Expression of pepsinogen A (PGA/PG I) in breast cancer and non-cancer tissues and its relationship with clinicopathological parameters of breast cancer

Human pepsinogens (PGs) are inactive pro-enzymes for the specic digestive enzyme--pepsin originating from the gastric mucosa, Pepsinogens A (PGA) is one of pepsinogens family members. In the past, relevant studies in PGA mainly focused on gastric diseases, but there were few reports outside the stomach. Further understanding of the relationship between PGA and extra-gastric diseases, especially cancer, is helpful to fully understand the role and function of PGA. Total of 362 patients with different breast disease after surgery were registered in the study. Immunohistochemical staining was used for PGA expression. GEO, and a series of software packages based on R language were used for further validation as well as function analysis of PGA in breast cancer. signaling on gastric diseases. There were few reports dealing with PGA expression outside the stomach. In the current research, we elucidated the expression status of PGA in breast cancer and non-cancer tissues with IHC and gured out its correlation with clinicopathological characteristics. Data from GEO was utilized for the verication of our results and for the conrmation of prognostic roles of PGA in breast cancer. And GO and KEGG analysis were performed to provide us a more comprehensive look at the functions of PGA. classication of breast cancer. Survival analysis revealed that the prognosis of patients with higher PGA mRNA expression was poorer. PGA may function through interactions with PPAR signaling pathway and AMPK signaling pathway in breast cancer. This study lays a theoretical and experimental foundation for a more comprehensive understanding of the physiological and pathological functions of PGA from extra-gastric organ.


Introduction
Human pepsinogens (PGs) are inactive precursors of pepsin, which is an speci c digestive enzyme.
Generally, PGs can be classi ed biochemically and immunochemically into pepsinogen A/I (PGA/PGI) and pepsinogen C/II (PGC/PGII). Mainly synthesized and secreted by the chief cells and the mucous neck cells of the gastric mucosa, PGs could be converted into pepsin under acidic conditions, and thus participate in the process of digestion [1]. In addition to the stomach, recently researchers have also detected the expression of pepsinogens in some non-gastrointestinal organs [2]. For example, several investigations have proved that there existed ectopic expression of PGC in breast, ovary, prostate and lung [3][4][5][6]. Unlike PGC, few studies are available concerning the ectopic expression of PGA. Up to now, only two reports have made mention of PGA expression outside the stomach. Meuwissen et al. proved the presence of PGA in Barrett's esophagus [7]. And another study conducted by Osman Mamat et al. demonstrated the expression of PGA in intraductal papillary mucinous neoplasms [8]. Further exploration of the relationship between PGA and extra-gastric diseases, especially cancer, could provide a more comprehensive understanding of the role and function of PGA.
Breast cancer in women has become the most common cancer worldwide and the fth leading cause of cancer-related death [9]. It has been reported that some breast cancer cells and breast cysts epithelial cells could produce PGC [10]. Furthermore, several researches have suggested that PGC ectopic expression is closely related to the differentiation, biological behavior and prognosis of breast cancer [11][12][13]. However, it still remains unknown whether PGA is expressed in breast cancer tissue and its correlation with clinicopathological parameters of breast cancer.
In this study, we compared PGA expression in breast cancer and non-cancer tissues including different benign breast diseases by immunohistochemical staining, and further validation as well as function analysis was carried out with data from GEO to explore the role of PGA in breast cancer and its relationship with clinicopathological parameters as well as prognosis of breast cancer.

Immunohistochemistry and its evaluation
The obtained tissue samples were xed with formalin and embedded with para n. Each para nembedded sample was cut into a 4-µm-thick section and mounted in a poly-L-lysine-coated glass slide for preservation [14]. Immunohistochemistry (IHC) was performed as previously described [15]. PGA antibody (Cat: 13082-R021, rabbit, 1:1500 dilution; Sino Biological Inc., Beijing, China) was used in the study. Other reagents including citrate buffer (PH=6.0), phosphate-buffered saline (PBS, PH=7.4), secondary antibody and DAB (DAB-0031) stain were obtained from Maixin Inc., Fujian, China.
During the study, we controlled the quality of immunohistochemistry from three different aspects. Firstly, we used negative controls (PBS instead of primary and secondary antibodies) and positive controls during the experiment to avoid false negative or positive results. Secondly, the color reaction with DAB was observed under the microscope to prevent overstaining or under-staining. Thirdly, a double-blind method was applied to grade PGA expression levels. According to the double-blind principle, two GO and KEGG enrichment analysis for PGA function and related pathway Gene ontology (GO) [17] and Kyoto Encyclopedia of Genes and Genomes (KEGG) [18,19] analysis are useful approaches to discover gene functions and related pathways. Firstly, we used R package limma to screen differentially expressed genes (DEGs) with data of GSE41119, which was separated into two groups according to the expression level of PGA by limma package too. And then GO and KEGG analysis was carried out with R package clusterPro ler and ggplot2 to illuminate the function and pathways of PGA.

