Identification of prognostic risk factors for pancreatic cancer using bioinformatics analysis

Introduction

Pancreatic cancer is one of the most common lethal tumors worldwide, and its overall 5-year survival rate is less than 5% in the United States. PDAC accounts for 95% of all pancreatic cancers (Siegel, Miller & Jemal, 2018). Moreover, it has been reported that only 10–15% of pancreatic cancer patients are eligible for tumor resection, a trend that is attributed to the lack of early diagnostic markers and advanced metastasis (Becker et al., 2016; Caruso Bavisotto et al., 2017). Mounting evidence indicates that abnormal expression and gene variants are related to the tumorigenesis and progression of pancreatic cancer. A study by Caldas C showed that K-ras activation was involved in early events in N-nitroso-bis (2-oxopropyl) amine-induced pancreatic carcinogenesis in hamsters (Caldas et al., 1994). In addition, it has been reported that inactivation of SMAD family member 4 (SMAD4) and cyclin dependent kinase inhibitor 2A (CDKN2A) is related to the development of pancreatic cancer (Pihlak et al., 2018). Obviously, mutations in tumor suppressor genes are linked to the progression of pancreatic cancer. Undoubtedly, an early diagnosis is beneficial for patients. Therefore, accurate knowledge of the molecular mechanisms involved in the tumorigenesis and proliferation of pancreatic cancer is vital.

Microarray technology and bioinformatic analysis have been extensively applied for screening the expression of genes, miRNAs, lncRNAs, and DNA methylation, helping to identify DEGs and functional pathways relevant to the tumorigenesis and progression of pancreatic cancer. In this work, we aimed to explore the pathogenesis of pancreatic cancer by a computational bioinformatics analysis of gene expression. Three mRNA microarray datasets from the GEO were extracted and analyzed to identify DEGs between PDAC tissues and noncancerous tissues. Subsequently, the functions of these DEGs were evaluated using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. A protein-protein interaction (PPI) network was visualized using Cytoscape, and the Literature Mining Gene Networks module in GenCLiP 2.0 showed that cyclin-dependent kinase 1 (CDK1) has strong interactions with other hub genes. Enrichment analysis of GenCLiP 2.0 suggested that cell division may be involved in the pathogenesis of pancreatic cancer. In the GEPIA database, the mRNA levels of all hub genes were higher in PAAD (pancreatic adenocarcinoma) tumor tissues than in normal tissues. Additionally, survival analysis indicated that abnormal spindle microtubule assembly (ASPM), CDK1, centromere protein F (CENPF), centrosomal protein 55 (CEP55), denticleless E3 ubiquitin protein ligase homolog (DTL), epithelial cell transforming 2 (ECT2), NIMA related kinase 2 (NEK2) and protein regulator of cytokinesis 1 (PRC1) may be associated with the tumorigenesis and development of pancreatic cancer. Overall, 210 DEGs and 13 hub genes that may be candidate biomarkers for pancreatic cancer were identified.

Material and Methods

Results

GO and KEGG pathway analyses of the PPI network

GO and KEGG pathway analyses were conducted to explore the potential functions and pathways of the upregulated DEGs using DAVID. According to GO analysis, the upregulated DEGs were enriched in cell migration, cell–cell adhesion and cell adhesion biological process (BP) categories (Table 1). The upregulated DEGs were primarily enriched in the extracellular exosome and cytoplasm cell component (CC) categories (Table 1); DEGs were mainly enriched in the cadherin binding involved in cell–cell adhesion and protein homodimerization activity molecular function (MF) categories (Table 1). Additionally, KEGG pathway analysis indicated that the upregulated DEGs were primarily enriched in the ECM-receptor interaction and pathways in cancer (Table 2).

Table 1:

GO analysis of upregulated DEGs in pancreatic ductal adenocarcinoma.

GO ID	Description	Gene count	P-value
GO-BP Terms
GO:0016477	Cell migration	13	1.83E−07
GO:0098609	Cell–cell adhesion	13	2.08E−05
GO:0007160	Cell–matrix adhesion	8	3.55E−05
GO:0022617	Extracellular matrix disassembly	7	1.20E−04
GO:0048333	Mesodermal cell differentiation	4	1.58E−04
GO:0031581	Hemidesmosome assembly	4	2.10E−04
GO:0090004	Positive regulation of establishment of protein localization to plasma membrane	5	2.26E−04
GO:0007155	Cell adhesion	15	2.45E−04
GO:0007229	Integrin-mediated signaling pathway	7	5.08E−04
GO-CC Terms
GO:0070062	Extracellular exosome	63	1.50E−10
GO:0005925	Focal adhesion	16	7.65E−06
GO:0005913	Cell–cell adherens junction	14	1.91E−05
GO:0005615	Extracellular space	28	3.05E−04
GO:0005886	Plasma membrane	61	5.23E−04
GO:0005737	Cytoplasm	72	9.52E−04
GO:0009986	Cell surface	15	9.53E−04
GO-MF Terms
GO:0098641	Cadherin binding involved in cell–cell adhesion	13	3.05E−05
GO:0042803	Protein homodimerization activity	18	8.70E−04
GO:0005509	Calcium ion binding	16	0.004808
GO:0005515	Protein binding	102	0.013495

