Comprehensive analysis of the association between tumor glycolysis and immune/inflammation function in breast cancer

Breast cancer is the leading cause of cancer death among women [1]. Breast cancer can be classified as Normal-like, Luminal A, Luminal B, HER2-enriched and Basal-like subtypes. In addition to surgery, systemic therapies including chemotherapy, hormonal therapy, and molecular targeted therapy can be chosen based on the molecular characteristics to combat cancer [2]. Although these systemic therapies have improved patients’ outcomes, many patients do not respond to these existing treatments, which leads to poor prognosis [3, 4]. On this account, some researches have been carried out to explore new effective therapies in breast cancer such as Immunotherapy and metabolic therapy [5, 6]. However, only a minority of patients benefit from these emerging therapies [7]. Exploring the interplay between tumor cells and tumor microenvironment could lead to deeper understanding of breast cancer initiation, progression, and therapeutic resistance, possibly provide prospective strategies for cancer prevention and treatment [8, 9].

Metabolic reprogramming is a key hallmark of cancer [10]. The most frequently mentioned way of metabolic reprogramming is aerobic glycolysis. Aerobic glycolysis, also known as the “Warburg effect”, is a general way of glucose metabolism in cancer cells. In this way, glucose is mainly processed into lactate even when oxidative capacity is intact. This will lead to a highly acidic microenvironment. According to current research, tumor aerobic glycolysis can contribute to malignant transformation and tumor progression [11]. Therefore, tumor aerobic glycolysis has possible implications for prognosis judgment and cancer treatment [12]. Exploiting tumor glycolysis for clinical application requires figuring out how intrinsic and extrinsic factors to be integrated to modify the metabolic phenotype [13].

Tumor microenvironment (TME), cancer cells’ supporting hotbed, has complicated and changeable composition including tumor infiltrating lymphocytes (TILs), other immune and inflammatory cells, fibroblasts, the blood and lymphatic vascular networks, the extracellular matrix (ECM) and so on [14]. Numerous evidences suggest that the immune cells infiltration in the TME could interact with tumor cells, which may affect tumor progression and the efficacy of existing anticancer therapies [15, 16]. The immune cells recruited to the tumor site have dual characters, some can restrain carcinogenesis and tumor progression while others may play a tumor-promoting role [17]. Thus, it is important to figure out the cellular heterogeneity composition of the immune cells infiltration and the cause behind it. The results may be significant for optimizing existing treatments and identifying novel therapeutic targets [18].

Several studies have investigated the relationship between tumor glycolysis and immune/inflammation function [19‐21]. A highly acidic microenvironment due to tumor glycolysis may differentially influence immune cells infiltration, ultimately leading to immune escape and cancer progression [22]. Exploration of the associations is providing a deeper look into cancer biological processes and can lead to more effective therapy selection. So far, however, there has been little comprehensive analysis focusing on the relationships between the tumor glycolysis, immune/inflammation function and the clinical features based on clinical data in the field of breast cancer. Given this, we implemented studies with transcriptome and clinical data of breast cancer from The Cancer Genome Atlas (TCGA) projects to explore the landscape of tumor glycolysis and immunity in breast cancer, to identify the relationship between the tumor glycolysis and immune and inflammatory cells infiltration, and to figure out the impact of the two on breast cancer prognosis.

