Over-expression of NFYB affects stromal cells reprogramming and predicts worse survival in gastric cancer patients

Introduction

Gastric cancer (GC) is a common gastrointestinal tumor. The number of new cases of GC in China accounts for 43.9% of the global total each year, posing a severe threat to the health of the Chinese people [1]. Radical surgical resection is still the cornerstone of gastric cancer treatment. The development of minimally invasive surgery, new targeted drugs, and immunotherapy have brought breakthroughs in treating gastric cancer patients. However, the overall therapeutic effect of gastric cancer is still not satisfactory. Both the individual heterogeneity of GC patients and the low response rate to immunotherapy affected the prognosis of GC patients. It is of great clinical significance to explore the new molecular mechanism of GC and to find new therapeutic targets for GC.

The extracellular matrix and various mesenchymal cells constitute the tumor stromal microenvironment [2]. Cancer-associated fibroblasts (CAFs) are the most abundant cell type of mesenchymal cells [3, 4]. Many CAFs in tumor tissue create a suitable environment for tumor development. CAFs play an essential role in cancer: CAFs can not only suppress the function of immune cells by secreting a variety of cytokines or metabolites to promote the development, invasion, and metastasis of tumors but also shape the external tumor stromal and form the permeation barrier to prevent the deep infiltration of drugs and immune cells into tumor tissues, thus reducing the therapeutic effect of tumor [3]. Therefore, Regulating CAFs or overcoming their barrier effect to control tumors is a new approach to tumor therapy [5]. Metastasis was the leading cause of poor prognosis and low survival rate in GC patients, which is responsible for about 60% of GC deaths [5]. Many studies have shown that CAFs can directly or indirectly promote the migration and invasion of GC cells by releasing growth factors or cytokines. CAFs can induce GC invasion and metastasis through multiple cascade pathways by regulating miRNA expression [6]. Wu et al.’s study showed that CAFs could secrete a large amount of IL-6 to induce EMT by activating the JAK2/STAT3 pathway, increase the migration of gastric cancer cells, and promote the development of GC [7]. Shen et al. demonstrated that vascular cell adhesion molecule 1(VCAM1) derived from CAFs could interact with integrin αVβ1/5 in GC cells to promote tumor invasion in vivo and in vitro [8]. CAFs are expected to be a potential diagnostic marker and therapeutic target for gastric cancer. Currently, the method to treat cancers is being explored by targeting CAFs [9]. However, there are still many challenges, such as the lack of specific markers of CAFs, which makes it difficult to make specific targeting strategies, and the functional heterogeneity and polarization dynamics of CAFs. So, it is necessary to have a deeper understanding of the therapeutic response to CAFs and more accurate targeting methods.

NF-Y is a ubiquitous heterotrimer transcription factor with a binding affinity with CCAAT consensus motifs and is one of the most common cis-acting elements in promoter and enhancer regions of eukaryotic genes [10, 11]. NF-Y consists of three subunits, namely the regulatory subunits NF-YA, NF-YB, and NF-YC, all of which require binding to CCAAT [12]. More and more studies have shown that the NF-Y gene family drives the transcription of many cell cycle regulation genes and plays a crucial role in proliferation regulation [13]. NFYB plays a vital role in many biological processes, including cell proliferation, senescence, and apoptosis. Studies have shown that NFYB can enhance the activity of STK33 and promote cisplatin resistance in diffuse large B cell lymphoma [14]. NFYB induces high expression of E2F1 and promotes oxaliplatin resistance in colorectal cancer by enhancing the CHK1 signaling pathway [15]. In head and neck squamous cell carcinoma, single-cell RNA-seq deconvolution revealed that NF-YA was associated with cancer-associated fibroblasts (CAFs) and p-EMT cells (populations with metastatic potential) [16]. The role of NFYB in the occurrence and development of GC remains unclear. Therefore, we used bioinformatics methods to analyze the expression of NFYB in the GC cohort and its correlation with clinicopathology and the prognosis of GC patients. Meanwhile, we analyzed the possible mechanism of NFYB affecting the infiltration of CAFs in GC.

