Down-regulation of TAK1 inhibits migration and invasion of p-NENs by regulating JNK signaling pathway through interaction with PDZD2

Down-regulation of TAK1 inhibits migration and invasion of p-NENs by regulating JNK signaling pathway through interaction with PDZD2 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Down-regulation of TAK1 inhibits migration and invasion of p-NENs by regulating JNK signaling pathway through interaction with PDZD2 Xiaoyu Yang, Jianwei Qiu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4325473/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Pancreatic neuroendocrine neoplasmas (p-NENs) is a rare type of disease among digestive malignancy tumors, but its mechanics are still unclear. It has been found that TAK1 plays a crucial role in the progression of many tumors. We discovered TAK1 was highly expressed in BON-1 compared to two other pancreatic cell lines（CFPAC-1and hTERT-HPNE). Inhibition of TAK1 could significantly suppress the migration and invasion of p-NENs cell line (BON-1). Furthermore, the down-regulation of TAK1 suppressed the process of epithelial mesenchymal transition[1],which increased the epithelial marker E-cadherin and decreased the mesenchymal marker vimentin and N-cadherin. Through mass spectrometry analysis, we found that TAK1 is potentially blinded to PDZ domain containing 2(PDZD2) and negatively regulates its expression. In addition, down-regulation of TAK1 resulted in decrease of p-JNK.JNK activator (Anisomycin) significantly enhanced invasion and migration in comparison with TAK1 knockdown groups without activator. And silence of PDZD2 could rescue the changes in cancer cells’ phenotype and JNK expression caused by TAK1. All in all, the results of this study indicate that TAK1 regulates the JNK pathway in controlling p-NENs migration and invasion through its interaction with PDZD2, providing us with a new target for the treatment of p-NENs. JNK signaling Invasion Pancreatic neuroendocrine neoplasmas PDZD2 TAK1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Pancreatic neuroendocrine neoplasms(p-NENs) are a kind of heterogeneous neoplasms derived from pluripotent stem cells of the neuroendocrine system, which is very rare. In recent decades, its incidence rate has gradually increasing, which has ranked the second most common tumor of the pancreas, accounting for about 2%-5% of pancreatic tumors[2]. According to SEER database, the incidence of p-NENs reached 1.0/100000 until 2016[3]. Atypical symptoms of p-NENs, which are easily overlooked by patients, often lead to actual autopsy rates much higher than the 0.8%-10% reported[4]. At present, surgery is still the preferred treatment for p-NENs. When the primary lesion is removed, patients' clinical symptoms can be reduced, their survival time is significantly extended, and even the disease is cured. In spite of this，a total of 49.2% of patients already had lymph node metastasis and 30% had liver metastasis when diagnosed[5]. For patients with extensive metastasis, or patients who lose surgical indications, drug treatment can also be selected. Commonly used drugs include somatostatin analogs (SSAs), cytotoxic drugs, inhibitor of mammalian target of rapamycin(mTOR) and tyrosine kinase inhibitors (TKIs), interferonα-2b(IFNα-2b) and so on. The efficacy of drugs depends on location, size, stage, and grade of the tumor[6-8]. Due to the heterogeneity of p-NENs, the medical curative effect is very limited. Once the disease progresses, there will be no more medicine could be selected. Due to a lack of cellular and biological research on p-NENs, the pathogenesis of its progression is obscure, resulting in an inability to diagnose or treat it effectively. Therefore, it's urging to find an effective target for p-NENs. Our study aims to provide a theoretical basis for finding new therapeutic and diagnostic strategies for p-NENs. TGF-β-activated kinase-1(TAK1) was originally identified as a mitogen-activated kinase kinase kinase (MAPKKK) activated by transforming growth factor-β(TGF-β). It is a key regulator of inflammatory and immune signaling pathways[9]. The role of TAK1 in tumorigenesis is becoming more and more apparent in recent studies. As a result of TAK1 inhibition, the intrinsic chemoresistance of pancreatic cancer may be reversed[10]. Oihana iriondo found that TAK1 participated in mediating tumor microenvironment and promoted lung migration in triple-negative breast cancer[11]. On the other hand, it has also been reported that TAK1 could suppress the development and progression of some tumors. The higher the Gleason grade, which corresponds to the degree of malignancy, the lower the expression of TAK1 in prostate cancer patients. And inhibition