DOI: 10.7717/peerj.9301/table-1

Notes:

GO

gene ontology

BP

biological process

CC

cellular component

MF

molecular function

Survival analyses of hub genes

Kaplan–Meier curve analysis was used to analyze correlations between overall survival and the hub genes. PDAC patients with alterations in anillin actin-binding protein (ANLN), ASPM, CDK1, CENPF, CEP55, DTL, ETC2, NEK2, TOP2A and PRC1 exhibited poor overall survival (Fig. 5). PDAC patients with ASPM, CDK1, CENPF, CEP55, DTL, ETC2, NEK2, PRC1, ribonucleotide reductase regulatory subunit M2 (RRM2), TOP2A, ZW10 interacting kinetochore protein (ZWINT), and ANLN alterations exhibited poor disease-free survival (Fig. 6). Among these genes, we selected CDK1 and CEP55 for further study with regard to overall survival and disease-free survival times.

Table 3:

Description of 13 hub genes of pancreatic ductal adenocarcinoma.

No.	Gene symbol	Full name	Function
1	TOP2A	DNA topoisomerase II alpha	Controlling and altering the topologic states of DNA during transcription. Involving in chromosome condensation, chromatid separation, and the relief of torsional stress.
2	CDK1	Cyclin dependent kinase 1	Essential for G1/S and G2/M phase transitions of eukaryotic cell cycle and mitotic cyclins.
3	RRM2	Ribonucleotide reductase regulatory subunit M2	Catalyzing the formation of deoxyribonucleotides from ribonucleotides.
4	PRC1	Protein regulator of cytokinesis 1	A substrate of several CDKs and necessary for polarizing parallel microtubules and concentrating the factors responsible for contractile ring assembly.
5	NEK2	NIMA related kinase 2	A serine/threonine-protein kinase that is involved in mitotic regulation.
6	ZWINT	ZW10 interacting kinetochore protein	Clearly involved in kinetochore function.
7	DTL	Denticleless E3 ubiquitin protein ligase homolog	Cellular response to DNA damage stimulus.
8	MELK	Maternal embryonic leucine zipper kinase	G2/M transition of mitotic cell cycle apoptotic process.
9	CENPF	Centromere protein F	Associating with the centromere-kinetochore complex and playing a role in chromosome segregation during mitotis.
10	CEP55	Centrosomal protein 55	Cranial skeletal system development and establishment of protein localization.
11	ANLN	Anillin actin binding protein	An actin-binding protein that plays a role in cell growth and migration, and in cytokinesis.
12	ASPM	Abnormal spindle microtubule assembly	Essential for normal mitotic spindle function in embryonic neuroblasts and regulating neurogenesis.
13	ECT2	Epithelial cell transforming 2	A guanine nucleotide exchange factor and transforming protein that is related to Rho-specific exchange factors and yeast cell cycle regulators.

DOI: 10.7717/peerj.9301/table-3

Notes:

CDKs

cyclin-dependent kinases

Oncomine analysis of CDK1 and CEP55 in cancer vs. normal tissue

Oncomine analysis of cancer vs. normal tissue indicated that CDK1 and CEP55 were significantly overexpressed in pancreatic cancer in different datasets (Figs. 7A and 7B). In the Grutzmann Pancreas dataset, CDK1 and CEP55 mRNA expression was higher in pancreatic cancer tissues than in normal pancreatic tissues (Figs. 7C and 7D). Additionally, higher mRNA levels of CDK1 and CEP55 were associated with tumor grade (Figs. 7E and 7F).

