DOI: https://doi.org/10.21203/rs.3.rs-985162/v1
Background
Studies have shown that kallikrein-related peptidase 7 (KLK7) is abnormally expressed in a various of tumours and plays a crucial role in tumour progression. The expression level and clinical significance of KLK7 in pancreatic cancer are still unclear. In this research, by detecting the expression of KLK7 in pancreatic cancer tissues and analyzing its relationship between clinicopathological characteristics and prognosis of patients with resectable pancreatic ductal adenocarcinoma (PDAC) as well as explore its clinical value in pancreatic cancer.
Methods
We compared the expression of KLK7 in public databases (TCGA and GTEx) between tumour tissues and normal pancreatic tissues. We used immunohistochemistry to detect the expression of KLK7 in 243 cases of PDAC tissues. For cancer patient data, the Kaplan-Meier survival curve and Cox risk model were using to analyzed the correlation between the KLK7 expression and the prognosis of pancreatic cancer patients.
Results
In the TCGA and GTEx data sets, the expression of KLK7 in normal pancreatic tissue was obviously lower than in pancreatic cancer tissue (P<0.05). In the TCGA database data sets, the survival time of KLK7 high expression group was obviously lower than the low expression group (P<0.05). In the TCGA data set, we found that the expression of KLK7 can be using as an independent prognostic factor in pancreatic cancer patients (HR=1.32, 95% CI=1.118~1.559, P=0.001). Among 243 patients with clinical pancreatic cancer, the positive expression rate of KLK7 in pancreatic tumour tissue was obviously higher than in adjacent pancreatic tissue (P<0.001). The time to live of PDAC patients with high KLK7 expression is obviously lower than patients with common KLK7 expression (P<0.05). KLK7 expression, low tumour differentiation, nodal metastasis and vessel invasion were independently associated with poor overall survival (P<0.05).
Conclusions
The expression of KLK7 in patients with pancreatic ductal adenocarcinoma increased obviously, and it is directly related to the poor prognosis of patients after radical resection of pancreatic cancer. The expression of KLK7 may used as a predictor of long-term prognosis after radical resection of pancreatic cancer.
Pancreatic cancer is a common malignant tumour of the digestive tract which have less than 8-9% of 5-year survival rate [1, 2]. Because pancreatic cancer is prone to metastasis or invade blood vessels early, only about 20% of pancreatic cancer patients can undergo radical resection of pancreatic cancer[3]. Therefore, surgical treatment is currently not the only treatment for pancreatic cancer. To extend life extension of patients with pancreatic cancer, a better gene therapy targets will help a lot. Disappointingly, the results of most clinical trials of targeted therapies are not satisfactory. Therefore, there is an urgent need to explore new targets for PDAC therapy.
Human tissue kallikrein is a secreted serine protease with various physiological and pathological functions [4]. Some researches have shown that KLKs are associated with poor prognosis in cancer patients[5]. As is reported that KLKs are involved in the developing of various of tumours [6, 7]. KLK7 was discovered firstly in the research of the skin keratoderma mechanism[8]. However, KLK7 can degrade extracellular matrix (ECM) and cell adhesion molecules (CAM) in tumour cells, such as fibronectin and E-cadherin. The adhesion between tumour cells reduced, which leads to the metastasis of pancreatic cancer cells [9 –12]. Several studies showed that the over-expression of KLK7 can use as an important prognostic indicator for various of malignant tumours [13–15]. Our previous research found that KLK7 was obviously over-expressed in pancreatic cancer tissues. Knockdown of KLK7 expression can substantially reduce the diffusion, metastasis and infiltrating of pancreatic cancer cells [16]. However, the relationship between the expression of KLK7 and the prognosis of patients with radical resection of pancreatic cancer still needs further research.
In this research, we analyzed the expression of KLK7 mRNA in pancreatic cancer tissues and normal pancreatic tissues through public databases which including The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) project. To verify the validity of public data, we tested the expression of KLK7 in 243 cases of radical resection of pancreatic cancer and we analyzed the relationship between KLK7 expression and clinicopathological characteristics and prognosis.
TCGA, GTEx datasets
The Gene Expression Profiling Interactive Analysis 2 (GEPIA2) database (http://gepia2.cancer-pku.cn) was using to compare the expression of KLK7 mRNA between the pancreatic cancer dataset in TCGA and the typical pancreas dataset in GTEx [17]. KM survival analysis compares the survival differences between the above two groups, and time ROC research compares the risk score and prediction accuracy of the KLK7 gene.