of TAK1 promoted invasive carcinoma and cell phenotype both in vivo and in vitro[12]. The tumor suppressor function of TAK1 in prostate cancer has been validated. TAK1 is involved in the activation of NF-κB and MAPK pathways，which are related to tumorigenesis and the progression of cancers. C-Jun N-terminal kinase (JNK) is a subfamily protein of the MAPK family, which can be activated by cytokines, pathogens and other stimuli. The activated JNK signaling pathway can regulate cell growth, immune response and is closely related to the occurrence and progression of tumors[13]. Additionally, Wu Min's team discovered that tumor tissues formed by TAK1 deletion exhibited reduced expression of p-JNK, suggesting that TAK1 may cause prostate cancer growth by controlling MAPK signaling[12]. PDZ domain containing 2(PDZD2) is a protein with multiple PDZ domains. The PDZ domain is a protein structure that is related to protein-protein interactions. It can specifically recognize and bind to the special amino acid sequence at the C-terminal of protein, regulate the interaction between protein and protein, and participate in the regulation of cellular signal transduction pathways[14]. Several studies have revealed that PDZD2 contributes to tumorigenesis in some ways. Such as, PDZD2 is down-regulated in small intestinal neuroendocrine tumors[15]. In addition, PDZD2 deficiency led to an increase in basal insulin secretion[16]. Tumor progression was also associated with it. It was confirmed that overexpression of circ_0000009 suppressed lung adenocarcinoma progression by upregulating PDZD2 expression[17]. In our study, we identified a link between TAK1, PDZD2 and JNK signaling pathways in p-NENs progression, providing theoretical groundwork for the possibility that TAK1-PDZD2-JNK may represent an important therapeutic target for the treatment of p-NENs in the future. 2. Materials and methods 2.1. Cell culture Human pancreatic neuroendocrine neoplasm cell line(BON-1) 、human normal pancreatic duct epithelial cell line(hTERT-HPNE) and human pancreatic cancer cell line(CFPAC-1) were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences(Shanghai, China). BON-1 was cultured in F12 medium containing 10% fetal bovine serum (Gibco, NY, USA). CFPAC-1 and hTERT-HPNE were cultured in DMEM medium with 10% fetal bovine serum. All cells were cultured at a constant temperature of 37°C with 5% CO 2 . 2.2. Cell transfection Cells were transfected when cell density reached 80–90%. In order to increase the expression of TAK1, the cells were transfected with the plasmid using Lipofectamine 2000 reagent (Life Technologies, New York, USA). pcDNA3.1 plasmid was used as scamble control. Small interfering RNAs (siRNAs) specifically targeting corresponding gene synthesized by Ribobio (Guangzhou, China) were transfected with Lipofectamine RNAimax reagent (Life Technologies, NY, USA) into cells to down-regulate the expression of corresponding gene. Si-control were used as negative control. The sequences of siRNAs were as follows, si-TAK1: 5’-GGAGTTGTTTGCAAAGCTA-3’.si-PDZD2-1:5’-GTGTCCCTAGGCTCATCTGC-3’,si-PDZD2-2:5’-ACTCACAAGGAGCCTGGAAA-3’,si-PDZD2-3:5’CTCGTGTGCAGAAGAAACCA-3’ . 2.3. RNA extraction and quantitative real-time PCR (qRT-PCR) Total RNA was extracted from cultured cells with Trizol reagent (Vazyme, Nanjing, China) according to the manufacturer's instructions. The cDNA was synthesized according to Reverse Transcription Kit (Takara, Dalian, China). And the quantitative Real-time PCR(Roche, Basel, Switzerland) was used to analyze the mRNA levels. GAPDH served as an internal control. The sequences of primers were as follows:GAPDH:5’-GAAGGTGAAGGTCGGAGT-3’(forward), 5’-GAAGATGGTGATGGGATTTC-3’(reverse);TAK1:5’-ATTGTAGAGCTTCGGCAGTTATC-3’(forward),5’-CTGTAAACACCAACTCATTGCG-3’(reverse);PDZD2:5’-TCTGTACTGTGTACCTCACCAA-3’(forward),5’-CCCTGCGCTTTTCACCATAG-3’(reverse). 2.4. Protein extraction and western blotting The total protein was extracted from cells was according to manufacturer’s recommending protocol (Vazyme, Nanjng, China) and then detected the concentration of protein by BCA reagent (Beyotime Institute of Biotechnology). Protein lysates were separated by electrophoresis(SDA-PAGE) and then transferred to PVDF membranes, 5% BSA was used to blocked with redundancy protein for 1 hour, the specific primary antibodies were incubated at 4 ℃ overnight, and then washing membrane with Tris buffer saline containing 0.1% Tween-20 (TBST) 3–4 times, the secondary antibodies were incubated at normal temperature (1:5000 dilution)for 1 h. Membranes were visualized by the ECL assay (Millipore, Sigma, USA) after washing by TBST, the gray value of protein was analyzed by ImageJ( https://imagej.nih.gov/ij/ ). Tubulin and GAPDH were used as the inner control. All antibodies were purchased from Abcam (Cambridge, MA, USA) or Cell Signaling Technology (Boston, MA, USA). Vimentin (ab8978), Snail (ab53591), E-cadherin (ab1416), N‐cadherin(ab18203), β‐catenin (ab16051), TAK1(CST5206), Actin(ab8224), and GAPDH (ab8245). Phosphor-JNK(CST#4668), JNK(CST#9252), phospho-ERK1/2(CST#4370), ERK1/2(CST#9102), Phosphor-p38(CST#4511), P38(CST#8690), PDZD2(ab196631). 