Discussion

In recent years, microarray technology has been extensively applied to reveal genetic alterations in tumors. Therefore, microarray analysis is a tool for revealing biomarkers for the diagnosis, treatment, and prognosis of pancreatic cancer. In this study, our results demonstrated that the upregulated DEGs were obviously enriched in the BP categories cell migration and cell–cell adhesion. Many studies have shown that migration and invasion are basic characteristics of pancreatic cancer (Zhuo et al., 2018). Moreover, cell–cell adhesion might be involved in cell stretching and movement, which are the molecular bases of the important physiological and pathological processes of tumor invasion and metastasis (Nobes & Hall, 1999; Serrill, Sander & Shih, 2018). For the CC category, the upregulated DEGs were primarily enriched in extracellular exosomes, which are nanoscale membrane vesicles with diameters ranging from 40 to 100 nm. A growing number of studies have shown that tumor-derived exosomes are associated with tumor development, metastasis, and drug resistance mechanisms (Jiao et al., 2018). Furthermore, in the MF category, upregulated DEGs were mainly enriched in cadherin binding involved in cell–cell adhesion and protein homodimerization activity, which are associated with invasion and metastasis. According to our KEGG pathway enrichment results, the upregulated DEGs were primarily enriched in the ECM-receptor interaction. During the development of tumors, the ECM experiences a remodeling process that is similar to the process of embryonic development. The most important feature of this remodeling is the change in the molecular composition of the ECM, whereby the reconstituted ECM creates a loose microenvironment for the proliferation and differentiation of tumor cells, leading to high rates of proliferation, poor differentiation, invasion and metastasis (Jin & Liu, 2018).

In our study, we showed that TOP2A, PRC1, ECT2, RRM2 and CEP55 can interact with CDK1 and TOP2A is the gene most closely related to CDK1. Previous studies have provided evidence about the expression of CDK1 and TOP2A in pancreatic cancer, but the correlation between them in pancreatic cancer has not been fully elucidated (Kalimutho et al., 2018; Li et al., 2017a; Shi et al., 2015; Xu et al., 2016). Cyclin-dependent kinases (CDKs) are important driving factors of the human cell cycle (Zhao et al., 2013). Topoisomerases, including topoisomerase 1 (TOP1) and topoisomerase 2 (TOP2), are key ribozymes that mainly participate in cell growth by breaking and reconnecting DNA strands to change DNA topology (Liu et al., 2018). We predict that the cyclin A2-CDK1-TOP2A axis plays an important role in tumor development. Furthermore, CDK1 might phosphorylate TOP2A to promote S phase transition and influence the progression of pancreatic cancer, but follow-up research is needed to determine the specific mechanism of the interaction (Kalimutho et al., 2018). Our enrichment analysis of 13 hub genes further verified the relationship between metastasis and the malignant progression of pancreatic cancer.

The transcription factors NFYC, TFDP3, POLE3 and E2F4 are closely linked to hub genes in pancreatic cancer. NFYC is a histone-fold domain-containing transcription factor engaged in chromatin remodeling, establishing permissive chromatin modifications at CCAAT motifs in promoters (Bieniossek et al., 2013). Deletion of NFYC halts cell cycle progression, predominantly by causing G2/M arrest, and concurrent gain of NFYC may serve to model an aberrant epigenome that promotes a proliferative and relatively undifferentiated state (Benatti et al., 2011). Most E2Fs are localized in the nucleus, but E2F4 shows cell cycle-specific localization and can be found in the nucleus of cycling cells in G0, early G1 and G2 phases (Lindeman et al., 1997). When present in the nucleus, E2F4 has functions necessary for the induction of mitosis; interestingly, E2F4 also appears to have a role in the cytoplasm during multiciliogenesis (Van Amerongen et al., 2010). TFDP3 is expressed in most cancer tissues and potentially plays a role in cell differentiation and proliferation. TFDP3 is a novel negative regulator of E2F that can enhance both the DNA binding and transcriptional activity of E2F through the formation of heterodimers; it also potentially plays an important role in the process of tumor development independent of pRb (Ma et al., 2014). POLE3 is similar to the first subclass of core histones with respect to regulation, and POLE3 expression is upregulated at the onset of S phase. E2F4 is often associated with promoters in G0, is minimally bound to the POLE3 gene in starved cells and maximally in cells expressing POLE3 at high levels. These findings indicate that these transcription factors may regulate the progression of pancreatic cancer, which provides direction for our future research (Bolognese et al., 2006).