Statistical analysis
Statistical analyses were performed by SPSS Statistics Version 20.0 (IBM, SPSS, and Chicago, IL, USA) and R software (R 4.0.4). The homogeneity of variance was tested for all data to determine the overall distribution type of data. Kruskal Wallis test was selected to estimate the differential expression of PGA among different groups. Receiver operating characteristic (ROC) curve was established to determine the diagnostic e ciency of PGA and to select the cut-off value that differentiated high and low expression.
The relationship between PGA protein expression and clinical characteristics of patients was evaluated by the chi-square test. Analysis with R packages was completed with the R software. P value no more than 0.05 was considered as signi cant.

The PGA expression in breast cancer and non-cancer tissues
In this experiment, we compared the expression difference of PGA between breast cancer and non-cancer groups. The speci c stained area was cytoplasm and representative photomicrographs of PGA expression in breast cancer and non-cancer groups were shown in Figure 1. The analysis results suggested that there was a signi cant statistical difference between the breast cancer and non-cancer group (P<0.001; Table 1).

Diagnostic e ciency of PGA expression for breast cancer
According to the analysis result of ROC curve, PGA protein expression (area=0.768; 95%CI = 0.720-0.816; P<0.001) was associated with incidence of breast cancer ( Figure 2). And further we obtained an appropriate cut-off value which was 3.5 from the curve to de ne relative high and low expression of PGA. A total of 190 cases (52.4%) were high expression which above the average score. The positive rate in non-cancer group (71 cases, 35.3%) was lower than that in the breast cancer group (119 cases, 73.9%).

Correlations between PGA expression and clinical parameters of breast cancer
In this study, we explored the association between PGA protein expression and clinical features. Basic features of the study set were shown in supplementary table 1. The analysis results showed a statistical difference in the molecular classi cation of breast cancer (Table 2). To be speci c, PGA expression in triple negative breast cancer (TNBC) was signi cantly different from the other three molecular types, of which were Luminal A (P = 0.009), Luminal B (P = 0.034), and HER2 (P = 0.037). We used GEO data to verify the correlations between PGA and general clinical parameters. Our results suggested that there existed signi cant correlation of PGA expression between T0 and T1 (P = 0.001), T2 (P = 0.004), T3 (P = 0.002), T4 (P = 0.020), respectively ( Figure 3D). As for tumor grade and molecular type, signi cant associations were found between grade 1 and grade 2 (P = 0.018; Fig. 3E), and between Luminal A type and unclassi ed type (P = 0.017; Fig. 3F). No signi cant correlation was found with age (P = 0.790; Fig. 3A), Er (P = 0.290; Fig. 3B), and Her2 (P = 0.980; Fig. 3C). In addition, we used GEO data to analyze the relationship between PGA expression and prognosis of breast cancer and the results showed that lower expression of PGA was associated with better prognosis of breast cancer (P = 0.048; Figure 4).

PGA function and related pathway
To further explore the possible functions and pathways of PGA in the breast, we further performed GO and KEGG enrichment analysis. First we identi ed DEGs associated with PGA expression levels with limma package. Heat maps of the rst 20 different genes were shown in Figure 5. Then GO and KEGG analysis were performed with PGA and these DEGs. The GO analysis showed that PGA may be involved in nuclear division, regulation of lipid metabolic process and organelle ssion ( Figure 6A). Detailed analysis results of GO was exhibited in supplementary table. In KEGG enrichment analysis, PGA was closely related to PPAR signaling pathway and AMPK signaling pathway ( Figure 6B).