2.5. Cell proliferation assay 8000 cells were planted in 96-well plates per well, each group had 3 replications. After treatment for 24h, 5-Ethynyl-29-deoxyuridine (EdU) assay was performed according to EdU kit instruction (Beyotime Institute of Biotechnology, Shanghai, China). Photos were taken by Olympus fluorescence microscope, and then analyze the cell proliferation by calculating the EdU positive rate between different groups. 2.6. Wound-healing assay Cells were cultivated in 6-well plates and transfected for 24 h. When cells achieved 90–100% confluence, wounds were created by a 200µl plastic pipette tip. Fresh serum-free medium was added, and the wound-closing process was evaluated and recorded with a microscope at 0h,24 h and 48h after wounding. Wound-healing rate was calculated to analyze the migration capability of cells. 2.7. Transwell migration and invasion assay After cells were treated for 24 hours, transwell assay was performed to detect mingration and invasion abilities. 2*10 5 cells with 200µl of serum-free medium were seeded into upper transwell chamber (Corning, NY, USA). And F12 containing 10% serum was added to the lower chamber. For invasive assay, the upper chamber was coated with Matrigel (BD, Franklin Lakes, NJ, USA) for 2 hours in incubator, and washed twice by PBS before seeding cells. After 24h of incubation, the cells were fixed with 4% PFA for 15min. After washing with PBS, the cells were stained with crystal violet for 30min. After washing with PBS twice, the cells in the upper chamber were gently wiped off with cotton swabs, the cells attached to the lower surface of the upper chamber were taken photos under a microscope. 2.8. Immunohistochemistry The tissues from the p-NENs’ patients were cut during endoscopic surgery in the people's hospital of Jiangsu province, and then fixed in formaldehyde. Tissues were embedded with paraffin and sliced with a thickness of 3 µm. Immunohistochemical staining was performed according to the kit instructions to detect the expression of TAK1 protein. Brown spots were deemed as positive expression of TAK1, different areas were photographed by microscope under 200 times field. 2.9. Immunoprecipitation assay The cells were harvested and added an appropriate amount of IP lysate, and then splitting for 30 minutes. Some of lysates were taken for western blot analysis, and the remaining were added with 1µg antibody and 20µl protein A-beads. The lysates were shaken softly and incubated overnight at 4℃. After the immunoprecipitation reaction, proteinA-beads were deposited to the bottom of the tube by centrifugation at 4℃, and then sucked off the supernatant. Protein A-beads was washed with 1ml lysate for 3 times, and then added 15µl SDS and boiled for 10 minutes. Then the samples were performed mass spectrometry in Shanghai Applied Protein Technology. 2.10. Statistical analysis All the trials were conducted at least three separate times. Statistical analysis was analyzed with GraphPad Prism software. The results were expressed as mean ± SEM, Student's t-test was used for measurement data between two groups, and one-way ANOVA analysis was used for comparison of sample mean between multiple groups. P < 0.05 indicated statistical significance. 3. Results 3.1. TAK1 was significantly over expressed compared to normal pancreatic epithelial cells and pancreatic cancer cells To evaluate the original expression of TAK1 in pancreatic neuroendocrine tumor cells, we analyzed the TAK1 mRNA and protein expression of BON-1 and compared it with human normal pancreatic epithelial cells(hTERT-HPNE) and human pancreatic cancer cells (CFPAC-1). We found that TAK1 was highly expressed than HPNE and CFPAC-1(Fig. 1 A, B). IHC were analyzed to detect TAK1 expression in tumor tissue and peri-tumorous normal tissue. Compared with adjacent normal tissue, tumor tissue had a deeper brown degree, and the brown area was larger. The difference between p-NENs tumor tissue and normal tissue was statistically significant, indicating higher TAK1 expression in p-NENs (Fig. 1 C). According to these, we suggested that pancreatic neuroendocrine tumors may lead to an increase in TAK1 expression. 