Transcription Levels of KPNAs in Patients with PAAD.
The transcriptional levels of seven KPNA family members were compared between cancers and those in normal tissue specimen by using ONCOMINE databases (Significant correlations are shown in Figure 1,Table 1).
The analysis revealed that the mRNA expression of KPNA1, KPNA2, KPNA3, KPNA4, and KPNA6 was upregulated in patients with PAAD, KPNA5 was downregulated. No data is available for KPNA7. In Ishikawa’s dataset [21], KPNA1 was upregulated in PAAD compared with that in the normal samples, with a fold change of 1.957 and p-value of 0.029 (Table 1). The transcription levels of KPNA2 were significant elevation in patients with PAAD in three datasets (Table 1). In Segara’s dataset, KPNA2 was upregulated in PAAD with a fold change of 4.194 and a p-value of 7.03E-6 [22]. In Pei’s and Badea’s dataset, KPNA2 was upregulated in PAAD compared with that in the normal samples, with a fold change of 2.485 (p–value=7.30E-5) and 2.644 (p–value=2.16E-8), respectively [23, 24]. The transcription levels of KPNA3 was significantly higher in patients with PAAD in Segara’s datasets[22]. The fold change of mRNA expression of KPNA3 was 2.194 and a p-value of 1.20E-4 (Table 1). The mRNA levels of KPNA4 (fold change =1.504 and p–value= 0.034) and KPNA5 (fold change = 1.536 and p–value= 5.54E-4) in patients with PAAD were significantly higher than those in the normal specimen in Ishikawa’s and Buchholz’s datasets, respectively (Table 1)[21, 25]. The transcriptional levels of KPNA6 in PAAD (fold change = 1.883 and p–value=0.017) were significantly different from those in the normal (Table 1) [21].
Table 1. The significant dysregulation of KPNA genes in transcriptional level between pancreatic cancer and normal tissues (Oncomine database)
|
Gene
|
Fold change
|
p-value
|
t-test
|
Source and /Reference
|
KPNA1
|
1.957
|
0.029
|
1.944
|
Ishikawa Pancreas[21]
|
KPNA2
|
4.194
|
7.03E-6
|
6.322
|
Segara Pancreas[22]
|
2.485
|
7.30E-5
|
4.74
|
Pei Pancreas[23]
|
2.644
|
2.16E-8
|
6.451
|
Badea Pancreas[24]
|
KPNA3
|
2.194
|
1.20E-4
|
4.957
|
Segara Pancreas[22]
|
KPNA4
|
1.504
|
0.034
|
1.866
|
Ishikawa Pancreas[21]
|
KPNA5*
|
1.536
|
5.54E-4
|
4.602
|
Buchholz Pancreas[25]
|
KPNA6
|
1.883
|
0.017
|
2.188
|
Ishikawa Pancreas[21]
|
KPNA7
|
NA
|
NA
|
NA
|
NA
|
NA, not available; * comparison between pancreatic cancer and pancreatic cancer precursor
|
In the GEPIA2 database, box plots revealed the expressional comparison between PAAD tissues and matched normal pancreatic tissues of each KPNA family member. Total 179 tumor cases and 171 normal samples were included. The results demonstrated that KPNA1-4 and KPNA6-7 were significantly upregulated in patients with PAAD (Figure 2a-2d, 2f-g). The transcription levels of KPNA5 were higher in patients with PAAD than normal control, but no significant differentiation (Figure 2e).
Translation Levels of KPNAs in Patients with PAAD
To verify the translational profiles, the immunohistochemistry (IHC) of KPNAs protein expression was compared between PAAD tissues and their counterparts in the Human Protein Atlas platform (HPA) (Figure 3). Representative images of immunohistochemically stained tissues indicated that KPNA1 and KPNA6 proteins were virtually no differential expression in normal pancreas and tumor tissues (Figure 3a, 3f), while KPNA3 was present at high levels in normal tissues (Figure 3c). In contrast, the proteins of KPNA2, KPNA4, KPNA5, KPNA7 were present in both normal and PAAD tissues, however, the expression levels were higher in the tumor sections (Figure 3b,3d, 3e,3g). Taken together, these results indicate that KPNA2, KPNA4, KPNA5, and KPNA7 are both transcriptionally and translationally upregulated in PAAD. KPNA1, KPNA3, KPNA6 are only transcriptionally upregulated in PAAD.