Discussion
As important members of human pepsinogens families, PGA and PGC participated in the process of human digestion. Unlike PGC, relevant studies in PGA mainly focused on gastric diseases. There were few reports dealing with PGA expression outside the stomach. In the current research, we elucidated the expression status of PGA in breast cancer and non-cancer tissues with IHC and gured out its correlation with clinicopathological characteristics. Data from GEO was utilized for the veri cation of our results and for the con rmation of prognostic roles of PGA in breast cancer. And GO and KEGG analysis were performed to provide us a more comprehensive look at the functions of PGA.
First of all, we observed the expression of PGA in breast cancer and non-cancer tissues including different benign breast diseases by immunohistochemical staining and grouped all the cases by score results based on HSCORE. Diagnostic value of PGA in breast cancer was further investigated by ROC models. As revealed in our study, the expression of PGA was higher in breast cancer when compared with non-cancer tissues. In consistent with the expression trend of PGA in breast, Serra et al. observed in their experiment that PGC expression was negative in non-cancer breast tissues, whereas in tumor tissues the expression staining was statistically positive [20]. Analysis results of in situ expression of PGA in the stomach revealed that PGA was usually decreased in gastric cancer [21]. Besides, an earlier research implemented by Waalewijn RA et al. showed that in gastric cancer mRNA expressed level of PGA was obviously decreased [22]. Further ROC analysis demonstrated that PGA had potential to serve as a diagnostic marker of breast cancer, differentiating benign and malignant breast diseases. All the ndings mentioned above indicated that there existed diverse expressional pro les of PGA in cancers originated from extragastric organs and stomach, and thus playing distinct roles in tumorigenesis of the two types of cancers.
Secondly, we analyzed the relationship between PGA expression and clinicopathological parameters and prognosis of breast cancer with IHC data and data from online database. IHC results showed that there was no signi cant statistical difference between PGA expression and general clinicopathological parameters of breast cancer except for the molecular classi cation. The overall positive rate of PGA expression was lower in Luminal A type and were at the middle level in Luminal B as well as HER-2 type, while the TNBC type showed more positive cases of PGA expression and more unique in comparison with other subtypes. Analysis with online data showed signi cant association of PGA with T stage, tumor grade and molecular type. Currently the molecular subtypes of breast cancer were identi ed mainly according to the expression of ER, PR, HER2, and Ki-67, which may be helpful for prognostic strati cation and treatment selection [23]. TNBC is a special type of breast cancer with negative expression of ER, PR and HER2 [24]. As an highly aggressive subtype of breast cancer, TNBC was reported to be associated with shorter survival time when compared with other subtypes [25][26][27][28]. Patients with TNBC had a more aggressive clinical course and a higher mortality rate [29,30]. It was obvious that hormone or hormone receptor status, showed a highly closed correlation with prognosis of breast cancer patients. Survival analysis with data from GEO further proved that patients with increased levels of PGA had worse OS.
Interestingly, PGC, which was also expressed as ectopic in the breast, had been reported that its expression in breast cancer was in uenced by multiple hormones and receptors, thus showing different expression intensity and correlation with prognosis [31]. A former research conducted by Vizoso et al. proposed that downregulated expression of PGC was observed in poorly differentiated breast cancer patients and could be served as a novel prognostic marker for breast cancer [12]. Hence it could be hypothesized that possible interaction may exist between expression of PGA, ER, PR and HER2 simultaneously and subsequently exert an adverse in uence on the prognosis of breast cancer patients. The inconsistent role of PGA and PGC in prognosis of breast cancer might due to the otherness lying in their regulation mechanisms in breast cancer. The ndings of this study disclosed that higher PGA was observed in TNBC type and could be selected as a prognostic biomarker of breast cancer.
In order to comprehensively understand the potential function of PGA in breast cancer, we conducted bioinformatics analysis of GO and KEGG. The results revealed that PGA was primarily involved in nuclear division, regulation of lipid metabolic process and organelle ssion. And KEGG further demonstrated that PGA was closely related to PPAR signaling pathway and AMPK signaling pathway. PPAR was a ligandactivated transcription factor belonging to the nuclear receptor superfamily [32]. Three subtypes of PPAR, α, β/δ and γ, were expressed in a tissue-speci c manner in multiple species [33]. Multiple cells in the tumor microenvironment, including macrophages and broblasts, activation of PPARγ induced a shift to a less aggressive phenotype that negatively affects breast cancer progression [34,35]. In epithelial breast cancer cells, activation of PPARγ resulted in decreased cell growth and motility, and increased autophagy and apoptosis [36]. Many studies had shown that autophagy can be activated by the AMPK signaling pathway, which was closely related to energy metabolism [37,38]. AMPK was a key energy receptor that regulates cell energy homeostasis and could be activated by a variety of stimuli, particularly by factors which affected cell metabolism [39]. Reductions in AMPK activity were associated with altered cell metabolic processes that drove tumor growth and development, suggesting that AMPK acts as a metabolic tumor suppressor by reprogramming cell metabolism and inhibiting cell growth [40]. Activated AMPK phosphorylates several downstream target pathways that were involved in the regulation of a variety of cellular functions, such as cell proliferation, cell cycle progression, metabolism, and autophagy [41][42][43][44][45]. In our study, the expression of PGA was correlated with the molecular typing of breast cancer, and showed a certain trend in tumor growth and differentiation. In consideration of all these ndings, we speculated that PGA may participate in the regulation of breast cancer through interactions with PPAR signaling pathway and AMPK signaling pathway.

Conclusion
In summary, in this study, for the rst time, PGA was found to be expressed in breast tissue. The expression of PGA in breast cancer was signi cantly higher than that of non-cancer tissues including different benign diseases, and it was related to molecular classi cation of breast cancer. Survival analysis revealed that the prognosis of patients with higher PGA mRNA expression was poorer. PGA may function through interactions with PPAR signaling pathway and AMPK signaling pathway in breast cancer. This study lays a theoretical and experimental foundation for a more comprehensive understanding of the physiological and pathological functions of PGA from extra-gastric organ.

Declarations Compliance with Ethical Standards
This study was approved by the Human Ethics Review Committee of the First Hospital of China Medical University (Shenyang, China), and written informed consent was obtained from each patients.

Consent for publication
A. breast cancer with negative PGA expression; B. breast cancer with positive PGA expression; C. Noncancer with negative PGA expression; D. Non-cancer with positive PGA expression. (bar = 40μm; magni cation ×200, by MOTIC digital slice scanning and application system) Figure 2 Diagnostic value of PGA in breast cancer.
ROC of PGA in breast cancer showed that elevated expression of PGA was correlated with incidence of breast cancer. Effect of PGA expression on prognosis of patients with breast cancer Figure 5 Heat map of DEGs associated with PGA expressed levels