Elevated Circulating Levels of N-Acetyl-Seryl-Aspartyl-Lysyl-Proline ( AcSDKP) Involved in Pancreatic Cancer Development


 Background: Pancreatic cancer is the fourth leading cause of patient mortality with poor early prognosis. Lack of symptoms as early detection of this cancer has become the major problem for its clinical investigation. Thymosin β-4 (Tβ4) has been associated with angiogenesis and metastasis in pancreatic cancer. Recently, N-Acetyl-Seryl-Aspartyl-Lysyl-Proline (AcSDKP), a cleavage and active form of Tβ4, is suggested related to systemic alteration to patients and involve in the Pancreatic Ductal Adenocarcinoma (PDAC) progression. However, the way this systemic change affected PDAC development is still not fully elucidated. This case-control study was conducted to evaluate the level of AcSDKP in health and pancreatic cancer patients during the progression of pancreatic cancer development. Methods: We analyzed 105 patients, including 15 healthy subjects and 90 pancreatic cancer subjects. The levels of AcSDKP were analyzed by Enzyme-Linked Immunosorbent Assay (ELISA) kit. Proliferation rate analysis was conducted using MTS assay in AsPC, HPAC, and PANC-1 cell line pre-treated by prolyl oligopeptidase (POP) inhibitor and AcSDKP polypeptide. Results: Compared to healthy patients (0.73 ± 0.13), the levels of AcSDKP significantly increased in pancreatic cancer subjects (1.34 ± 0.14). AcSDKP induced pancreatic cell proliferation while inversely inhibited with POP inhibitor by in vitro analysis. Moreover, levels of AcSDKP were strongly positively correlated with pancreatic cancer status (r = 0.278, p = 0.026), betatrophin (r = 0.270, p = 0.025), ALP (r = 0.267, p = 0.034), ALT (r = 0.336, p = 0.007), and AST (r = 0.255, p = 0.042), while inversely correlated with cholesterol levels (r = - 0.250, p = 0.046). Furthermore, in univariate and multivariate binary logistic regression analysis, the levels of AcSDKP showed a strong association with pancreatic cancer [(p = 0.031, OR±95%CI = 3.342 (1.117-10.002)] and [(p = 0.024, OR±95%CI = 4.324 (1.030-18.148)]. Quintile analysis showed that the level of AcSDKP has a strong association with betatrophin [(p = 0.033, OR±95%CI = 5.623 (1.142-24.266)]. Conclusions: AcSDKP can contribute to the progression of pancreatic cancer incidence and may indirectly be associated with liver lipid metabolism hormone as well as tumor cell proliferation. AcSDKP might be a potential additional predictor for pancreatic cancer.

been associated with pancreatic cancer. Therefore, surgical resection is only the standard curative therapy in pancreatic cancer due to a lack of prognosis marker in the early stages of this disease development. The future target is to improve the survival rate of pancreatic cancer patients. Therefore, identi cation of individuals at high risk for pancreatic cancer and early diagnosis could have a marked impact on reducing morbidity and mortality.
Furthermore, it is suggested that thymosin beta-4 (Tβ4) regulate the increase of cell proliferation rate through enhancing β-catenin as an activator of c-Myc, and downregulate E-cadherin that results in loss of cell-cell adhesion [12]. Recently, AcSDKP is also associated with human malignant tumor as a potent stimulator of angiogenesis during tumor development, and under regulations of prolyl oligopeptidase (POP), this N-terminus sequence of Tβ4 induce cell survival through Phosphatidylinositol-3-Kinase/Akt (PI3KCA/Akt) pathway [13,14]. Even though some previous studies have investigated the pro les of amino acids in some cancers and the role of thymosin beta-4 with the cleavage of this polypeptide AcSDKP, an advanced investigation on the fundamental function of AcSDKP activity in particular for pancreatic cancer subjects is still required.
This study aimed to investigate circulating AcSDKP in pancreatic cancer patients and to determine whether this small cleavage polypeptide can become a potential predictor for pancreatic cancer development. Moreover, this case-control study also aimed to evaluate the possible interaction between AcSDKP and other serologic parameters for PDAC. Therefore, we investigated the level of AcSDKP among healthy and pancreatic cancer patients to determine whether this polypeptide involved directly in PDAC pathogenesis.