Expression analysis of PAAD tissues and normal tissues based on the TCGA database also indicated higher levels of TOP2A (Jiao et al., 2019), CDK1 (Jing et al., 2019), RRM2 (Zhao et al., 2019), PRC1 (Mao et al., 2019), NEK2 (Deng et al., 2019), ZWINT (Obuse et al., 2004), DTL(Cui et al., 2019), MELK (Meel et al., 2019), CENPF (Chen et al., 2019), CEP55 (Hauptman et al., 2019), ANLN (Wang et al., 2019), ASPM (Hsu et al., 2019), and ECT2 (Daulat et al., 2019) in tumor tissues. Previous studies have provided abundant evidence about the function of the thirteen identified hub genes in cancer development. Mutations in ANLN, ASPM, CDK1, CENPF, CEP55, DTL, ETC2, NEK2, TOP2A and PRC1 affect overall survival and disease-free survival in pancreatic cancer, whereas mutations in RRM2 and ZWINT affect disease-free survival. Hence, we reasoned that ANLN, ASPM, CDK1, CENPF, CEP55, DTL, ETC2, NEK2, TOP2A and PRC1 might be preferable prognostic factors that are positively related to pancreatic cancer. Additionally, Lu, Li & Liao (2019) analyzed the microarray datasets GSE32676, GSE15471, GSE71989 and GSE19650 to identify five upregulated hub genes, including MELK, MET, THBS1, TOP2A and SDC1; Li et al. (2018a) analyzed the microarray datasets GSE71989, GSE15471, GSE16515, GSE32676, GSE41368 and GSE28735 and found that five genes (BIRC5, CKS2, ITGA3, ITGA6 and RALA) were significantly associated with survival time in patients with pancreatic duct adenocarcinoma; Zhu et al. (2017) studied the five GEO datasets (GSE15471, GSE16515, GSE18670, GSE32676, GSE71989) and reported that GJB2 and ERO1LB dysregulation was associated with tumorigenesis in pancreatic adenocarcinoma. Within addition to these hub genes, we supplied some new hub genes that may be associated with the tumorigenesis and development of pancreatic cancer.

Our results showed that among these hub genes, TOP2A is the most closely related gene to CDK1, TOP2A and CDK1, and it has been previously reported as a biomarker for pancreatic cancer (Kokkinakis, Liu & Neuner, 2005); however, to date, little attention has been paid to CEP55 and its possible relationship with CDK1. Indeed, only one study reported that CEP55 can inhibit CDK1 phosphorylation and proteolysis mediated by the anaphase-promoting complex to induce anaphase I in oocytes (Zhou et al., 2019). Nonetheless, the relationship between CEP55 and CDK1 and the mechanism involved have not been studied in pancreatic cancer cells. It is well known that pancreatic cancer has a high degree of malignancy and a short disease course, which is largely attributed to the fact that it readily metastasizes to and invades adjacent organs. CEP55 belongs to the centrosomal family of proteins, which plays an important role in critical cell functions. Mounting evidence shows that CEP55 is highly expressed in multiple cancers, such as colon cancer, hepatocellular carcinoma and bladder cancer (Gao & Wang, 2015; Li et al., 2018b; Singh et al., 2015). Moreover, overexpression of CEP55 accelerates the cell cycle transition in gastric cancer, and low expression of CEP55 inhibits cell growth in breast cancer and gastric cancer (Kalimutho et al., 2018; Tao et al., 2014). These results demonstrate that CEP55 may serve an oncogenic role and a potential target for tumor treatment. CEP55 can also promote aggressive behavior in pancreatic cancer cells by activating the NF-κB pathway (Peng et al., 2017). Hence, the underlying mechanism of the proinvasive and prometastatic effects of CEP55 in pancreatic cancer needs to be further studied. Last, we preliminarily validated the ChIP analyses by detecting CDK1 and CEP55 protein levels in eight snap-frozen tumorous and adjacent noncancerous adjacent tissues of pancreatic cancer patients. The results showed that CDK1 and CEP55 were significantly overexpressed in pancreatic tumorous tissues compared to normal tissues, which is consistent with the results in different datasets. However, our sample size was relatively small, so we could not verify the link between clinical samples and prognosis. Fortunately, Piao et al., (2019) validated that high expression of CDK1 was correlated with the short survival of pancreatic cancer patients by analyzing 99 cases of surgically resected pancreatic cancer samples and 71 cases of normal pancreases. Peng et al. (2017) reported that CEP55 expression was an independent prognostic factor of patient outcome and that CEP55 protein expression levels in pancreatic cancer specimens were inversely correlated with survival time by analyzing 126 archived paraffin-embedded pancreatic cancer specimens with immunohistochemical staining using an antibody against human CEP55. The results were in accordance with the Kaplan–Meier curve analysis online.

Conclusion

In summary, by analyzing multiple datasets from the GEO database and validating the results with the TCGA and Oncomine databases, our present work identifies dominant genes, their interaction network and possible transcription factors involved during the progression and metastasis of pancreatic cancer. Some relationships between hub genes and transcription factors have never been reported to influence the progression of pancreatic cancer and may serve as potential targets for pancreatic cancer therapy. However, due to the low quantity of gene probes in our selected datasets, the number of discovered DEGs was strikingly limited. More genes and noncoding RNAs should be detected to enrich the network for a more comprehensive and integrated understanding of pancreatic cancer development. In addition, we did not analyze the expression of hub genes, such as CDK1, in pancreatic patients with or without lymphatic metastasis or use ROC analysis to explain the prognosis and diagnostic value of the genes. We will collect these data in our future research to complete our analysis of the prognosis and diagnostic value of these genes.

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