Sufficient energy and metabolic intermediates for biosynthesis are the foundation of tumor cells initiation, proliferation and metastasis. Thus, many types of cancer are characterized by enhanced level of glycolysis and suppressed mitochondrial metabolism. In this way, tumor cells gain an advantage over stromal and immune cells in the competition to share limited nutrients and acquire ATP and intermediate metabolites faster [11]. The present study demonstrated that the key enzymes in glycolytic pathways had higher expression in breast cancer tissue, which indicated increased glycolysis activity in tumors. Among these glycolysis related genes, PGK1 has attracted our attention for its significant correlation with advanced tumor stage, and poor prognosis. By regulating ATP and 3-phosphoglycerate levels, PGK1 plays a critical role in coordinating energy production with biosynthesis and redox balance [34]. A recent comprehensive analysis of pan-cancer metabolic transformation also indicated that PGK1 was the most frequently upregulated gene in glycolytic pathway and had 100% frequency of upregulation in different types of cancer [28]. It has been demonstrated that high expression and activity of PGK1 was associated with poor prognosis in several types of cancer [35, 36]. Metabolism remodeling is an exquisite process regulated by oncogenic signaling pathways and transcription factors [11]. PGK1 functions as downstream targets of multiple oncogenic signal pathways and directly regulated by MYC,HIF-1α and other major regulator in metabolic reprogramming [37]. What’s more, PGK1 can translocate to Mitochondria and function as a protein kinase to inhibit pyruvate dehydrogenase complex, and promote tumor progress by increasing glycolysis and suppressing mitochondrial tricarboxylic acid cycle [38]. All of the above evidence indicated that PGK1 played a key role in metabolism reprogramming. Our study confirmed this hypothesis as glycolytic pathway-specific genes and pH-regulating genes had remarkable higher expression and glucose metabolism pathways and cell proliferation-related pathways such as cell cycle and DNA replication significantly upregulated in high PGK1 expression group. Therefore, PGK1 expression was regarded to represent the activity of glycolysis in the present study.

Much work so far has focused on the tumor glycolysis. However, the biological events affected by the tumor glycolysis, especially its immune/inflammation regulatory role, haven’t been well clarified. we compared gene expression profile, immune cell type enrichment scores and TIL percentage between low and high glycolysis groups and conducted functional enrichment analysis. The results suggested that numerous cytokines, chemokines, related receptors and immune/inflammation-related pathways were upregulated in the high glycolysis group. Consistent with this, high glycolysis group had a higher immunescore and higher TIL percentage. However, NKT, CD8+ T-cells, CD8+ Tcm, CD4+ Tem, cDC cells which may have an anti-tumor effect in breast cancer weren’t enriched in high glycolysis group. What’s more, immunosuppressive factors including IDO1, PD1, PDL1, CTLA4 and FOXP3 showed remarkable higher expression in high glycolysis group.

The upregulated immune factors and increased infiltration of immune cells did not bring a good outcome to the patients in the high glycolysis group. It seemed to be an inflammatory condition. Inflammation is another key hallmark for cancer initiation and progression, which refers to the local immune response involving cytokines, chemokines, small inflammatory protein mediators, and infiltrating immune cells in the TME [15]. The presence of tumor-antagonizing CTLs and NK cells is not surprising, however the list of tumor-promoting inflammatory cells now includes monocytes, macrophages, DCs, mast cells and neutrophils, as well as T and B lymphocytes [39]. Our study also indicated that high glycolysis contributed to an immunosuppressive tumor microenvironment. The phenomenon that tumor glycolysis and immune/inflammation function influence each other was also observed in other studies. One study found that IL-6 secreted by macrophages can phosphorylate PGK1 to promote glycolysis and tumorigenesis [40]. Several excellent studies have revealed that the metabolic microenvironment of tumor cells including glucose deprivation, lactic acid accumulation and acidification of tumor microenvironment may selectively disable tumor-killing T and NK cell activation and promote immune evasion, while attracting tumor-promoting inflammatory cells [19, 22, 41‐45]. Lactic acid accumulation was demonstrated to induce macrophages toward an inflammatory pro-tumor phenotype [46]. In addition to the one-way effect, some studies have shown that cytokines from inflammatory cells and lactic acid from tumor cells can form a positive feedback loop to promote tumor progression [20, 47].