Study population
This study employed an ongoing hospital-based case-control study conducted by a liation between molecular, metabolic medicine laboratory, Department of Biological Science and Technology, National Chiao Tung University Hsinchu-Taiwan, and National Taiwan University Hospital Hsin-Chu Branch, Hsinchu, Taiwan. One hundred and ve Taiwanese subjects were recruited to involve during this case-control study. In brief, 15 patients (healthy) and 90 patients (pancreatic cancer) were enrolled for clinical data collection. In addition, patients with severe conditions who could not ful ll the essential criteria of the samples were excluded during data collection. This study was approved by the institutional review board of the National Chiao Tung University Hsinchu, Taiwan, and written informed consent were obtained from all participants.

Serological Test
Blood and urine samples were acquired after overnight fasting and kept in an icebox immediately after collection. The serum was separated from erythrocytes by centrifuging at 1,500g for 10 min at 4°C. Furthermore, the serum and urine samples were frozen at minus 80°C and prepared for serologic and urine measurement of some parameters. The serum level of some variables was analyzed at the Department of Medical and Chemical Laboratory Diagnostics at the National Taiwan University Hospital Hsin-Chu Branch used routine procedures. The level of AcSDKP was quanti ed using a commercially available EnzymeLinked Immunosorbent Assay (ELISA) kit (SPI BIO, Bertin Group, France; catalog number A05881). In addition, the level of carbohydrate antigen 19-9 (CA 19-9) was measured by using the human ELISA kit (IBL America, catalog no. IB19124, Minneapolis, USA).
For the control group, the cell was treated with ddH2O and 0.1% DMSO, while for the experimental group all of that pancreatic cancer cell line ere induced with 100 µg/mL POP inhibitor, 100 µg/mL POP inhibitor + 10 -7 M AcSDKP, and 100 µg/mL POP inhibitor + 10 -9 M AcSDKP. After incubation for 24 hours, a ten μL MTS solution (MTS Cell Proliferation Colorimetric Assay Kit, Cat.#K300-500, BioVision) was added to each well, and the incubation continued for 4 hours. The visible absorbance of each well was quanti ed at 490 nm using a microplate photometer.

Statistical analysis
All statistical analyses were performed using SPSS version 20.0 (IBM). The data were presented as mean ± SEM for continuous variables. Moreover, to check the normality of data distribution, the Kolmogorov-Smirnov test was used, followed by parametric statistical analysis to compare differences and associations among all experimental groups. Overall, to compare differences between groups, unpaired ttest analyses were used. In addition, to assess the relationships between AcSDKP and other predictors, the correlation between all parameters were determined by Pearson's correlation (normally distributed data). Binary logistic regression was used to test the in uence of clinical data and the levels of AcSDKP on the pancreatic status of patients. A P value less than 0.05 was considered statistically signi cant.

Subject characteristics
The baseline characteristic of healthy and pancreatic cancer subjects are shown in Table 1. Indeed, fasting plasma blood glucose, triglyceride, alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), amylase, and lipase were markedly increased in pancreatic cancer subjects compared to healthy patients. In particular, the levels of AcSDKP signi cantly increased, starting from (0.73 ± 0.13) in healthy subjects to (1.34 ± 0.14) for pancreatic cancer patients ( Figure 1A). A similar pattern of serum cancer antigen 19-9 (CA 19-9) levels was observed in PDAC subjects compared to healthy control ( Figure 1B). However, the level of hemoglobin and cholesterol was decreased steadily in pancreatic cancer subjects.

