and C4–2 cells, LNCaP cells are a more reasonable and reliable choice for the study of castration resistance.
To confirm the establishment of LNCaP-AI cells, we observed changes in the cell morphology, proliferation rate and PSA levels of LNCaP cells. During the whole induction process, the features of neuroendocrine (NE) cells emerged as a hallmark of the conversion to androgen-independence (Fig. 1d). The NE phenotype is widely accepted to be the most prominent characteristic of LNCaP-AI cells and promotes aggressive phenotypic transformation [13, 22]. CCK-8 analysis confirmed that LNCaP-AI cells could adapt to the androgen-depleted environment (Fig. 1f). LNCaP-AI cells proliferated more rapid than LNCaP in the same medium (Fig. 1e). ELISA confirmed that the PSA secretion of LNCaP-AI cells rose gradually and exceeded that of LNCaP cells on the 6th day. But in first 6 days, the proliferation rate of LNCaP-AI was similar to that of LNCaP, and the PSA levels was lower than that of LNCaP cells. Previous studies showed that the PSA secretion of LNCaP-AI cells was greater than that of LNCaP cells . The reason for this difference may be that charcoal/dextran can’t completely strip hormones in FBS . LNCaP cells can continue to use the remaining androgen to maintain the above biological behavior. Another possible reason is that LNCaP-AI cells appear to be stem cell-like cells with the feature of a low PSA level and the morphology of androgen-independent cells [22, 24]. Proliferation rate and PSA levels gradually increased when LNCaP-AI cells were continuously differentiated. In general, the establishment of LNCaP-AI cells is a continuous process that provides a good model for studying the mechanism underlying CRPC development in vitro.
It is well known that the enhanced proliferation of cancer cells is due to cancer cells having higher glycolytic activity than benign cells, which increases glucose uptake and lactate secretion. Our results indicated that the glucose consumption and lactate secretion of LNCaP-AI cells were increased after androgen-independent growth. This result is the same as the finding of our previous study, which indicated that androgen-independent PC-3 cells had higher glycolytic levels than androgen-dependent LNCaP cells . In this study, we further demonstrated that glucose metabolism might be an important difference between two tumor types and therefore might be a potential target for the treatment of CRPC. Evaluation of the change in glycolytic metabolism during androgen-independent growth is a relatively good mimic of the glycolytic process occurring in CRPC and could provide the metabolic basis for the study of the unique biological behaviors of CRPC.
Importantly, tumor metabolism is extremely complex. Cancer cells undergo energy metabolism reprogramming to adapt to the changing microenvironment , including changes to glycolytic metabolism, transcriptional regulation and signal transduction . These changes require the involvement of enzymes. Based on current research on PFKFB4, this enzyme regulates glucose metabolism in tumor cells, which has been widely recognized. PFKFB4 controls glycolysis and the pentose phosphate pathway (PPP) by regulating F-2,6-BP. ATP and lactic acid production in glycolysis are associated with proliferation and drug resistance in tumor cells. NADPH in the PPP is associated with stabilizing redox homeostasis and lipid synthesis in tumor cells, which are associated with tumor cell survival [7, 16]. Our previous research demonstrated that PFKFB4 was highly expressed in androgen-independent cells DU145 and PC-3 cells compared with androgen-dependent LNCaP cells and could regulate the proliferation, invasion, and migration of PC-3 cells [3, 8]. In the present study, PFKFB4 expression increased significantly with androgen-independent growth, suggesting that PFKFB4 is involved in the behavior of PCa cells during the phenotypic switch from adenocarcinoma to CRPC. Therefore, based on the consensus on PFKFB4 and our previous studies, we consider that this phenomenon likely occurs through the regulation of glucose metabolism. This speculation is not without foundation. The findings from another study showed that the expression of PFKFB4 was significantly higher in small cell neuroendocrine carcinoma than in adenocarcinoma, which suggested that increasingly aggressive PCa might be related to increasingly high PFKFB4 expression . Moreover, the expression of PFKFB4 is higher in metastatic PCa than in primary lesions . However, PFKFB4 was also involved in tumor progression through redox homeostasis, transcriptional regulation, autophagy and so on . Therefore, PFKFB4 was related to androgen-independent growth and might be a potential therapeutic target for CRPC treatment. We had a preliminary understanding of the role of PFKFB4 in androgen-independent state, so the next step was to determine association and mechanism between PFKFB4 and proliferation and migration of androgen independent cells, furthermore, the role of PFKFB4 as glycolytic protein in androgen independent growth.
Western blotting also showed that the expression of CD44 and PDK1 increased when LNCaP cells transformed into LNCaP-AI cells, which might be involved in the high glycolytic activity accompanying PFKFB4 expression. PDK1 and CD44 are regulatory enzymes in glucose metabolism [3, 8]. PDK1 can promote lactate production in tumor cells by increasing glycolysis and making the microenvironment acidic, which increases the invasive behavior of malignant cells . PDK1 also regulated the proliferation, invasion, and migration of PC-3 cells in our previous studies, which was similar to the results for PDK1-regulated androgen-independent growth found in this study. CD44 is a multifunctional enzyme. CD44 promotes antioxidant activity and drug resistance in cancer cells by modulating glucose metabolism . We have identified that CD44 regulates glycolysis in PC-3 and LNCaP cells. Research has shown that CD44 is expressed in PC-3 cells but not in LNCaP cells . However, according to our Western blot results, CD44 was expressed in LNCaP-AI and LNCaP cells. The possible reason is that CD44 expression is associated with cells of the NE phenotype in human PCa cell lines . LNCaP cells exhibited NE features in our results. Our laboratory has established that CD44 regulates PDK1 and PFKFB4 expression in PCa cells, PDK1 and PFKFB4 expression is higher in LNCaP-AI cells than in LNCaP cells, and glucose consumption is increased during androgen-independent growth. Therefore, the results of the present study suggest that CD44 may be involved in the androgen-independent transformation of LNCaP-AI cells through the upregulation of glucose metabolism by PFKFB4 and PDK1.
The immunohistochemical staining results showed that the staining intensity of PCa tissue was significantly stronger than that of BPH tissue. It is consistent with our previous research results, the intensity of PFKFB4 expression is proportional to the type of PCa . It was known ADT could induce the CRPC , based on these findings and our results, it was reasonable that PFKFB4 expression would increase accompany with ADT. However, the staining of ADT tissue in present study showed no statistically difference before and after ADT. The main reason was the duration of neoadjuvant ADT, usually no more than 3 months. In hormone-sensitive stage, ADT inhibited proliferation and glycolytic activity of cancer cells which potentially inhibited PFKFB4 as well. As we all know, development of CRPC in ADT-treated tumor can take years to emerge clinically . So, after short term neoadjuvant ADT, tumor cells were also hormone-sensitive, which could explain the findings of immunohistochemical staining. Taken together, high expression of PFKFB4 was the hallmark of tumor proliferation and androgen-independent growth, which suggested PFKFB4 might be a novel biomarker in prostate cancer.
There are several possible limitations to this study. We performed only glucose consumption and lactate secretion assays for metabolic evaluation. The glucose metabolism and PFKFB4 expression of LNCaP-AI cells need to be compared with those of other androgen-independent cell lines. The PFKFB4-focused in vivo experiments with LNCaP-AI cells need further improvement. The human PCa tissue sample size needs to be enlarged, and the expression pattern of PFKFB4 in CRPC tissue remains unclear.