1. Introduction
Prostate cancer (PCa) is the most commonly diagnosed malignant tumor of the male genitourinary system, ranking second and fifth in morbidity and mortality worldwide [
1]. According to GLOBOCAN 2020, an estimated 191,930 new patients with PCa were diagnosed globally [
2]. Radical prostatectomy (RP) and radical radiotherapy (RT) are the recommended curative treatments for clinically organ-confined PCa, and technological advances have improved the efficacy of both RP and RT. However, approximately 20–60% of patients who receive radical treatment encounter biochemical recurrence (BCR) within 10 years [
3]. Biochemical recurrence is when prostate-specific antigen (PSA) levels rise after the treatment of PCa, with certain PSA not reaching a consistent level. Routinely, we define PSA > 0.2 ng/mL after radical surgery or PSA > 2 ng/mL higher than the post-radiation PSA nadir as BCR [
4,
5]. It is reported that 24–34% of PCa patients with BCR exhibit distant metastasis [
6]. Once the patients demonstrate signs of BCR, the treatment becomes very tricky. Antonarakis et al. reported that the median time for metastasis-free survival of patients with BCR was 10 years [
7]. Studies have shown that clinicopathological factors, such as clinical staging, PSA, Gleason score, and surgical margin, cannot accurately predict BCR [
8]. Therefore, it is urgent to seek new and more accurate BCR predictive markers and explore the pathogenesis of PCa.
Mitochondria are the core site of cellular energy production in the form of adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS) of glucose [
9]. Recent studies have shown that targeting the energy metabolism of cancer cells might be a new and promising area for selective tumor treatment [
10]. Emerging evidence suggests that mitochondria are involved in fatty acid metabolism, reactive oxygen species (ROS) production, and cell apoptosis, which can change cancer cell progression and survival [
11,
12]. In 1956, Warburg first observed a close relationship between mitochondrial respiratory defects, aerobic glycolysis, and cancer [
13]. A recent study has verified that impairment of mitochondrial respiration can inhibit the migration and invasion of breast cancer, mouse melanoma, and PCa cells [
14]. Among patients with colorectal cancer, a high mitochondrial respiration level in the tumor samples has been associated with poor survival [
10]. Roy et al. showed that the mitochondrial respiration profile might serve as a biomarker for the identification of leukemic cells [
15]. Mitochondrial DNA (mtDNA) encodes essential subunits of the mitochondrial respiratory chain that act as energy sources and facilitate tumor proliferation and invasion. Kalsbeek et al. used next-generation sequencing to examine mitochondrial genomes from PCa tissue biopsies, which showed a positive correlation of the total burden of acquired mtDNA variants with the elevated Gleason score at diagnosis and BCR [
9]. Furthermore, it was found that mitochondrial damage and genome change were related to tumor growth, metastasis, and BCR [
16]. Although studies have established that mitochondrial respiration plays a critical role in tumor development and the anti-tumor process, the relationship between mitochondrial respiration and BCR in PCa is still unclear.
In our present study, we constructed a risk signature based on four mitochondrial respiration-related genes (MRGs) using The Cancer Genome Atlas (TCGA) sequencing data of PCa, and the GSE116918 dataset was used to verify the stability of this model. Furthermore, the correlation of MRGs with clinical features and the tumor immune microenvironment was analyzed. Finally, a nomogram was constructed based on four MRGs and clinicopathological factors which demonstrated good performance for predicting prognosis in patients with PCa.
4. Discussion
The occurrence of BCR in patients with PCa after radical treatment indicates the likelihood of distant metastasis and the development of castration-resistant prostate cancer (CRPC) [
8]. Although the advancement in salvage treatment regimens, including radiation therapy, androgen deprivation therapy, chemotherapy, and even intensive multimodal therapy, has improved the prognosis of patients with PCa, most still die within 2 to 4 years [
22,
23]. PSA and the Gleason score are well-known indicators used to predict BCR in PCa patients and grade the risk of BCR after clinical treatment [
24]. However, PCa is a highly heterogeneous disease, and determining the prognosis of certain patients could be challenging. Therefore, better and more accurate prognostic indicators are needed to avoid unnecessary over-medical treatment to identify high-risk patients with BCR and guide individual clinical treatment.