KPNAs Frequently Altered Genes and their association with survival in Patients with PAAD
A total of 370 patients’ data from two sources (TCGA Firehose Legacy and TCGA Pancancer Atlas) was included. Genes of KPNA family members are altered in 34 (9%) of query patients (Figure 4). The genetic alterations, such as missense mutations, amplification, and deep deletions were found in almost each KPNA members (Figure 4a). However, two or more alterations were detected only in KPNA4 and KPNA5, and the alternation samples were less than 3% (Figure 4a). Moreover, the Kaplan–Meier estimate displayed that the overall survival (OS), disease-free (DFS), progression-free (PFS), and disease-specific (DSS) survival were not statistically different in pancreatic adenocarcinoma patients with or without alterations of KPNAs (Figure 4b, 4c, 4d, 4e).
The mRNA expression of KPNAs and the Clinicopathological Parameters of Patients with PAAD.
The correlation between mRNA expression of each KPNA family member with tumor stage was demonstrated with violin plots in the database GEPIA2. The results displayed the distribution of KPNA4, KPNA5, and KPNA7 mRNA expression correlated with tumor stage significantly, whereas KPNA1, KPNA2, KPNA3, and KPNA6 groups were not (Figure 5 a-h).
Then, the correlation between transcription of KPNAs and tumor grade of patients with PAAD was analyzed in the UALCAN database. Based on individual PAAD cancer grade, the expression panels for KPNAs are compared between 4 normal individuals and 176 PAAD patients. The mRNA expression of KPNA1, 4, 7 were found to be significantly correlated with the different grades, while other members in the KPNA family did not significantly differ (Figure 6 a-g). Taking all of our results together, the mRNA expression levels of KPNA4 and KPNA7 have indicated an association with both cancer stage and pathological grade in PAAD patients.
Prognostic values of KPNA mRNA expression in patients with PAAD
We further explored the critical efficiency of KPNAs in the survival of patients with pancreatic adenocarcinoma. The survival curves were plotted with data from patients with PAAD included in Kaplan–Meier plotter website. The results revealed all of the significant OS and RFS curves associated with the KPNA family members (Figure 7 a-g OS; i-o RFS). The upregulated mRNA levels of KPNA4 and KPNA5 conferred a worse overall survival of patients with PAAD when the threshold of the log-rank p values was set at 0.01 (Figure 7d and 7e). High levels of KPNA3 and KPNA4 resulted in a worse relapse-free survival of patients with PAAD when the threshold of log-rank p values was set at 0.05 (Figure 7k and 7l).
Collectively, KPNA4 demonstrated the most obviously prognostic value for the patients than the other KPNAs members (Figure 7h). Then receiver operating characteristic curve (ROC) of KPNA4 is analyzed by R, the area under the curve (AUC) 0.978 implies that high expression of KPNA4 may be a diagnostic biomarker for patients with PAAD (Figure 7p).
The clinical parameters of KPNA4 in patients with PAAD were also analyzed statistically (Table 2). The differential mRNA expression of KPNA4 is significantly associated with tumor T stage and histologic grade. All these results suggest that KPNA4 might be used to predict prognosis in PAAD.
Table 2. The relationship between the mRNA level of KPNA4 and clinical characteristics for patients with PAAD based on TCGA-PPAD RNAseq-FPKM
|
Characteristic
|
Low expression of KPNA4
|
High expression of KPNA4
|
p
|
statistic
|
method
|
n
|
89
|
89
|
|
|
|
Gender, n (%)
|
|
0.88
|
0.02
|
Chisq.test
|
Female
|
39 (21.9%)
|
41 (23%)
|
|
|
|
Male
|
50 (28.1%)
|
48 (27%)
|
|
|
|
Age, n (%)
|
|
|
0.764
|
0.09
|
Chisq.test
|
<=65
|
45 (25.3%)
|
48 (27%)
|
|
|
|
>65
|
44 (24.7%)
|
41 (23%)
|
|
|
|
Age, mean ± SD
|
65.43 ± 10.53
|
64.07 ± 11.07
|
0.403
|
0.84
|
T test
|
T stage, n (%)
|
|
0.041
|
|
Fisher.test
|
T1
|
5 (2.8%)
|
2 (1.1%)
|
|
|
|
T2
|
17 (9.7%)
|
7 (4%)
|
|
|
|
T3
|
63 (35.8%)
|
79 (44.9%)
|
|