Results

Discussion

GC is one of the most common malignant tumors, and its mortality and morbidity are at the forefront of malignant tumors [17]. It is of great clinical significance to explore the new molecular mechanism of GC and to find new therapeutic targets for GC. NFYB is a subunit of nuclear transcription factor Y, Also known as HAP3, CBF-A, CBF-B, and NF-YB. Its location is 12q23.3. NFYB was highly expressed in multiple tumors and promotes tumor invasion, metastasis, and drug resistance [14–16, 18]. However, the role of NFYB in gastric cancer has not been reported, so studying the effect of NFYB on GC may further clarify the possible mechanism of the occurrence and development of GC. In this study, by mining TCGA and GEO public databases, we found that NFYB is highly expressed in GC tissues and is associated with a poor prognosis of GC. There was no significant difference in the expression of NFYB in T1 gastric cancer compared with normal tissues. However, with the increase in the T stage, the expression of NFYB also increased significantly, suggesting that NFYB may be associated with the invasion and metastasis of GC. The expression of NFYB in 21-40 year-old gastric cancer patients was significantly higher than that in older gastric cancer patients, suggesting that NFYB may play a role in the occurrence and development of early-onset gastric cancer. Meanwhile, the analysis showed that the expression of NFYB was higher in poorly differentiated GC tissues and higher in diffuse gastric cancer than in intestinal gastric cancer. These results suggest that high NFYB expression may be associated with the poor prognosis of GC patients. It has been reported that CAFs are related to the occurrence, development, and prognosis of gastric cancer, but the specific mechanism of the difference in the infiltration degree of CAFs in gastric cancer patients remains unclear. Our analysis showed that the expression of NFYB was positively correlated with the degree of CAFs infiltration in gastric cancer patients. The expression of NFYB was correlated with the expression of various markers of CAFs. According to the expression of NFYB, GC patients were divided into two groups: the low NFYB expression group and the high NFYB expression group. And we found no significant correlation between the infiltration degree of CAFs and the prognosis of GC patients in the low NFYB expression group, while in the high NFYB expression group, the prognosis of GC patients with high infiltration of CAFs was worse. These results indicated that the overexpression of NFYB was closely related to the function of CAFs. Altogether, our study provides insights into understanding the potential role of NFYB in CAFs and its use as potential anti-cancer targets.

We used the TIMER database to analyze TCGA’s pan-cancer RNA-SEQ expression data and explore the expression of NFYB in various tumors. The results showed that NFYB expression was significantly higher in Cholangiocarcinoma (CHOL), colon cancer, esophageal cancer, head and neck cancer, hepatocellular carcinoma, and gastric cancer than in adjacent normal tissues. It was significantly lower expressed in breast cancer, glioma, kidney cancer, pheochromocytoma and paraganglioma, prostate cancer, skin melanoma, and endometrial cancer than in normal adjacent tissues. This suggests that NFYB may have different functions in different types of cancer. It has been reported that NFYB is highly expressed in the head and neck squamous cell carcinoma [16], lung cancer [19], cervical cancer [20], and liver cancer [18], which is consistent with our analysis results. We then analyzed the correlation between NFYB expression and clinicopathology of gastric cancer. The results showed that the expression of NFYB was correlated with T stage, age of onset, histological grade, and laurun type of gastric cancer. The expression of NFYB was relatively higher in patients with a higher T stage, suggesting that NFYB overexpression may be related to the invasion of GC. Previous studies have shown that NF-Y drives the transcription of many cell cycle regulatory genes and plays a crucial role in proliferation regulation [13]. Uncontrolled proliferation is a hallmark of cancer cells, and proper control of cell growth is crucial to preventing cancer. NFYC has been shown to promote the growth of prostate cancer cells [21]. The expression of NFYB in 21-40 year-old gastric cancer patients was significantly higher than that in older gastric cancer patients, suggesting that NFYB may play a role in the occurrence and development of early-onset gastric cancer. The potential etiology of early-onset gastric cancer is still unknown and is a severe social burden [22]. Many patients with early-onset gastric cancer were already at an advanced stage when diagnosed and could not benefit from treatment [23]. Therefore, exploring the molecular markers of early-onset gastric cancer is of great significance and realizing the early diagnosis of early-onset GC. Our results also showed that NFYB expression was correlated with diffuse gastric cancer and poor differentiation of gastric cancer cells. These results suggest that NFYB expression may be associated with a poorer prognosis in patients with gastric cancer. Hence, we used the TCGA-STAD cohort and GEO GC dataset to analyze the affection of NFYB expression levels on the prognosis of GC patients. The results showed that high expression of NFYB was associated with poor OS and DFS but not with PFS in gastric cancer patients. These results suggest that NFYB may serve as a prognostic molecular marker for gastric cancer.