With the development of bioinformatics, studies have reported several prognostic models based on gene signatures to predict BCR. Signatures based on multiple gene expressions, including metabolic [
25], immune-associated [
26], and ferroptosis-related genes [
27], were highly associated with the BCR of patients with PCa. However, too many genes in the above signatures limit their clinical application. There is still a lack of a precision molecular targeting index to effectively predict BCR in patients with PCa. In recent years, the alterations of mitochondrial metabolism in the tumor have been the focus of our research. Mitochondria are highly evolved intracellular organelles that control cell energy production, signaling transduction, and cell death [
28]. Many core metabolic pathways in the mitochondria, including those of amino acids, lipids, and carbohydrates, as well as oxidative phosphorylation (OXPHOS), are essential for cancer cell proliferation [
29]. The rapid proliferation of cancer requires metabolic adaptations to meet the increasing energy demand and to cope with the oxygen-deprived microenvironment [
30]. As essential intermediates produced by mitochondria, NADH, NADPH, and FADH2 fuel the electron transport chain (ETC) and OXPHOS to produce energy [
11]. One well-recognized strategy is to shift the metabolic flow from OXPHOS or respiration in the mitochondria to glycolysis in the cytosol, also known as the Warburg effect. However, some cancers do not follow Warburg’s rule. Studies have found that OXPHOS in the ETC provides the major sources of energy to promote cancer proliferation, such as colon cancer [
31], PCa [
30], and chronic lymphocytic leukemia (CLL) [
15]. Thus, the ETC may act as a potential therapeutic target for cancer. Previous research has shown that metastatic cancer maintains high rates of O
2 consumption compared with normal tissues and stimulates mitochondrial biogenesis [
31,
32]. Rebane-Klemm et al. [
29] used high-resolution respirometry to observe mitochondrial respiration in 48 patients with mutated
KRAS and
BRAF in colorectal cancer (CRC). The results indicated that CRC patients have a higher level of mitochondrial respiration with poor survival. Furthermore, Roy et al. (15) found that the zeta-chain-associated protein of 70 kD (ZAP-70), a mitochondrial respiration-related prognostic marker, predicted increased maximal respiration in patients with CLL and increased sensitivity of ZAP-70+ cells to Ibrutinib treatment. Mutations in oncogenes, tumor suppressor genes (including TP53 and bcl-2), and mtDNA variation could directly affect mitochondrial respiration and metabolism in PCa [
33,
34].
Additionally, zinc ion plays a vital role in the energy metabolism of prostate epithelial cells. Studies also showed that a decrease in zinc concentration could power OXPHOS in the ETC during the early development of PCa [
35]. Mitochondrial metabolism-related enzymes, such as SUMO-deficient hexokinase 2, bound to mitochondria, could reduce mitochondrial respiration and result in cancer cell proliferation [
36]. The previous literature also reported that high expression of OXPHOS-related sulfite oxidase was associated with post-operative BCR in patients with PCa, possibly by inducing PCa cell proliferation [
37]. Together, the above findings indicate that mitochondrial respiration is cross-linked with cancer occurrence, development, and recurrence. Hence, we constructed an MRG prognostic model to predict the BCR in patients with PCa.
Our study used machine learning algorithms (univariate and LASSO) to identify the prognostic signatures associated with mitochondrial respiration, consisting of
APOE,
DNAH8,
EME2, and
KIF5A, which have valuable and independent significance for predicting BCR. Apolipoprotein E (
APOE), including E2, E3, and E4 isoforms, have pivotal roles in mediating cholesterol and lipid uptake by cells [
38]. In addition,
APOE is also involved in carcinogenesis since it can modulate angiogenesis, cell growth, and metastasis in tumors [
39]. Genetic polymorphisms of
APOE have been reported to influence the growth and progression of many cancers, including colon cancer [
39], breast carcinoma [
40], and primary brain tumor [
41]. High plasma cholesterol concentration was observed in patients with PCa, and APOE mRNA was highly expressed in PCa cell lines and prostatectomy specimens [
42,
43]. Further studies found that different APOE isotypes are associated with varying aggressiveness of PCa cells, such as non-aggressive PCa cell lines carry the E3/E4 isotypes while aggressive ones carry the E2/E4 isotypes [
44]. Utermann et al. [
45] observed that the frequency of homozygosity for the
APOE ε4 allele was increased in PCa compared with normal tissues. Furthermore, Yencilek et al. [
38] demonstrated that the
APOE E3/E3 genotype might be a potential risk factor for PCa and high Gleason scoring.
