In recent years, an increasing number of studies have focused on the development of agents that target the TME for the treatment and prevention of PCa bone metastasis[27, 28]. The TME, also known as the tumor stroma, is mainly composed of tumor cells and a variety of stromal cells, inflammatory cytokines and chemokines, and is the microenvironment between tumor cells and normal tissue cells. The TME plays a critical role in tumor growth and metastasis[4]. We have previously shown that TME-based key targets were significant indicators of tumor progression[29]. Alterations in the TME are mainly controlled by hypoxia, glycolysis, and altered extracellular acidity. Hypoperfusion of tumor cells at an early stage leads to an hypoxic state in the TME, and hypoxia induces the release of hypoxia inducible factor-1 (HIF-1)[30], which further promotes secretion of the pro-angiogenic factor VEGF, leading to the generation of new blood vessels. The metabolism of tumor cells under hypoxic conditions occurs mainly through glycolysis, and the lactate produced by glycolysis further decreases the pH in the tumor extracellular microenvironment. An acidic microenvironment is unfavorable for the survival of immune killer cells, and leads to tumor cell growth and metastasis. Studies have also shown that an acidic TME had a drug-resistant effect on most anti-tumor drugs, while HIF-1 initiated transcription of the drug-resistance gene P glycoprotein, prompting a decrease in drug concentration within tumor cells[31]. Therefore, studying the regulation of the TME will facilitate the identification of novel targeted agents for the treatment of PCa bone metastasis.
Previous studies have shown that an acidic TME promoted tumor cell growth, invasion, and metastasis. Tian et al. revealed that an acidic microenvironment upregulated exosomal miR-21 and miR-10b in early hepatocellular carcinoma, thereby promoting cancer cell proliferation and metastasis[6]. Similarly, Boedtkjer et al. examined the role of the acidic TME on tumor genetic stability, epigenetics, cell metabolic proliferation and invasion[7], and found that an acidic microenvironment promoted proliferation of tumor cells and the occurrence of metastasis.
In this study, we first verified the importance of the acidic microenvironment on PCa. Using the TCGA public database, we screened acidity-related genes in PCa through machine learning classification, and constructed a prognostic risk assessment model based on the acidic microenvironment. Our model revealed that the acidic microenvironment significantly predicted the progression, metastasis and prognosis of PCa. In addition, in multiple immunoassays, our study revealed that an acidic microenvironment was indicative of poorer immune cell infiltration in PCa.
We next confirmed that an acidic microenvironment promoted PCa proliferation in vitro, as well as the migratory and invasive abilities of PC-3 cells. In addition, our cell-matrix adhesion assay confirmed that the number of PC-3 cells adhering to fibronectin was higher under acidic conditions than control NM conditions, indicating that the acidic environment promoted adhesion of PC-3 cells to the extracellular matrix, which may lead to tumor cell metastasis. Together, our findings demonstrated that the acidic TME promoted proliferation and metastasis of PC-3 cells in vitro.
Next, we investigated the effects of potential drug intervention treatments, specifically AS, in the acidic TME of PCa. AS is a potent active component extracted from the traditional Chinese herb Centella asiatica, which possesses potential anti-tumor properties. AS has been shown to exert inhibitory effects on a variety of tumor cells. For example, Ying et al. demonstrated that AS inhibits hepatocellular carcinoma cell proliferation and reduces chemoresistance[18], while Zhou et al. showed that AS inhibits the growth of colon cancer cells both in vitro and in mouse models in a dose-dependent manner[19]. However, to date, there have been no studies examining the effects of AS on PCa.
CSCs have been shown to play a key role in tumor growth and metastasis in several types of human malignant tumors[32–34]. As a heterogeneous sub-population of cancer cells that possess self-renewal and multidirectional differentiation potential, CSCs bring many uncertainties to the occurrence and development of tumors and are considered to be the major cause of resistance in chemotherapy and radiotherapy[35, 36]. Thus, targeting CSCs is considered to be a crucial intervention strategy for cancer therapy. We have previously shown that CSCs promoted angiogenesis[14]. In the current study, we proposed that treatment with AS could be a novel strategy for targeting CSCs.
Here, we found that AS significantly inhibited the proliferation of PC-3 cells in a dose-dependent manner. Furthermore, AS treatment inhibited PC-3 cell migration and invasion in the acidic microenvironment, as well as reduced cell adhesion between cells and fibronectin. In addition, we demonstrated that AS treatment led to a significant dose-dependent reduction in the sphere formation and colony formation of PC-3 cells under acidic conditions. Moreover, western blot analysis revealed increased expression of the CSC-related markers CD44, KLF4, OCT4 and AGO2 under acidic conditions. To determine whether the therapeutic potential of AS on PCa within the acidic microenvironment involved CSCs, we next co-cultured PC-3 and BM-EPC cells to simulate the angiogenesis of tumors. We found that AS significantly inhibited angiogenesis. In addition, we found that AS treatment led to a reduction in VEGF levels. Because VEGF is a key factor in BM-EPC differentiation and angiogenesis, our findings further indicated that AS may inhibit tumor angiogenesis. MMP-2 and MMP-9 are members of the MMP family, which are associated with CSCs and play an important role in the invasion and metastasis of tumor cells[37, 38]. Upon addition of AS, the secreted levels of VEGF, related cytokines (IL-1β, IL-6, IL-8 and TNF-α) and tumor-associated metastatic factors (MMP-2, MMP-9) were all decreased to various degrees, indicating that AS treatment could affect the secretion of factors associated with the acidic TME.
Overall, we verified the importance of the acidic microenvironment on PCa by bioinformatics analysis, then constructed a prognostic risk assessment model based on the acidic microenvironment which could effectively predict the progression, metastasis and prognosis of PCa. We also confirmed that the acidic microenvironment promoted PCa proliferation and migration/invasion in vitro. Most importantly, our findings highlighted the therapeutic role of AS in the treatment of PCa, and demonstrated that AS reduced tumor angiogenesis in PCa through the inhibition of CSCs.