AcSDKP induced proliferation rate in pancreatic cancer
Further analysis was addressed to clarify the primary involvement of AcSDKP and its inhibitor during tumor growth. Here in our in vitro study, we found that pretreatment of AsPC, HPAC, and PANC-1 cell line using S17092, a speci c prolyl oligopeptidase inhibitor (POPi) for AcSDKP synthesis, signi cantly decreased cell proliferation in all this pancreatic cancer cell line ( Figure 2.A-C). Also, we found that cotreatment with AcSDKP peptide inversely increased cell number through stimulating proliferation rate.

Univariate correlations of AcSDKP with the serologic marker in pancreatic cancer
In this study, the fundamental association of AcSDKP and other serologic parameters are presented in Table 2. Overall, the level of AcSDKP had a positive correlation with pancreatic cancer status (r = 0.278, p = 0.026), betatrophin (r = 0.270, p = 0.025), ALP (r = 0.267, p = 0.034), ALT (r = 0.336, p = 0.007), and AST (r = 0.255, p = 0.042), while negatively correlated with cholesterol. On the other hand, there was no signi cant association between AcSDKP and CA 19-9 serum levels in PDAC subjects. Moreover, there was also no signi cant correlation with other serologic parameters in healthy and pancreatic cancer groups.

Univariate binary logistic regression and quintile analysis in the total samples
To determine whether the levels of AcSDKP are associated with the pancreatic cancer development and able to become independent predictor, we further analyzed the levels of AcSDKP and some serologic markers to predict pancreatic cancer progression in all samples.
AcSDKP was able to become an independent predictor for pancreatic cancer development. Univariate and multivariate binary logistic regression (Table 3a and