3.2. No significant phenotype was observed in BON-1 after TAK1 was over-expressed It was reported that disruption of TAK1 contributed to carcinogenesis and cancer development[ 18 ]. We were curious to know whether TAK1 is involved in the development or progression of p-NENs. We increased the expression of TAK1 by transfecting with a plasmid targeting TAK1 into BON-1, and received a high expression after transfection (Fig. 2 A, B). Next, we examined BON-1 cells' phenotype. Edu assay was conducted to assess cell proliferation ability. Cells that are EDU positive are undergoing proliferation, and the proportion of positive cells did not differ from the control group (Fig. 2 C). To examine BON-1's migration ability, we also performed a transwell assay where no significant difference was found between the normal control group and the TAK1-overexpressed group(Fig. 2 D). As a consequence of all these observations, overexpression of TAK1 has no impact on BON-1 progression. We speculate that because TAK1 was already highly expressed in p-NENs, it is unobvious to observe significant changes in cell phenotype when increasing TAK1 in BON-1. 3.3. Down-regulation of TAK1 significantly suppressed migration and invasion of BON-1 and altered EMT-related proteins. In order to detect the effect of TAK1 on tumor growth, we transfected siRNA-3 which was reported had the highest efficiency targeting TAK1 on BON-1 to reduce the expression of TAK1. To verify TAK1 knockdown efficiency, we detected the expression of TAK1 mRNA and protein levels (Fig. 3 A). And then we investigated the cells’ vitality by EdU assay. Similarly, TAK1 decreased did not affect cell proliferation (Fig. 3 B). The ability of migration and invasion is an important indicator of distant metastasis of tumors. It was reported that inhibition of TAK1 blocked cancer cell invasion, and metastasis[ 19 ], so we were curious about whether dysregulation of TAK1 could influence the growth of p-NENs cells. We performed wound-healing assay to evaluate the migratory ability of BON-1. At 24h or 48h, the wound area compared to the initial area represented the wound-healing rate. According to our findings, wound-healing rates in the TAK1 deficient group were aberrantly higher than in the control group (Fig. 3 C), indicating that cell migration is inhibited. As a confirmation, we conducted transwell experiments, which showed a significant drop in cell crossing from upper chamber to lower chamber when TAK1 was knocked down. The same results were observed with matrigel coated chambers (Fig. 3 D), indicating an inhibition of invasion after decreasing TAK1. Therefore, TAK1 is not capable of suppressing cell proliferation, but it can suppress BON-1 migration and invasion. Due to the fact that EMT plays a key role in tumor metastasis, we detected proteins that represent EMT. The characteristic protein of epithelial cells, E-cadherin, was significantly increased compared with the control group. And the mesenchymal markers, vimentin and N-cadherin, were significantly reduced after TAK1 was down-regulated (Fig. 3 E). This result was consistent with our previous observation that TAK1 attenuated the EMT process in cells which resulted in the reduction of metastasis to distant sites. 3.4. TAK1 affected tumor progression by inhibiting the expression of PDZD2 To find out which downstream molecule TAK1 interacted with, we performed immunoprecipitation experiments, followed by mass spectrometry, and found that PDZD2 could bind with TAK1 (Fig. 4 A). The PDZ domain-containing protein 2 (PDZD2) is a multi-PDZ protein that has been shown to be downregulated in lung adenocarcinoma[ 17 ]. As mentioned above, the expression of TAK1 was highest in BON-1, so we also tested the expression of PDZD2 in these three cells, and found that the expression of TAK1 was the highest in BON-1, but the expression of PDZD2 was the lowest oppositely (Fig. 4 B). It seemed that TAK1 and PDZD2 have opposite effects on the progression of p-NENs. Therefore, we speculate whether TAK1 negatively regulates PDZD2. In order to explore the relationship between TAK1 and PDZD2, we detected the expression of PDZD2 after down-regulation of TAK1 at mRNA and protein levels. As shown in Fig. 4 C, TAK1 knockdown significantly increased expression of PDZD2. To identify PDZD2's role in p-NENs cell progression, we generated three pairs of siRNAs targeting PDZD2 to reduce its expression and analyze its effect on cell phenotype. Among the three pairs of siRNAs, siRNA-1 knocked down PDZD2 most effectively (Fig. 4 D). Following siRNA-1 treatment, we conducted wound-healing assays and transwell assays to test BON-1's migratory and invasive abilities. It showed that the wound-healing rate and the number of cells that migrated or invaded to the lower chambers both augmented after PDZD2 was decreased (Fig. 