In our study, the most enriched immune/inflammation-related pathway in high glycolysis group was IL-17 signaling pathway. Emerging evidence indicated the tumor-promoting role of IL-17 in colorectal cancer, pancreatic cancer and lung cancer [48‐50]. The upregulation of IL-17 signaling pathway recruited myeloid suppressive cells, which could induce angiogenesis and restrain anti-tumor immunity [51]. Interestingly enough one study showed that increased glucose uptake and metabolism reprogramming mediated by IL-17 signaling pathway was necessary for the activation of lymph node stromal cells [52]. Our study revealed that IL-17 mediated metabolism reprogramming existed not only in lymph node stromal cells but also in breast cancer tissues. In addition to macrophages which have been the research focus of pro-tumor inflammation, T helper cells especially Th2 cells showed remarkable correlation with tumor glycolysis in our study (r = 0.480, P < 0.001). Previous studies indicated that tumor-associated antigen and HLA-G expressed in tumors can promote immune evasion by strongly favoring Th2 development [53, 54]. However, the relationships between Th2 cells and tumor glycolysis have not been elucidated. Since our study was entirely based on public database and bioinformatic methods, the results should be interpreted with caution. Further in vitro and in vivo studies are needed to confirm our point.

Taken together, these evidences indicated that tumor glycolysis and pro-tumor immunity interdependently promoted each other. Thus, targeting glycolysis may refresh anti-tumor immunity and improve responsiveness to existing treatment such as chemotherapy and Her2-targeted therapy [55]. Since high glycolysis group had higher expression of immune checkpoint and higher TILs density which were both promising predictor for immunotherapy [56], they may exhibit a better immunotherapy response. In line with our research, tumor metabolic parameters on fluorodeoxyglucose positron emission tomography (FDG-PET) showed positive correlation with PD1/PDL1 expression and presence of TILs in non-small lung cancer [57, 58]. Some literature have also suggested the value of FDG-PET in predicting response to immune checkpoint inhibitors in non-small cell lung cancer [59], which partially supported our hypotheses. In our study, Her2-enriched and basal subtypes had higher glycolysis activity and higher immune inflammation cells infiltration in our study, which was consistent with its higher proliferative ability and higher malignant potential [2]. Thus, FDG-PET as the noninvasive detection method of tumor glycolysis may have the potential value to identify molecular subtypes of breast cancer and predict response rate to neoadjuvant chemotherapy since TIL is already known as a good predictor of response to neoadjuvant chemotherapy [60‐62].

Given the critical role of PGK1 in metabolism remodeling and tumorigenesis, it may be a potential therapeutic target. However, targeting key metabolic enzyme can lead to severe systemic toxicity, because they are also essential for sustaining the normal cell function [6]. Attenuating the enzymatic activity of PGK1 by targeting its post-translational modifications (including phosphorylation, acetylation, ubiquitination, etc.) which have been shown significantly altered in cancer might reduce systemic toxicity and is an alternative approach [35, 40, 63, 64]. Despite all this, there is an urgent requirement for clinical application to establish a selective targeted delivery system to inhibit PGK1 activity in cancer cells precisely [6].

Numerous studies have revealed that the initiation and progression of cancer are dependent on a complex interplay between tumor cells and tumor microenvironment. Our study demonstrated that tumor glycolysis and immune inflammatory response may be the entry points for exploring the communication between tumor cells and tumor microenvironment. Deeper insight of the correlation between tumor glycolysis and immune inflammatory infiltration may lead to a revolution in cancer prevention and treatment.

In conclusion, the enhanced glycolysis activity of breast cancer was associated with enriched inflammatory cell infiltration and enhanced inflammation response but higher expression of immunosuppressive factors and inhibition of antitumor immunity. The interaction between tumor glycolysis and immune/inflammation function may be mediated through IL-17 signaling pathway. Thus, drugs targeting metabolism may have an anti-inflammatory effect and vice versa. The level of glycolysis activity may guide the selection of checkpoint inhibitor immunotherapy. PGK1, as an important enzyme in glycolytic pathways, had obvious influence on breast cancer patients’ clinical outcome, which revealed that PGK1 played a critical role in the progression of breast cancer and may be a potential therapeutic target.