Discussion
AcSDKP linked lipid pro le and metabolic disturbances to induce PDAC Metabolic reprogramming is one of the essential hallmarks of cancer through increase oncogenic signaling pathways and directly enhances nutrient acquisition to promote lipids, proteins, and nucleic acid assimilation [15]. The net result of this process affects cancer cell growth and proliferation. Pancreatic ductal adenocarcinoma (PDAC) is a kind of tumor with hypovascularization to induce oxygen and nutrient delivery to the tumor. This tumor enhances PDAC aggressiveness during metabolic reprogramming. We hypothesized that AcSDKP might enhance pancreatic cancer associated with a metabolic imbalance in particular related to lipid and glucose metabolism. Moreover, it is suggested that AcSDKP activity accelerates pancreatic cancer progression through involving in metabolic reprogramming and enhancing neoangiogenesis within the tumor site.
Here in our study, we found that the level of fasting blood glucose tended to increase in pancreatic cancer patients. Furthermore, we also found that the level of triglyceride signi cantly increased in our samples. In line with the previous nding, in cancer development, oncogenic ras stimulates both glucose uptake via glucose transporter-1 (GLUT-1) and utilization of glucose by anabolic pathways. Linear to by Daemen et al. study, in this PDAC clinical samples, the lipogenic subtype is more sensitive to the epithelial cell. In contrast, the glycolytic subtype is associated with mesenchymal cells to induce PDAC progression [16].
The signi cant feature of lipid and glucose pro le of our subjects may stimulate the acceleration of tumor growth, facilitate cancer cell proliferation, metastasis, and aggressiveness [17]. In brief, it clari es that the increase in obesity incidence is associated with the early stage of pancreatic cancer development. We speculate that increased AcSDKP expression induces liver lipid metabolic hormone activity and corroborate pancreatic injury. However, further investigation is required to improve our hypothesis of whether AcSDKP related fatty pancreatic and pancreatic brosis incidence caused by hypertriglyceridemia.
AcSDKP may accelerate PDAC through enhancing neovascularization, cancer cell proliferation, and pancreatic brosis PDAC is a common cause of death from cancer in the world that ranks rst in the mortality rate of all cancer [7,18], characterized by hypovascular and highly desmoplastic cancer [19], and the incidence is increasing in age groups of > 50 years. Due to the late symptoms, resection is associated with improved survival rate, but this is only possible in approximately 10% of patients [20]. According to the cancer therapy perspective, the identi cation of an angiogenic network is crucial to develop an antiangiogenic therapy [21]. Even though some biomarkers or prognostic factors of PDAC has been explored, further investigation to trace a sensitive and speci c tumor predictor that may be more valuable in diagnosis, improving prognosis, and to improve clinical outcome in PDAC patients is still required.
This study attempted to explore the role of AcSDKP in pancreatic cancer development. Here in our study, we proposed an additional novel biomarker for pancreatic cancer, AcSDKP, a cleavage tetrapeptide of Thymosin β4 as an inducer of cancer cell proliferation, migration, angiogenesis, and metastasis in many cancer cases. It has been established that growth and metastasis of pancreatic cancer depend on the activation of VEGF and other angiogenic factors. Signaling complex between cancer cells and adjacent endothelial cells is also suggested to promote aberrant vascularization/tumor angiogenesis [22]. Also, since pancreatic carcinoma shows active tumor neoangiogenesis, overexpression of a critical mediator of angiogenesis in pancreatic cancer (VEGF) becomes the target to develop anti-angiogenic therapies at present. Inhibition of neo-angiogenesis is a reliable and attractive target for tumor therapy. Indeed, the failure of the antiangiogenic agent is due to a single inhibitor that cannot inhibit the interconnection/network of angiogenesis signaling pathways [21]. Cross-inhibition of angiogenesis and apoptosis probably provide an extended bene t of combination cancer therapies consisting of antiangiogenics. Furthermore, establishing speci c biomarker that plays a pivotal role in angiogenesis during PDAC development is essential to increase the patient survival rate.
Importantly, the study of Oh et al., 2010 has shown that TB4 paclitaxel treatment increases ROS production and triggers a signi cant increase in tumor density through hypoxia-inducible factor-1α (HIF-1α) stabilization in HeLa human cervical tumor cells. This linear nding also similar to a fundamental characteristic of PDAC with hypovascularization and resistance to antiangiogenic inhibitor in speci c pathways. Moreover, a study claims that tolfenamic acid is a potential nonsteroidal anti-in ammatory drug (NSAID) to reduce angiogenesis through VEGF signaling pathway by degrading transcription factor Sp1, Sp3, and Sp4; and decrease tumor growth and metastasis [23]. On the other hand, another study suggests that the lower e cacy of VEGF-antiangiogenic drugs is due to the main focus of these treatments related to angiogenesis inhibition without combination to reduce tumor malignancy [24].
Previous studies have shown that Tβ4 is a potent inducer of angiogenesis in cancer development that presents in blood at nanomolar concentration, and relates to angiogenesis in particular for pancreatic cancer [7,14]. Tβ4 also increases pancreatic cancer incidence (through inducing the elevation of proin ammatory cytokine production), increases the number of metastasis tumors, represses the anticancer immunity, controls tumor cell migration in angiogenesis, and controls tumor metastasis through HIF-1α [8,25,26]. Tβ4 is a small tetrapeptide that can be a novel target for the therapeutic approach of cancer, mainly tumor dormancy [25]. Besides, it also indicated accelerated severity and decreased the survival rate in multiple myeloma [10]. The solid tumor shows a switching process by changing the balance between angiogenesis inducer and countervailing inhibitor. Interestingly, the AcSDKP concentration is signi cantly increased within the intratumor blood of patients under the regulation of POP (prolyl oligopeptidase) [14,27]. Indeed, AcSDKP has a unique function related to a particular pathologic condition. The amino-terminal site contains four amino acid that able to block in ammation and reduce brosis Transforming Growth Factor-β (TGFβ) signaling pathways [28][29][30], while another site of this polypeptide (amino terminus with 15 amino acids) promotes cell survival and inhibits apoptosis, angiogenesis, cell migration, and wound healing [30,31].
Importantly, our study found that the level of AcSDKP signi cantly increased in pancreatic cancer patients, suggested that this pathological condition stimulates the increase of POP activity and angiogenesis to support tumor growth. However, the limitation of our study, we still cannot provide a complete molecular signaling network between AcSDKP and other biomarkers involved in pancreatic cancer. It is only hypothesized that the possible mechanism of AcSDKP activity during pancreatic cancer development by triggering the activation of pro-angiogenic molecules, cancer cell proliferation, and induced migration. Moreover, AcSDKP may be associated with other oncometabolomic molecules involved in nutrient supply, metabolic disturbances, and pancreatic brosis during cancer development. Therefore, further investigation should be addressed to the molecular pathways of AcSDKP in pancreatic cancer through EMT markers activity, and the downstream of pro-angiogenic inducer, in particular, VEGF as the potential targets for cancer therapy.

