3.3. Analysis of Significant Candidate Genes
Using our method, we predicted thirteen genes that may participate in PC. Based on the principle underlying our method, the candidate genes are specifically connected with PC-related genes. Moreover, such genes have all been reported as genes that are relevant to PC and may exhibit diverse functions in tumor initiation and invasion, especially in PC.
Among the candidate genes, three are inhibitory genes that may participate in PC through their respective mechanisms. The first candidate gene is NFKBIA (see row 1 of Table 1, with betweenness 73 and permutation FDR 0.002), which is expressed in pancreatic tissues [31]. NFKBIA encodes a member of the NF-kappa-B inhibitor family, which has been confirmed to further participate in interactions with REL dimers to inhibit the NF-kappa-B pathway in processes of inflammation immune response and tumorigenesis [32, 33]. Targeted by a specific microRNA, miR196a, NFKBIA has been proved to be associated with PC, especially in the metastasis process [31]. As we have mentioned above, in most tumors including pancreatic cancer, NF-kappa-B pathway has been widely reported to be overactivated and has a close connection with the patients’ prognosis, indicating the underlying relationship between NFKBIA and the pancreatic cancer [34–37]. Mutations have been widely reported to contribute to specific functional alteration of crucial proteins including NFKBIA in diseases especially in various cancer subtypes like pancreatic cancer [38–41]. What is more, as one of the crucial inhibitory components of NF-kappa-B pathway, NFKBIA has been reported to be downregulated in cancer [42, 43]. The overexpression of NFKBIA has also been reported to be associated with a better prognosis of various treatment methods in different human tumor subtypes, especially for the prognosis of patients that have taken alpha 1-adrenoceptor antagonists and radiotherapy [44, 45]. Further, considering the inflammation associated function of such gene, such mutations and expression alteration may contribute to the process of tumorigenesis through two individual regulation mechanisms: proliferation associated pathways that involve NF-kappa-B and specific immune response associated pathways in tumor microenvironment [46]. In addition to the NF-kappa-B pathway, the JNK pathway is also a specific pathway in tumor initiation and progression, including PC [47, 48]. A specific regulator of Rho protein exchange reactions which is crucial for JNK pathway, ARHGDIA (see row 2 of Table 1, with betweenness 94 and permutation FDR 0.012), was also identified using our method and functions in several types of tumors [49, 50]. In addition, this apoptosis inhibitory protein has been confirmed to control Rho protein homeostasis and participate in the initiation and progression of PC through the apoptosis associated GDP/GTP exchange reaction via RhoA-Rho pathway [51]. Apoptosis inhibition widely exists in tumor tissues and is an effective way to induce malignant cells, implying the potential relationship between ARHGDIA and pancreatic cancer [52, 53]. As we all know, pathways that contribute to the proliferation and invasion of cancer cells (JNK-STAT, RhoA-Rho signaling pathway, etc.) have all been confirmed to involve phosphorylation and dephosphorylation process which may be regulated by the survival associated GDP/GTP exchange reaction, further validating the potential relationship between ARHGDIA and pancreatic cancer [54–58]. What is more, regarded as a therapeutic target of pancreatic cancer, ARHGDIA (also known as RhoGDI) has been actually proved to be associated with the proliferation and invasion process of the pancreatic tumorigenesis which may further affect the prognosis of various patients [51]. The expression quantity of such gene, ARHGDIA, as the specific negative regulator of Rho protein exchange reactions, has been widely confirmed to be downregulated in tumors, which further induce the activation of survival associated Rho GTPases and promote the progression of various tumors [55–57]. As for specific mutations that may affect the biological function of such gene, coincidentally, specific triple Y156F/S101A/S174A-RhoGDI mutation has been confirmed to be related to the progression of pancreatic cancer [58]. Furthermore, we also obtained the gene XIAP as one of the candidate genes in our list (see row 3 of Table 1, with betweenness 64 and permutation FDR 0.006) that encodes a functional apoptosis suppressor protein. As the most potent apoptosis suppressor, XIAP interacts with several caspases directly and prevents the apoptosis process [59–61]. In PC, XIAP has been proved to be critical for the progression and prognosis of this disease via several classical pathways (Erk, PTEN/PI3K/AKT, etc.) [62–64]. What is more, the overexpression of XIAP has been confirmed to be associated with poor prognosis of pancreatic cancer. Therefore, such protein has already been applied as a useful laboratory test parameter and a functional therapeutic target of pancreatic cancer [64, 65]. As an immune associated functional gene, the polymorphism of XIAP may contribute to the complex and personalized immune response to pancreatic cancers. Some of the functional variants have already been proved to be directly associated with the prognosis of various subtypes of pancreatic cancer, indicating the potential significant role of XIAP in pancreatic cancer [62, 66].