Genomic variations of dynein axonemal heavy chain (
DNAH) family members have been frequently reported in multitudes of malignant tumors. A variant of the
DNAH11 gene, rs2285947, is a potential risk factor for ovarian and breast cancer progression [
46]. In addition, gene mutations in
DNAH increase the sensitivity of patients with gastric cancer to chemotherapy [
47]. According to a genome-wide RNAi screen, Wang et al. [
48] have found that the high expression of
DNAH8 contributes to a greater risk of relapse and poor survival after prostatectomy, possibly by activating the androgen receptor signaling pathway.
EME2 can restart a stalled fork and regulate the homologous recombination repair pathway [
49,
50]. In the present study, we found that
EME2 may regulate mitochondrial respiration and affect the BCR in patients with PCa. Therefore,
EME2 could be a potential therapeutic target for PCa. Further,
KIF5A is a member of the kinesin family, which can modulate the cell cycle, proliferation, and differentiation [
51]. Many studies have demonstrated that high expression of
KIF5A is associated with cancer progression and a poor prognosis, such as in bladder, lung, and breast cancers [
52,
53,
54]. In addition, exome sequencing for 64 tumor samples from 55 PCa patients demonstrated that the
KIF5A mutation was related to aggressive diseases [
55]. Notably, our qRT-PCR result showed that the expression of
KIF5A is downregulated in PCa cells compared to normal prostate epithelial cells, which is contrary to the result of the TCGA database and may be related to tumor heterogeneity. Finally, we established a nomogram using these signatures and clinical data and evaluated its performance to facilitate clinical decision-making. Through the above analysis, we could suggest that these four MRGs might serve as potential novel target genes for PCa treatment.
Additionally, GO and KEGG analysis revealed that the risk model was closely related to the Peroxisome proliferator-activated receptor (PPAR) signaling pathway, primary bile acid biosynthesis, the cholesterol catabolic process, the organic hydroxy compound biosynthetic process, the small molecule catabolic process, and the steroid catabolic process. Peroxisome proliferator-activated receptors (PPARs) are nuclear transcription factors that play a vital role in regulating growth and differentiation within normal prostate and PCa cells [
56]. The activation of the FABP12/PPARγ pathway induces epithelial-to-mesenchymal transition and lipid-derived energy production to promote PCa metastasis [
57]. Olokpa et al. [
58] found that reduced androgen receptor function could increase the expression of PPARγ and the anti-tumor effects of PPARγ agonists in PCa. In addition, PPARγ derived PCa growth and metastasis by upregulating AKT3 could increase mitochondrial biogenesis levels [
59]. Studies have found that the amount of cholesterol is higher in PCa cells compared to normal cells, influencing cancer development and progression [
60]. Henrich et al. [
61] revealed that reducing cholesterol in bone marrow myeloid cells can render the transduction of PCa extracellular vesicle signaling, thus hindering the bone metastasis of PCa. It was reported that sex steroid hormones, especially androgens (testosterone and dihydrotestosterone), contribute to the growth and progression of PCa [
62]. Ahlering et al. [
63] demonstrated that testosterone replacement therapy after radical prostatectomy significantly reduced BCR in patients with PCa and delayed the time to BCR. Furthermore, a new steroid compound (steroid-based copper transporter 1 inhibitors) has also been discovered, which can suppress PCa cell proliferation and tumor growth by reducing copper uptake and may act as a novel anti-cancer drug for PCa [
64]. However, further research is needed to understand its mechanisms in PCa better.
Activating the immune response to treat cancer has become the cornerstone of modern oncology therapy. Emerging studies have explored the roles of immune cells in PCa [
65,
66]. Here we showed that Tregs were the most significantly enriched immune cell in the high-risk group. In addition, activated memory CD4+ T cells and M2 macrophage cells were higher in the high-risk group than in the low-risk group. Various immune cells, namely, CD4+ T cells, CD8+ T cells, natural killer (NK) cells, and macrophages, are enriched in the prostate tumor microenvironment [
67]. Research showed that the reduction of T cells was correlated with BCR and poor survival in patients with PCa [
68,
69], which is partly consistent with our results.