4 E, F). We found that the expression trend of PDZD2 in BON-1 was opposite to that of TAK1, and the phenotype of BON-1 was also opposite. We speculated that TAK1 may attenuate PDZD2 expression in p-NENs, therefore promoting the development of p-NENs. To validate the above speculation, we conducted co-transfection of PDZD2 and TAK1 siRNAs. Originally, down-regulation of TAK1 alone attenuated cell migration. Cell migration was partially reversed when TAK1 and PDZD2 were reduced together. As a result of co-knockdown of TAK1 and PDZD2, the percentage of cells crossing into the lower chambers was somewhat increased versus TAK1 reduction alone (Fig. 4 E, F). Apparently, it can reverse some of the effects of TAK1 on p-NENs. These studies demonstrated that PDZD2 was a target gene for TAK1. 3.5 A down-regulation of TAK1 suppressed JNK signaling activity, while PDZD2 rescued this effect. As we all know, TAK1 plays a crucial role in the MAPK signaling pathway which participates in the modulation of cell growth, differentiation, inflammatory response and other processes. Accumulated evidence recovered that the aberrant MAPK pathway was related to tumorigenesis and the development of cancers. It has been reported that down-regulation of TAK1 inhibited the progression of pancreatic ductal adenocarcinoma by inactivating the NF-KB pathway and the MAPK pathway[ 20 ]. We also wanted to explore whether TAK1 affected tumor development by modulating the activity of the MAPK signaling pathway. We detected related proteins of the MAPK pathway after TAK1 was silenced in BON-1. We found no significant difference in p-ERK and p-P38 between the control and knockdown groups. However, the p-JNK notably decreased after TAK1 was silenced (Fig. 5 A). We investigated the influence of BON-1 phenotype following anisomycin treatment, which can activate JNK signaling. The results of wound-healing assay and transwell assay both showed that migration and invasion of BON-1 were significantly suppressed after the inactivation of JNK (Fig. 5 B, C). Based on these findings, it was concluded that depleting TAK1 inhibited migration and invasion of BON-1 by inhibiting the JNK signaling pathway. According to previous studies, TAK1 could bind to PDZD2 and inhibit tumor growth. Furthermore, the silence of TAK1 inhibit the activation of the downstream p-JNK signaling pathway, suppressing the progression of p-NENs. We wondered whether there was any connection between PDZD2 and the JNK signaling pathway. A decrease in p-JNK is seen in BON-1 after TAK1 is downregulated. Interestingly, the decrease of p-JNK reversed after co-transfecting with si-TAK1 and si-PDZD2 on BON-1, suggesting that PDZD2 could rescue the effect of TAK1to p-JNK partly (Fig. 5 D). 4. Discussion The incidence rate of neuroendocrine neoplasms (NENs) has steadily increased over the past forty years, increasing 6.4-fold from 1973 to 2012, mostly in early-stage tumors[ 21 , 22 ].The gastroenteric tract and pancreas are the most prevalent sites for neuroendocrine tumors. In recent years, with the popularization of endoscopic techniques, the improvement of diagnostic level and the gradual enhancement of people's awareness of physical examination, the incidence of gastroenteropancreatic neuroendocrine tumors ( GEP-NENs ) has gradually increased, which was nearly 3.8 times in 30 years in the United States[ 23 ]. In China, the pancreas is the most common site of GEP-NENs, accounting for 49.8%[ 24 ]. But the heterogeneity of p-NENs’ clinical features and vague pathogenesis still result in unsatisfactory therapies. So effective biomarkers are necessary for early diagnosis, prognosis prediction and monitoring therapeutic outcome[ 25 ]. Currently, chromogranin A (CgA), peppeptide (PP) and neuron specific enolase (NSE) have been commonly used in clinic[ 26 ], but their sensitivity and specificity are still limited. Our study established a link between molecules and tumor progression, providing a theoretical basis for clinical diagnosis and treatment. TGF-β is a vital cytokine that regulates the progression of cancer, which may slow tumor progression in early stage by inhibiting cell cycle and apoptosis. And in the late stage of cancer, tumor cells resist the inhibition of TGF-β, which can promote tumor migration and invasion[ 27 ]. TAK1, which could be activated by TGF-β, is also an essential kinase participates in physical and pathological process. Furthermore, accumulating research have reported that dysregulation of TAK1 was closely associated with the initiation and progression of numerous tumors. TAK1 was demonstrated to be both a tumor promoter and suppressor. In triple-negative breast cancer, TAK1 is highly expressed and aberrantly activated, which