In addition to inhibitory genes, a group of scaffold genes was also among the thirteen candidate genes. MAPK8IP3 (see row 4 of Table 1, with betweenness 64 and permutation FDR <0.001), which is also known as JIP3, is a scaffold protein involved in the JNK pathway [67]. Associated with the MAP kinase cascade, it may be related to PC via potential downstream mechanisms [67–69]. With high tissue specificity, such gene has been reported to actually induce the excessive proliferation of pancreatic cells, which may further initiate the biological process of pancreatic cancer [70]. MAPK8IP3 is also one of the crucial components of JNK pathway which has been overactivated in tumors including pancreatic tumor [71, 72]. During the initiation and invasion stages of pancreatic cancer, MAPK8IP3 has been reported to be overexpressed which may further contribute to the excessive proliferation of pancreatic cells in clinical cases [70]. Mutations have also been identified in MAPK8IP3 which has been reported to partially interfere with its own functions under pathological conditions [73, 74]. Some of the specific mutations of MAPK8IP3 have been reported to influence the adhesion and invasion process of various cancer cells including the pancreatic cancer cells, validating the crucial role of MAPK8IP3 in pancreatic cancer [75, 76]. As another functional scaffold protein that participates in the interaction between the cell membrane and cytoskeleton, our candidate gene SLC9A3R1 (see row 5 of Table 1, with betweenness 128 and permutation FDR 0.031) regulates the subcellular location and function of SLC9A3 and plays a specific role in the Wnt pathway [77–79]. Known as NHERF1, SLC9A3R1 acts as a scaffold protein in many types of tumors depending on its specific role in Wnt pathway, especially in PC [78, 80, 81]. During the course of pancreatic cancer raising, SLC9A3R1 has been confirmed to be overexpressed and involve the poor prognosis of such disease [81, 82]. Just like other candidates encoding scaffold proteins, SLC9A3R1 has also been proved to contain specific nonsynonymous mutations in various cancer subtypes, especially in pancreatic cancer [83, 84]. Coding an adaptor protein, the candidate gene PARVB (see row 6 of Table 1, with betweenness 64 and permutation FDR <0.001) is critical to certain specific biological processes in cancer, such as ERK signaling pathway and focal adhesion which both have been identified in pancreatic cancer [85, 86]. Therefore, this gene is critical for tumorigenesis and may have a specific role in PC [87]. As a crucial adaptor protein, the overexpression of PARVB has been proved to be associated with high cell proliferation rate which indicates worsening clinical prognosis of tumors, including pancreatic cancer [86, 88]. The polymerase of such gene, PARVB, has also been identified and confirmed to be associated with a group of severe diseases including cancers [87]. Such abnormal expression level and high polymerase in tumor cells suggest that PARVB may play a crucial role in the specific pathogenic process of pancreatic cancer.