Tregs, distinguished by specific markers (CD25, CD4, CD127, and FOXP3), play a vital role in maintaining immune homeostasis. It is reported that Tregs are significantly enriched in PCa tissues and associated with the progression of cancer cells [
70]. Vidotto et al. [
71] observed that increased FoxP3+ Tregs were associated with PTEN deficiency and lymph node metastasis in patients with PCa. Several possible mechanisms are involved in cancer progression, such as Tregs inhibiting T lymphocytes’ function, NK cells, DCs, and macrophages, or weakening the immune response by secreting immunosuppressive cytokines such as TGF-β and interleukin-10 (IL-10) [
67]. Previously, Hu et al. [
72] reported that CD4+ T cells contribute to PCa immune evasion and progression. Their team further discovered that infiltrating CD4+ T cells could promote PCa chemotherapy resistance by modulating the CCL5/STAT3 signaling pathway [
73]. Due to nutritional deficiencies caused by energy competition between tumor cells and immune cells, some immune cells use lactic acid as an energy substrate. However, studies found that lactate resulting from stromal metabolic reprogramming could modulate CD4+T cell polarization and induces immunosuppressive behavior to promote PCa progression [
74]. M2 macrophages are a class of differentiated tumor-associated macrophages (TAMs) associated with poor clinical outcomes in several cancers [
75,
76]. A recent study found a significant correlation between M2 macrophages and Tregs; M2 macrophages can stimulate lymphocytes to develop into Tregs to promote an immunosuppressive environment in aggressive PCa [
77]. Meanwhile, we further analyzed the correlation between immune cell infiltration and biomarkers and found that
APOE had a significant positive correlation with M2 macrophages and a negative correlation with resting memory CD4+T cells. In anti-atherogenic,
APOE can induce macrophage conversion from M1 to M2 [
78]. Furthermore, Zheng et al. [
76] demonstrated that M2 macrophages could transfer functional
APOE exosomes to neighboring gastric cancer (GC) cells and activate the PI3K-Akt signaling pathway to promote GC migration. Therefore,
APOE could become the potential target gene of immunotherapy for PCa.
Tumor Immune Dysfunction and Exclusion (TIDE) is a method to predict the immune checkpoint blockade response using gene expression profiles. Patients with cancer and higher TIDE scores could undergo anti-tumor immune escape [
79]. Compared to PA-L1 and tumor mutation burden (TMB) indicators, the TIDE score is more accurate in predicting the survival outcome of patients who received immune checkpoint blockade treatment [
80]. We observed that the TIDE score was significantly lower in the high-risk group than in the low-risk group, and the finding suggested that patients with PCa are more sensitive to ICB treatment. Moreover, we also evaluated the association between the risk score and the IC50 of the cancer drugs. Our results indicated that PCa patients with high risk scores could be more resistant to the 12 administered chemotherapies (Lapatinib, Bicalutamide, Embelin, Erlotinib, Bexarotene, A.770041, Z.LLNle.CHO, FH535, Imatinib, Cyclopamine, AZD8055, and MG.132). Resistance to chemotherapy may be due to the poor prognosis of patients with PCa. The IC50 of JNK.Inhibitor.VIII and ABT.888 were negatively correlated with the risk score, indicating that these drugs could benefit patients with high DE-MRGs-based risk scores. However, large samples of randomized controlled trials are needed to further validate the effectiveness of the two drugs (NK.Inhibitor.VIII and ABT.888).
Although our study has achieved encouraging results, there are still some limitations. Firstly, this is a retrospective analysis, and selection bias may exist in this study. Secondly, the clinical information of some patients with PCa from the GEO dataset was incomplete. Thirdly, although we performed a multi-faceted, multi-database validation, the amount of data in this study was relatively small, and therefore, the analysis may be biased. Finally, although qRT-PCR has been used to detect the expression of the four mitochondrial respiration-related genes, further experiments in vitro and in vivo are needed to explore the underlying mechanism behind the risk scores and BCR in PCa.