Patients and specimens
This research approved by the Ethics Committee of the First Clinical Medical College of Three Gorges University, all patients involved were providing informed consent for their data adopted for research purposes. Tumour specimens consisted of 243 pancreatic cancer tissues obtained from patients with pancreatic ductal adenocarcinoma who went through radical resection from 2009 to 2016 in the First Clinical Medical College of Three Gorges University, China. The clinical pathology and essential characteristics including age, gender, tumour size, tumour differentiation, tumour stage, perineural infiltration state, vascular invasion, lymph node metastasis ect. There were 163 males and 80 females who were aged from 31 to 80 years old and the median age is 59 years old. All specimens were confirmed to be pancreatic ductal adenocarcinoma by pathology after surgery. Sixty-eight cases were high or intermediate-differentiated, and 175 cases were poor differentiated. In TNM staging[18], 50 cases were I~IIA ranked, and 193 cases were IIB~III ranked. None of the enrolled patients received any treatment for pancreatic cancer before surgery.
Immunohistochemistry
The formalin-fixed paraffin block of the clinical sample was sectioned (4μm), and perform immunohistochemical staining in a two-step method. The quick steps are as follows: After the tissue sections baking at 65℃, they deparaffinized with xylene and alcohol gradients. Peroxidase inactivated by 3% hydrogen peroxide at room temperature for 15 minutes. Sodium citrate was used for antigen retrieval and rinsing. After blocking with 3% fetal bovine serum protein for 30 minutes and kept it at 4℃ Incubate overnight, after rinse for three times，incubate at 37℃ for 2 hours in the dark，we add the KLK7 goat anti-human primary antibody (1:800) and rabbit anti-goat secondary antibody (1:1000) as well as colour reagent, stop staining when light yellow appears but hematoxylin will regain dye to set up positive and negative controls.
Microscopy imaging
Observe the sections under a microscope (Leica DMI3000B). Use Adobe Photoshop CS6 to process images-Pro, and use Image Pro Plus v6.0 to measure integrated optical density (IOD). Calculate the average IOD based on the four images of each slice, which is using to indicate the expression level of KLK7 protein in the tissue. In this experiment, two observers have no idea about the clinical data.
Statistical analysis
The log-rank is using to test the KM survival analysis for the purpose of comparing the survival difference between the KLK7 high expression group and the low expression group. Clinical data analyzing through SPSS 23.0 statistical software， data counting is expressed by the cases (percentage) [n(%)] through χ2 test or Fisher exact probability method, the multivariate Cox regression analysis method was adopted to compare the effects of KLK7 on prognosis. P <0.05 means that the difference is statistically significant. The above analysis methods and R software packages applied by v4.0.3 version R software (R Foundation for Statistical Computing, 2021).
KLK7 expression is upregulated in PDAC
We analyze the KLK7 mRNA expression of KLK7 in pancreatic cancer tissues and normal pancreatic tissues through GTEx and TCGA databases. As shown in Figure 1, according to the TCGA and GTEx data sets, we found that the expression of KLK7 in pancreatic cancer tissue was significantly higher than that in normal tissues (P<0.001).
Prognostic analysis of gene signature in the TCGA set
We calculated the risk score of each patient and divided them into high-risk and low-risk groups (Figure 2a). Figure 2b shows the survival status of all patients, and Figure 2c shows the expression heat map of KLK7. According to the KM curve, it showed that the overall survival period of high-risk groups are shorter than low-risk groups (Figure 2d). In addition, time-dependent ROC analysis showed that KLK7 expression had better predictive power in 3 and 5 years of OS (Figure 2e).
KLK7 is over-expressed in pancreatic cancer tissues
The immunohistochemistry was adopted to detect the expression of KLK7 in 243 patients with radically resected pancreatic cancer tissue samples. KLK7 expression in normal pancreatic tissue adjacent to cancer is lower than that in pancreatic cancer tissue (Figure. 3). According to the average optical density (IOD), the expression of KLK7 in pancreatic cancer tissue increased obviously, Still, the word in normal pancreatic tissue adjacent to the cancer is not apparent (P<0.001) (Figure. 4).
Relationship between KLK7 expression and clinicopathological characteristics in patients with resectable pancreatic cancer
We detected the expression of KLK7 in 243 cases of pancreatic cancer tissues and analyzed its relationship with clinicopathological data. The relationship between KLK7 expression and characteristics of pancreatic cancer patients analyzed by Pearson χ 2 test or Fisher's exact test. The results showed in Table 1 indicate that the expression level of KLK7 in PDAC positively related with the clinical stage (P=0.0001), gender of pancreatic cancer (P=0.005) and T stage (0.005). However, there were no significant differences in the distribution of age, tumour size, nerve invasion, vascular invasion, and distant metastasis between the two groups.