To explore NFYB interacting proteins and their possible functions, we performed protein-protein interaction network analysis using the String database. The minimum interaction score was set as 0.9, and the PPI Enrichment p-value was less than 0.05. The results showed that the NFYB protein interaction network contained 11 genes with 23 edges, and the PPI enrichment p-value was 0.0033 (Figure 4A). These genes include NFYA POLE4, NFYB, EP300, NFYC, TP53, MYC, XBP1, REPIN1, CHRAC1, and CIITA. Among them, TP53, MYC, and EP300 are widely studied oncogenic genes that affect cancer’s occurrence and development in various ways [24–26]. We performed the GO and KEGG pathways enrichment analysis of NFYB interacting proteins. The results showed these genes are closely related to gene transcription regulation and lipid metabolism. It is also closely related to the Wnt signaling pathway, TGFβ signaling pathway, and JAK-Stat signaling pathway. These results suggested that NFYB may be related to tumor immune or with CAFs infiltration.

High NFYB expression was associated with GC stromal score, and about 50% of stromal cells in the tumor microenvironment were CAFs [4]. The role of CAFs in various types of cancer has been proven. And CAFs can promote cancer progression through multiple mechanisms [27, 28]. However, depletion of fibroblasts accelerates tumor growth in pancreatic cancer, and specific subpopulations of CAFs exhibit cancer suppressive effects, indicating the molecular and functional heterogeneity of CAFs [29, 30]. Nevertheless, most current studies support CAFs as a tumor-promoting agent and consider targeting CAFs may be a promising cancer treatment strategy. At present, many markers of CAFs have been identified; common markers include α-smooth muscle actin (α-SMA) and fibroblast activation protein, FAP, fibroblast specific protein1, FSP1, platelet-derived growth factor receptor beta (PDGFR - beta), etc. [31]. Specific labeled CAFs subgroups can affect the survival and prognosis of tumor patients. For example, the increase of α-SMA labeled CAFs cells in breast cancer is significantly associated with poor prognosis, and FAP+ CAFs are abundant in invasive breast cancer tumor tissue [32, 33]. PDGFR-β -labeled CAFs subpopulations are associated with a higher risk of recurrence and a poor prognosis for ductal carcinoma in situ [34]. Our analysis showed that the expression of NFYB was positively correlated with the CAFs infiltration in GC. However, it has been reported that NFYB can promote T cell infiltration in the tumor microenvironment [35]. Therefore, NFYB may not promote tumor development through the immunosuppressive effect of CAFs. In pancreatic ductal adenocarcinoma (PDAC), some researchers found two subsets CAFs, which were muscle fibroblasts CAFs (myCAFs) and inflammatory CAFs (iCAFs) [36]. myCAFs are characterized by high expression of α-SMA, secretion of extracellular matrix components, and rapid proliferation around tumor cells [36]. In contrast, iCAFs are characterized by low α-SMA expression, sufficient secretion of inflammatory factors, slow proliferation, and distance from tumor cells [36]. In terms of function, myCAFs mainly mediate the production of extracellular matrix components to promote tumor metastasis and invasion, while iCAFs can secrete some cytokines such as IL-1 and IL-6 to promote tumor growth and mediate immunosuppression. Therefore, we further analyzed the correlation between NFYB and CAFs molecular markers. The expression of NFYB was positively correlated with most of the molecular markers of CAFs. The correlation with FAP, α-SMA, and Vimentin was stronger. This suggested that NFYB may promote the infiltration of myCAFs in GC and promote the metastasis and invasion of GC. In the high NFYB expression group, the prognosis of GC patients with high infiltration of CAFs was worse.

In conclusion, NFYB is differentially expressed in a variety of cancers. In GC, high NFYB expression was associated with high T stage, high histological grade, and diffuse gastric cancer. Meanwhile, NFYB may be a marker molecule of early-onset gastric cancer. High expression of NFYB is associated with poor prognosis of GC and promotes CAFs infiltration in the GC microenvironment. Therefore, NFYB may serve as a potential prognostic biomarker in patients with GC.

Materials and Methods