|
|
T4
|
2 (1.1%)
|
1 (0.6%)
|
|
|
|
N stage, n (%)
|
|
0.222
|
1.49
|
Chisq.test
|
N0
|
29 (16.8%)
|
21 (12.1%)
|
|
|
|
N1
|
57 (32.9%)
|
66 (38.2%)
|
|
|
|
M stage, n (%)
|
|
0.644
|
|
Fisher.test
|
M0
|
32 (38.1%)
|
47 (56%)
|
|
|
|
M1
|
1 (1.2%)
|
4 (4.8%)
|
|
|
|
Pathologic stage, n (%)
|
|
0.069
|
|
Fisher.test
|
Stage I
|
15 (8.6%)
|
6 (3.4%)
|
|
|
|
Stage II
|
68 (38.9%)
|
78 (44.6%)
|
|
|
|
Stage III
|
2 (1.1%)
|
1 (0.6%)
|
|
|
|
Stage IV
|
1 (0.6%)
|
4 (2.3%)
|
|
|
|
Histologic grade, n (%)
|
|
0.048
|
|
Fisher.test
|
G1
|
21 (11.9%)
|
10 (5.7%)
|
|
|
|
G2
|
45 (25.6%)
|
50 (28.4%)
|
|
|
|
G3
|
20 (11.4%)
|
28 (15.9%)
|
|
|
|
G4
|
2 (1.1%)
|
0 (0%)
|
|
|
|
Genes Co-expressed with KPNA4 in patients with PAAD
To find the biological significance of KPNA4 in PAAD, KPNA4 co-expression mode in the PAAD cohort was predicted by the function module of LinkedOmics (Figure 8). Total 19696 genes were revealed a significant correlation with KPNA4. 8941 genes (dark red dots) were displayed significant positive correlations with KPNA4, whereas 10754 genes (dark green dots) were found significant negative correlations (false discovery rate, FDR < 0.01) (Figure 8a). The top 50 significant genes positively and negatively correlated with KPNA4 were shown in the heat map (Figure 8b). The biological process (BP), cellular components (CPs), and molecular functions (MFs) were predicted with Gene Ontology (GO) analysis. The results showed that these correlated genes participate primarily in biological regulation and metabolic process (BP), membrane and nucleus (CPs), protein binding, and ion binding (MFs) (Figure 8c).
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis with GSEA demonstrated enrichment of these genes is correlated primarily with adherens junction, and focal adhesion. (Figure 8d) (Figure 9).
Adherens junction is one part of cell adhesion junction, the other two parts are desmosomes and tight junction. Each part contains classes of proteins composed of groups of genes or gene families. Three primary molecular families: cadherins, armadillo proteins, and plakins constitute the adherens junction [26]. To a certain extent, the metastasis and epithelial-mesenchymal transition (EMT) of cancer cells are associated with loss of cell adhesion and increase of cellular mobility [26, 27]. Therefore, we explored whether KPNA4 may impact the metastasis of PAAD or not. 19696 genes correlated with KPNA4 and 64 genes involved in cell adhesion junction and EMT were compared and evaluated (Table 3). The genes associated with EMT mainly include SNAIL, SLUG, TWIST, Zeb1-3, and TGFB1. 49 overlap genes were found in these two gene sets (Figure 10). The Venn diagram was created by website tools (http://bioinformatics.psb.ugent.be/webtools/Venn/).

Correlation Analysis between KPNA4 and genes associated with cell adhesion junction in Patients With PAAD
The overlap 49 genes associated with cell adhesion and EMT were explored in GEPIA2 further (Table 3 and Figure 11). Pearson correlation coefficient more than 0.2 and p-value less than 0.01 were set as the threshold, respectively. 43 genes were demonstrated as a linear association with KPNA4. CTNNA1, CTNNB1, TJP1, and MUPP1 were showed an extremely strong correlation with KPNA4 (Table 3 and Figure 11a). CTNNA1and CTNNB1 belong to the adherens junction cluster. The plot is based on TCGA PAAD tumor, TCGA PAAD normal and GTX pancreas dataset.
Further network investigation was conducted between KPNA4 and those linear correlated genes in Metascape. The process and pathway enrichment analysis, protein interaction network of associated genes with KPNA4 were displayed in figure 11b and 11c. These genes are primarily related to the cell-cell junction organization. The pathway enrichment demonstrated that these genes significantly enriched in pathway neoplastic cell transformation and neoplasm invasiveness (Figure 11d). These results suggested that abnormal expression of KPNA4 may have a major effect on the metastasis of PAAD.