Six remaining genes may also be associated with PC in their respective way. RARA (see row 7 of Table 1, with betweenness 24 and permutation FDR 0.005) encodes a specific receptor for retinoic acid, which may further regulate germ cell development during spermatogenesis and the expression of specific genes through recruiting the chromatin complex (KMT2E, MLL5, etc.) [89–91]. As we all know, the stem-cell-like features of tumor cells may be associated with the potential invasion and proliferation ability of such tumor cells [92–94]. In pancreatic cancer, it has been confirmed that the more “stem-like” the cancer cell is, the more malignant the cancer is [95]. Similar to its specific function in germ cells, RARA is related to the regulation of stem- cell-like features in PC, which implies that it may further affect the prognosis of such a severe malignancy [96]. The overexpression of RARA has been reported to be related to specific biological behaviors of tumors such as EMT (epithelial-to-mesenchymal) and invasion, which indicates poor prognosis of pancreatic cancers in clinical cases [97]. Although few mutations have been identified in RARA, crucial fusion gene variants have been widely identified in cancers which may be analyzed later. As we have mentioned above, SLC9A3R1 has been reported to regulate cellular morphology as a scaffold protein [78]. Similarly, the candidate gene ILK1 (see row 8 of Table 1, with betweenness 64 and permutation FDR 0.005) also mediates cell architecture, signaling transduction and cell adhesion via integrin-mediated signaling transduction, especially in tumor tissues [98, 99]. Although studies have not explained clearly how ILK1 actually contributes to the process of pancreatic cancer, it has been shown to participate in several types of tumors and may be associated with several unique molecules (GSK, AKT, PTEN, etc.) that all have been confirmed to be critical to PC [98–103]. Recently, such gene has been reported to be associated with the expression of TGF-α, which may partially explain the underlying carcinogenic mechanism of such gene [104]. What is more, such gene has been confirmed to be overexpressed during the whole biological process of tumorigenesis including the proliferation, migration, and invasion of pancreatic cancers [104–106]. Further, specific mutations have also been identified in such gene. Integrin-linked kinase 1 (ILK1) and HSP90 (HSP90AA1, which is also in our list and will be analyzed later) have both been confirmed to contain quite a lot of tumor-associated variants. Such mutations of ILK1 have been reported to be functional pancreatic cancer biomarkers [107–109]. All in all, ILK1 has been proved to regulate the tumorigenesis process of PC through a specific TGF-α-associated mechanism. As a functional SLC9A3R1-associated protein, a specific protein ezrin is encoded by one of our candidate genes EZR (see row 9 of Table 1, with betweenness 128 and permutation FDR 0.026) [110]. Crucial for cytoplasmic peripheral membrane, EZR is associated with the actin cytoskeleton and regulates the surface expression of actin [111, 112]. It has been widely reported that ezrin is critical for the proliferation, metastasis, and invasion process of several tumor subtypes, especially of pancreatic cancer [113, 114]. The overexpression of EZR has been widely reported to be associated with the initiation, proliferation, and metastasis processes of tumor, which strongly affect the patients’ prognosis especially in pancreatic cancer [115–117]. EZR, as the crucial scaffold protein which may interact with SLC9A3R1 that we have mentioned above, has also been reported to contain specific mutations that may contribute to the tumorigenesis process of various cancer subtypes including pancreatic cancer [118]. All in all, EZR may be a potential pancreatic-cancer-associated gene and may contribute to similar pathways with SL9A3R1 as we have mentioned above [110].