Table 1 Correlation between KLK7 expression and clinicopathological variables
|
Variables |
KLK7 expression |
P value |
|
|
Low(n=122) |
High(n=121) |
||
|
Age,n(%) |
|
|
0.138 |
|
＜60 years |
57(46.3%) |
66(53.7%) |
|
|
≥60 years |
65(54.2%) |
55(45.8%) |
|
|
Sex,n(%) |
|
|
0.005* |
|
Male |
72(44.2%) |
91(55.8%) |
|
|
Female |
50(62.5%) |
30(37.5%) |
|
|
Tumor grade,n(%) |
|
|
0.063 |
|
High and intermediate |
40(58.8%) |
28(41.2%) |
|
|
Low |
82(46.9%) |
93(53.1%) |
|
|
Tumor size,n(%) |
|
|
0.472 |
|
＜4.0cm |
23(46.0%) |
27(54.0%) |
|
|
≥4.0cm |
99(51.6%) |
93(48.4%) |
|
|
Stage |
|
|
0.0001* |
|
Ⅰ |
21(80.8%) |
5(19.2%) |
|
|
Ⅱ |
64(54.7%) |
53(45.3%) |
|
|
Ⅲ |
37(37.0%) |
63(63.0%) |
|
|
T |
|
|
0.005* |
|
T1 |
11(84.6%) |
2(15.4%) |
|
|
T2 |
14(60.9%) |
9(39.1%) |
|
|
T3 |
59(54.1%) |
50(45.9%) |
|
|
T4 |
38(46.5%) |
60(53.5%) |
|
|
N |
|
|
0.143 |
|
N0 |
41(58.6%) |
29(41.4%) |
|
|
N1 |
80(46.5%) |
92(53.5%) |
|
|
N2 |
1(100%) |
0(0%) |
|
|
Perineural infiltration,n(%) |
|
|
0.120 |
|
Absence |
46(56.1%) |
36(43.9%) |
|
|
Presence |
76(47.2%) |
85(52.8%) |
|
|
Vessel infiltration,n(%) |
|
|
0.228 |
|
Absence |
49(53.8%) |
42(46.2%) |
|
|
Presence |
73(48.0%) |
79(52.0%) |
|
|
KLK7 kallikrein-related peptidase 7 |
|||
|
* P value＜0.05 |
|||
Relationships between KLK7 expression and overall survival (OS) in PDAC patients
Kaplan-Meier methods were using to analyzing the potential relationship between the KLK7 and overall survival of PDAC patients. The results of the Kaplan-Meier analysis showed that PDAC patients with higher KLK7 expression had poor OS (P<0.001), as shown in Figure.5. The median survival time of KLK7 high expression group and low expression group was 1.4 years and 2.1 years, respectively (Figure 5).
KLK7 expression is an independent predictor in pancreatic cancer patients
The multivariate-adjusted Cox regression model including age, gender, stage, T stage, N stage, tumour differentiation, vascular invasion, and perineural invasion (Figure 6). When comparing the high and low expression of KLK7, the mortality HR was 1.108 (95% CI = 1.026 to 1.195, P = 0.009) which indicating that the high expression of KLK7 correlated independently with the OS after surgical resection in pancreatic cancer patients. Low tumour differentiation,nodal metastasis and vessel invasion were also independently associated with poor overall survival, and the HR for mortality was 4.051 (95% CI =2.181 to 7.526, P = 0.001)、0.413(95% CI = 0.250 to 0.683, P < 0.001) and 1.726 (95% CI =1.119 to 2.662 , P = 0.013).
Kallikrein plays a role in various physiological functions, including maintaining blood pressure stability, tissue remodelling, hormone processing and skin stratum corneum metabolism. In cancer, kallikrein is also involved in the physiological process of tumours, and the high expression of kallikrein may be involved in tumour cell diffusion, metastasis, and infiltrating.[19, 20]. Our previous research found that the expression of KLK7 increased in pancreatic cancer cells. When the expression of KLK7 inhibited by lentiviral transfection, the diffusion, infiltrating and metastasis ability of pancreatic cancer cells was obviously reduced.[16]. This mechanism may be related to the cleavage of ECM protein and attachment/adhesion molecules by KLK7 [11, 12]. The high expression of KLK7 in pancreatic cancer leads to tumour proliferation and increased invasiveness. This biological behaviour may be closely connected with the poor prognosis of pancreatic cancer.