AcSDKP associated with betatrophin in pancreatic cancer
We also studied the correlation between AcSDKP to other parameters in pancreatic cancer progression. From our previous study, we found that the elevation of betatrophin concentration also correlates with pancreatic cancer incidence in patients with diabetes [32]. Here, our study showed that an attractive feature, whereas the level of AcSDKP is strongly associated with the serum level of betatrophin. This nding provides a novel molecular cross interaction between AcSDKP and betatrophin. Furthermore, the increase of AcSDKP concentration is in line with the elevation of liver injury markers (ALT, AST, and ALP). AcSDKP and betatrophin are produced in the liver, and both of these molecules suggested to regulate pancreatic cancer development that results in liver injury. Here in our study, the level of triglyceride and cholesterol also increases signi cantly in pancreatic cancer subjects. It is established that betatrophin regulates the degree of triglyceride by activating lipoprotein lipase (LPL) as the primary enzyme of triglycerides synthesis from lipid metabolism [33,34]. In vivo study showed that knockout of betatrophin reveals about 70% reduction in plasma levels of [35]. Furthermore, it remains unknown whether pancreatic cancer patients stimulate the increase of hepatic AcSDKP expression to induce the elevation of betatrophin. In addition, our study also demonstrated that the serum levels of betatrophin were signi cantly correlated with AcSDKP levels.
In further analysis of this study, we found an exciting feature, whereas the serum levels of betatrophin can predict the pancreatic cancer progression by increase the odds-ratio of AcSDKP by quintile analysis.
We speculate that AcSDKP corroborates pancreatic cancer development through cross molecular connection with betatrophin-LPL activity induce the increase of triglyceride levels. As a result, this pathological initiation process causes fatty pancreas as the pre-cancerous lesions during PDAC development. However, the underlying pattern of the molecular mechanism of this phenomenon is still unknown, and whether AcSDKP-betatrophin molecular interaction in pancreatic cancer associated with pancreatic brosis is not entirely elucidated.

Conclusions
In summary, circulating AcSDKP is signi cantly associated with pancreatic ductal adenocarcinoma development. AcSDKP was strongly correlated with serum levels of betatrophin and had a positive correlation to liver injury marker. Therefore, the levels of AcSDKP may serve as a potential additional prognostic biomarker for the early diagnosis of PDAC progression. This work has been approved by the Research Ethics Committee for Human Subject Protection National Chiao Tung University with the application number NCTU-REC-103-021. All participants have decided to participate in this study after reading all the considerations within the informed consent form and ll the form without prejudice.

Consent for publication
All the authors have been agreed that all the information (text and any pictures) within this manuscript may be seen by the general public. All authors declare that the paper is not under consideration for publication in another journal and will not be submitted elsewhere until the editorial refereeing process is completed.

Availability of data and material
The datasets generated during and/or analyzed during the current clinical study are available on reasonable request to the corresponding author.

Competing interest
The author declares that there is no con ict of interest in the eld covered by this manuscript.  Data are means ± SEM with P-value < +0.05 significantly different between any groups. ^ pancreatic cancer vs. Healthy subjects (H)  Odds-ratio (OR) and p values are given. * significant with p < 0.05. Odds-ratio (OR), p values, and p value adjusted (false discovery rate = FDR) are given. * significant with p < 0.05.  Figure 1 The level of AcSDKP and CA 19-9 in pancreatic cancer Data are means ± SEM with a P-value < 0.05 signi cantly different between any groups.

Figure 2
AcSDKP induced pancreatic cancer cell proliferation Data are means ± SD with a P-value < 0.05 signi cantly different between any groups.