contributes to tumor metastasis and progression[ 28 ]. The depletion of TAK1 in tumor endothelial cells suppressed tumor progression by inhibiting blood vessel growth[ 29 ]. Acted as a tumor suppressor, TAK1 also inhibited development of prostate cancer. Despite this, TAK1 plays a complicated role in some tumor cells. Similarly, Sayaka Inokuchi discovered that deletion of TAK1 in hepatocytes resulted in inflammation and fibrosis of liver cells, resulting in hepatocellular carcinoma in vivo[ 30 ]. However, a recent study found that TAK1 is overexpressed in mice HCC models and tissues of human HCC, and high levels of this gene are associated with poor outcomes[ 31 ]. It is the first time that we revealed the function of TAK1 in p-NENs. Over-expression of TAK1 had no significant difference in cell phenotype. As TAK1 is highly expressed in p-NENs cells already, we hypothesized that up-regulation would not have a discernible effect. According to some of the published studies about TAK1, almost all of them performed knockout or knockdown experiments on TAK1 rather than overexpressing it. Undeniably, the suppressive function of si-TAK1 on BON-1 was significantly observed. Transwell assay and wound-healing assay both demonstrated that migration and invasion was suppressed after TAK1 was decreased. It is thought that EMT was a critical event during tumor metastasis, resulting in the down-regulation of expression of some epithelial proteins, such as E-cadherin. As mesenchymal characteristic proteins, such as vimentin, N-cadherin, and snail, are increased significantly, allowing tumor cells to invade surrounding tissues and migrate to distant organs more effectively[ 32 ]. Our results showed that after TAK1 was inhibited, epithelial marker (E-cadherin) was significantly up-regulated and mesenchymal marker, vimentin and N-cadherin were significantly down-regulated, indicating that the EMT process was suppressed. As a result of downregulated TAK1 expression, BON-1 exhibits less migratory and invasive abilities due to inhibition of EMT, resulting in fewer tumor cells that metastasize to distant organs. TAK1 is involved in the regulation of many signaling pathways, which are involved in processes of cell growth, inflammation, and tumor development, such as NF-κB, MAPK, TGF-β signaling pathways. Nuclear Met promotes HCC metastasis and invasion by phosphorylating TAK1 and activating its downstream NF-κB signaling pathway[ 33 ]. It has been illuminated that hepatocyte dual-specificity phosphatase 14 (DUSP14) reduced activation of TAK1 and its downstream signaling molecules JNK, p38, and NF-κB, maintaining metabolic homeostasis and preventing inflammation in the liver[ 34 ]. Therefore, we hypothesized that TAK1 regulates p-NENs progression through MAPK signaling. And our results showed that p-JNK was significantly decreased after TAK1 was silenced. Similar to the results of down-regulating TAK1, inhibiting JNK signaling suppressed BON-1's migratory and invasive abilities. These observations suggested that inhibition of TAK1 caused the JNK signaling pathway to be inactivated, which suppressed migration and invasion of p-NENs. An important role for the PDZ domain is in protein-protein interactions, which regulate signal transduction in cells. PDZD2 is expressed in multiple tissues such as the heart, brain, lungs, pancreas. Since very few reports are available about PDZD2, its function and detailed mechanism remain unknown. Our research found that TAK1 could blind PDZD2 and significantly repress the expression of PDZD2 in BON-1. In a reduction in PDZD2, the effect of TAK1 on BON-1 might be partially rescued. It is obvious that the two acted antagonistically on p-NENs. As reduction of TAK1 could inhibit the activation of JNK, we also demonstrated that p-JNK activity partially increased when BON-1 was co-transfected with si-TAK1 and si-PDZD2 compared to TAK1 was reduced alone. The simultaneous attenuation of TAK1 and PDZD2 rescued the inhibition of BON-1 migration and invasion mainly by activating the JNK pathway. Therefore, we speculated that TAK1 could suppress the function of PDZD2 and affect the progression of p-NENs through the JNK signaling pathway. In addition, the down-regulation of TAK1 caused an inhibition of p-JNK activation that could be restored by silencing PDZD2. Thus, it is likely that the TAK1-PDZD2-JNK axis may be a potential therapy target for p-NENs in the future. Declarations Author Contributions XY and JQ contributed to the experiment design, XY performed all experiments，analyzed the data and prepared the manuscript text. JQ was responsible for revising articles. All authors approved the publication of the final manuscript. Funding No funding was received. Data availability The data used and analyzed during the present study are available from the corresponding author on reasonable request. 