To preliminarily verify this possibility, we comprehensively analyzed the relationship of the expression and prognosis of KLK7 in pancreatic cancer and normal pancreatic tissues in TCGA and GTEx. We further detected the expression of KLK7 in 243 cases of pancreatic cancer and adjacent tissues by immunohistochemistry and analyzed the relationship between the expression and clinicopathological indicators.
The public databases and immunohistochemistry show that KLK7 expressed in pancreatic cancer tissues highly, but low in paracancerous tissues, Patients with high KLK7 expression have lower long-term survival. These results indicate that KLK7 can be used as an independent predictor to judge the long-term prognosis of patients with pancreatic cancer. Traditional prognostic factors such as tumour size and tumour differentiation are a form of results and not integrated into the occurrence and development of pancreatic cancer. However, gene expression can provide more accurate predictions for the prognosis of pancreatic cancer, and it can also used as a new target for cancer therapy.
Many studies about the relationship between gene expression and prognosis, the research subjects did not distinguish the surgical and non-surgical patients, they could not accurately predict the prognosis after radical resection of pancreatic cancer. The innovation of our research is that the research object is patients after radical resection of pancreatic cancer, which is more meaningful for evaluating the long-term prognosis after surgery. In addition, we have comprehensively analyzed the research results of public databases and our clinical research centre, which is more accurate than only clinical data analysis or biometric analysis. However, the limitation of our research is that the data does not include key information such as whether patients have undergone standard chemotherapy or targeted therapy after surgery or whether they have undergone a second operation and the specific cause of death which may affect the accuracy of our results. In addition, the multivariate regression analysis found that the tumour staging and perineural infiltration were not statistically significant which raised the question of validity. The specific mechanism which KLK7 up-regulation promotes the progression and aggressiveness of PDAC remains to be further studied.
In summary, the expression of KLK7 related to the prognosis of resectable pancreatic cancer closely. KLK7 can not only be using as an essential indicator for judging the prognosis of resectable pancreatic cancer but also hopefully become a new target for the treatment of pancreatic cancer.
PDAC: Pancreatic ductal adenocarcinoma;
KLK7: kallikrein-related peptidase 7;
TCGA: The Cancer Genome Atlas;
PAAD: Pancreatic adenocarcinoma;
OS：overall survival
GTEx: Genotype-Tissue Expression;
TNM: Tumor-node-metastasis;
ROC: Receiver operating characteristic;
HR: Hazard ratio;
IHC: Immunohistochemistry
IOD: integrated optical density
Acknowledgements
The present study was mainly supported by The First College of Clinical Medical Science, China Three Gorges University.
Authors’ contributions
All authors contributed to the study conception and design. Kecheng Yao, Jun Hu and Jun Zheng: conceived the ideas and designed the experiments. Dawei Cheng and Yuanjie Li: resources and investigation and data curation. Xiaosong Li，Jun Hu and Kecheng Yao: Immunohistochemistry test and data analysis. Kecheng Yao, Jun Hu and Jun Zheng: review and editing. Rucheng Yao and Pei Zhou: formal analysis and software and writing—original draft. All authors read and approved the final manuscript.
Funding
This work was supported by grants from Health Commission of Hubei Province scientific research project (WJ2021M067)、(WJ2021M065), Medical and Health Research Project of Yichang (A21-2-024).
Availability of data and materials
Not applicable.
Competing interest
The authors have no competing interest to declare.
Ethics approval and consent to participate
This study was approved by the Ethics Board of The First College of Clinical Medical Science, China Three Gorges University, and all patients involved in this study provided informed consent for the use of their personal data for research purposes. Informed consent was obtained from all individual participants included in the study.
Consent for publication
Written informed consent for publication was obtained from all participants.
Author details
1 Department of Hepatobiliary and Pancreatic Surgery, The First College of Clinical Medical Science, China Three Gorges University, Yichang, Hubei 443000, China
2 Institute of Hepatopancreatobilary Surgery, China Three Gorges University, Yichang, Hubei 443000, China
3 Department of Hepatobiliary and Pancreatic Surgery, Zhongnan Hospital of Wuhan University, Wuhan 430072, China.
4 Department of Geriatrics, The People's Hospital of China Three Gorges University/the First People's Hospital of Yichang, Yichang, , Hubei 443000, China.