Further, we also predicted one of the most famous oncogenes HRAS (see row 10 of Table 1, with betweenness 113 and permutation FDR 0.033), which binds GDP/GTP and exhibits intrinsic GTPase activity [119]. Associated with several tumors and similar to its homologues (r-RAS, k-Ras, etc.), HRAS is a powerful oncogene that can initiate tumors through inducing excessive growth factor activation in cells and promoting malignant proliferation of tumor cells, especially in tumors in situ [120–122]. In PC, such gene has also been regarded as a main oncogene and is widely reported to be overexpressed during the entire clinical course of pancreatic cancer, validating our prediction of the potential pancreatic cancer-associated genes [123]. Several genetic alterations in HRAS (mutations, CNVs, etc.) have been identified and reported to be associated with such a severe type of cancer [124–126]. Recently, some of the crucial mutations have already been confirmed to directly contribute to the tumorigenesis of pancreatic cancer, indicating the underlying relationship between mutations of candidate genes and the process of tumorigenesis [127]. Heat shock proteins compose a unique group of proteins produced by cells to resist harmful and stressful conditions, including the tumor microenvironment, which contains less oxygen and a lower pH [128–130]. This cluster of proteins has been confirmed to be functional in tumor microenvironment and promote the progression of various tumor subtypes [131, 132]. One of the candidate genes, HSP90AA1 (see row 11 of Table 1, with betweenness 230 and permutation FDR 0.007), also encodes a specific heat shock protein, heat shock protein 90 kDa alpha, which has been reported to be expressed in the cytoplasm. Several types of heat shock proteins (such as HSP20, HSP70, and HSP90), including our predicted protein, are not only related to tumor genesis but specifically contribute to PC [133–135]. Heat shock proteins have also been confirmed to be overexpressed in pancreatic cancer, including HSP90 which is encoded by our predicted gene [136–140]. The overexpression of heat shock proteins has been confirmed to actually contribute to the tumorigenesis process of pancreatic cancer which validates our predication of HSP90AA1 as a functional candidate gene [133]. As a hormone-associated gene, our candidate gene INS-IGF2 (see row 12 of Table 1, with betweenness 64 and permutation FDR 0.031) is a readthrough gene of INS and IGF2. This gene may act as a posttranscriptional regulatory factor for the two genes INS and IGF2, which has been shown to be associated with several pancreatic-associated diseases, such as diabetes [141–143]. As a specific fusion gene, INS-IGF2 participates in several cancer types, especially in prostate cancer [144–146]. The fusion of a tumor-associated gene and a pancreas-related gene, INS-IGF2, probably participates in PC progression and invasion specifically. Recently, such fusion gene INS-IGF2 is reported to be specifically expressed in pancreatic islets especially in patients with autoimmune diseases under pathological conditions [147, 148]. Since immune response is quite significant for tumor surveillance, INS-IGF2 is definitely associated with the unique tumor-associated immune response and may further contribute to the prognosis of pancreatic cancer [148]. Although genes like RARA (mostly identified as FIP1L1/PML-RARA fusion gene) (we have mentioned above) and INS-IGF2 have not been reported to contain specific mutations that may be associated with pancreatic cancer, such fusion genes have both been confirmed to have abnormal gene functions which may directly contribute to the tumorigenesis of various cancer subtypes, including pancreatic cancer [149, 150]. The last candidate gene PRKCA (see row 13 of Table 1, with betweenness 175 and permutation FDR <0.001) encodes a specific kinase that has been reported in several types of tumors [151, 152]. As a widely reported oncogene, PRKCA contributes to the phosphorylation process of crucial tumor-associated proteins [153]. The overexpression of PRKCA has been proved to enhance the transformed proliferation and invasion process of pancreatic cancer which further definitely influences the prognosis of such disease [154, 155]. While at the same time, genes like PRKCA which encodes functional kinase may be strongly affected by genetic alterations especially by some specific nonsynonymous mutations [75, 156]. Therefore, the polymerase of gene PRKCA may definitely affect the function of protein kinase C which has also been identified in pancreatic cancer [156, 157]. However, recent studies show that, at least in vitro, PRKCA has a specific function in tumor suppression [156]. The complex role of PKC in cancer may reflect the complicated interactive relationship between PRKCA and PC [156, 158]. All in all, our predicted gene PRKCA has been confirmed to be definitely associated with pancreatic cancer, while, at the same time, the various functions of PRKCA imply the complex role of such gene in pancreatic cancer.
Consequently, all candidate genes obtained using our method play a critical role in tumor initiation and progression. Because the detailed regulation mechanisms of only few genes remain unclear, those candidate genes may be genes that are critical to PC. PC features genetic heterogeneity, and many familial hereditary related genes remain ambiguous [16]. Therefore, in addition to the confirmed genes, such as KRAS and p16/INK41 [8, 9], more genes must be screened and validated to clearly demonstrate the mechanisms of pancreatic adenocarcinoma. In conclusion, based on reported PC-related genes, the proposed method was effective at predicting candidate tumor-associated genes in PC.