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Endokrynologia Polska, 2022. 73 (3):491-548. http://doi.org/10.5603/EP.a2022.0050 Malireddi, R.K.S., et al., Innate immune priming in the absence of TAK1 drives RIPK1 kinase activity–independent pyroptosis, apoptosis, necroptosis, and inflammatory disease. Journal of Experimental Medicine, 2020. 217 (3). http://doi.org/10.1084/jem.20191644 Melisi, D., et al., Modulation of Pancreatic Cancer Chemoresistance by Inhibition of TAK1. J Natl Cancer Inst, 2011. 103 (15):1190-1204. http://doi.org/10.1093/jnci/djr243 Iriondo, O., et al., TAK1 mediates microenvironment-triggered autocrine signals and promotes triple-negative breast cancer lung metastasis. Nature Communications, 2018. 9 (1). http://doi.org/10.1038/s41467-018-04460-w Wu, M., et al., Suppression of Tak1 promotes prostate tumorigenesis. Cancer Research, 2012. 72 (11):2833-43. http://doi.org/10.1158/0008-5472.Can-11-2724 Xu, R. and J. Hu, The role of JNK in prostate cancer progression and therapeutic strategies. 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Trends in Pharmacological Sciences, 2012. 33 (10):522-530. http://doi.org/10.1016/j.tips.2012.06.007 Ray, D.M., et al., Inhibition of transforming growth factor-β-activated kinase-1 blocks cancer cell adhesion, invasion, and metastasis. British Journal of Cancer, 2012. 107 (1):129-136. http://doi.org/10.1038/bjc.2012.214 Huang, F.T., et al., MiR-143 Targeting TAK1 Attenuates Pancreatic Ductal Adenocarcinoma Progression via MAPK and NF-κB Pathway In Vitro. Digestive diseases and sciences, 2017. 62 (4):944-957. http://doi.org/10.1007/s10620-017-4472-7 Ito, T., et al., Epidemiological trends of pancreatic and gastrointestinal neuroendocrine tumors in Japan: a nationwide survey analysis. Journal of Gastroenterology, 2014. 50 (1):58-64. http://doi.org/10.1007/s00535-014-0934-2 Dasari, A., et al., Trends in the Incidence, Prevalence, and Survival Outcomes in Patients With Neuroendocrine Tumors in the United States. Jama Oncology, 2017. 3 (10):1335–1342. http://doi.org/10.1001/jamaoncol.2017.0589. Yao, J.C., et al., One Hundred Years After "Carcinoid": Epidemiology of and Prognostic Factors for Neuroendocrine Tumors in 35,825 Cases in the United States. Journal of the American Society of Clinical Oncology, 2008. 26 (18):3063. http://doi.org/10.1200/JCO.2007.15.4377 郭林杰, 中国胃肠胰神经内分泌肿瘤临床研究现状分析. 胃肠病学, 2012. 17 (5):276-278. http://doi.org/10.3969/j.issn.1008-7125.2012.05.005. Young, K., N. Starling, and A.J.S.i.c.b. Sadanandam, The molecular biology of pancreatic neuroendocrine neoplasms: Challenges and translational opportunities. Seminars in cancer biology, 2020. 61 :132-138. http://doi.org/10.1016/j.semcancer.2019.09.024 Herrera-Martínez, A.D., et al., Neuroendocrine neoplasms: current and potential diagnostic, predictive and prognostic markers. Endocr Relat Cancer, 2019. 26 (3):R157-r179. http://doi.org/10.1530/erc-18-0354 Katz, L.H., et al., Targeting TGF-β signaling in cancer. Expert Opinion on Therapeutic Targets, 2013. 17 (7):743-760. http://doi.org/10.1517/14728222.2013.782287 Sun, E.G., et al., Suppression of triple-negative breast cancer aggressiveness by LGALS3BP via inhibition of the TNF-α-TAK1-MMP9 axis. Cell Death Discovery, 2023. 9 (1):122. http://doi.org/10.1038/s41420-023-01419-9 Naito, H., et al., TAK1 Prevents Endothelial Apoptosis and Maintains Vascular Integrity. Developmental cell, 2019. 48 (2):151-166. http://doi.org/10.1016/j.devcel.2018.12.002 Inokuchi, S., et al., Disruption of TAK1 in hepatocytes causes hepatic injury, inflammation, fibrosis, and carcinogenesis. Proceedings of the National Academy of Sciences of the United States of America, 2010. 107 (2):844-849. http://doi.org/10.1073/pnas.0909781107 Ridder, D.A., et al., Transforming Growth Factor-β Activated Kinase 1 (Tak1) Is Activated in Hepatocellular Carcinoma, Mediates Tumor Progression, and Predicts Unfavorable Outcome. Cancers (Basel), 2022. 14 (2). http://doi.org/10.3390/cancers14020430 Santamaria, P.G., et al., EMT: Present and future in clinical oncology. Molecular Oncology, 2017. 11 (7):718-738. http://doi.org/10.1002/1878-0261.12091 Tey, S.K., et al., Nuclear Met promotes hepatocellular carcinoma tumorigenesis and metastasis by upregulation of TAK1 and activation of NF-κB pathway. Cancer Letters, 2017. 411 :150-161. http://doi.org/10.1016/j.canlet.2017.09.047 Wang, S., et al., Hepatocyte DUSP14 maintains metabolic homeostasis and suppresses inflammation in the liver. Hepatology, 2018. 67 (4):1320-1338. http://doi.org/10.1002/hep.29616 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4325473","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":296591298,"identity":"b7e2a854-5606-45a0-aa9c-f6e74b6b7ea3","order_by":0,"name":"Xiaoyu Yang","email":"","orcid":"","institution":"Nantong First People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyu","middleName":"","lastName":"Yang","suffix":""},{"id":296591299,"identity":"3a01ec91-c249-4fc9-b696-86c37d9bc327","order_by":1,"name":"Jianwei Qiu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAApElEQVRIiWNgGAWjYFACxgdAwoaHn72BaC3MBkAiTUay5wBpWg7bGNxwIFKDuXQz42OeP+d5GG4wMH74mEOEFss5h5kNZ7bd5mGc3cAsOXMbEVoMbuQfk/jYcJuHWeYAGzMvcVqS2SQS/pzjAZKkaPnAdoCHh2gtUL8k80jwHGwmzi/QELOztz/efPDDR6IcJgFnMjYQoR5VyygYBaNgFIwCHAAAkq0yxSR9xh0AAAAASUVORK5CYII=","orcid":"","institution":"Nantong First People's Hospital","correspondingAuthor":true,"prefix":"","firstName":"Jianwei","middleName":"","lastName":"Qiu","suffix":""}],"badges":[],"createdAt":"2024-04-25 16:56:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4325473/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4325473/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":56021147,"identity":"03360a0c-bd81-4ac4-aa15-97b880e00b21","added_by":"auto","created_at":"2024-05-07 16:12:08","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":280601,"visible":true,"origin":"","legend":"\u003cp\u003eTAK1 expressed highest in BON-1 and highly expressed in p-NENs tumor tissue.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eA, B\u003c/strong\u003e) TAK1 mRNA and protein expression of p-NENs cells (BON-1), human normal pancreatic duct epithelial cells(hTERT-HPNE) and human pancreatic cancer cells (CFPAC-1). \u003cstrong\u003eC \u003c/strong\u003eIHC for TAK1 in p-NENs tumor tissue and peritumor normal tissue. **P\u0026lt;0.01, ***P\u0026lt;0.001\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4325473/v1/8e1846e9a68e283a0399cbad.jpg"},{"id":56021149,"identity":"a9294451-b579-4423-8abc-ba16960e2b35","added_by":"auto","created_at":"2024-05-07 16:12:08","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":502121,"visible":true,"origin":"","legend":"\u003cp\u003eOver-expression of TAK1 had no significance in the phenotype of BON-1. (\u003cstrong\u003eA, B\u003c/strong\u003e) The expression of TAK1 mRNA and protein after over-expression by transfecting plasmid in BON-1. \u003cstrong\u003eC\u003c/strong\u003e EdU assay was conducted to detect cell vitality and count the rate of EdU positive rate (red cells/blue cells). \u003cstrong\u003eD \u003c/strong\u003eTranswell assay was used to observe the ability of migration and invasion after TAK1 was overexpressed. ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4325473/v1/72456c4f644ee2c0628d901b.jpg"},{"id":56021897,"identity":"64099037-4849-4bce-b784-b8218595fbb8","added_by":"auto","created_at":"2024-05-07 16:20:08","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":781797,"visible":true,"origin":"","legend":"\u003cp\u003eDown-regulation of TAK1 significantly repressed migration, invasion and EMT process in BON-1. \u003cstrong\u003eA\u003c/strong\u003e The expression of TAK1 mRNA and protein after transfecting siRNA targeting TAK1 in BON-1. \u003cstrong\u003eB \u003c/strong\u003eEdU assay was conducted to detect cell proliferation ability and counted the EdU positive rate. \u003cstrong\u003eC \u003c/strong\u003eThe wound-healing assay showed area of the wound at 24h and 48h, indicating the change of migratory ability. \u003cstrong\u003eD \u003c/strong\u003eThe ability of migration and invasion were detected by transwell assay and the number of cells crossed to the lower chamber was calculated. \u003cstrong\u003eE\u003c/strong\u003eExpression of EMT-related protein was compared between the si-NC and the si-TAK1 group. *P\u0026lt;0.05,**P\u0026lt;0.01,***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4325473/v1/80eeac9f21933727df26c41c.jpg"},{"id":56021150,"identity":"ae1b8e31-8dea-4b59-ab40-98accb1667a7","added_by":"auto","created_at":"2024-05-07 16:12:08","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1048110,"visible":true,"origin":"","legend":"\u003cp\u003eThe silence of PDZD2 rescued the inhibition of migration and invasion resulted from down-regulation of TAK1 alone. \u003cstrong\u003eA\u003c/strong\u003e Mass spectrum of PDZD2 after cells was transfected with plasmid targeting TAK1 and immunoprecipitation. \u003cstrong\u003eB\u003c/strong\u003e The PDZD2 mRNA expression of three cell lines: hTERT, CFPAC-1, BON-1. \u003cstrong\u003eC \u003c/strong\u003eThe mRNA and protein expression of PDZD2 after TAK1 was silenced. \u003cstrong\u003eD \u003c/strong\u003eThe expression of PDZD2 mRNA after BON-1 was transfected with 3 pairs of siRNAs targeting PDZD2. \u003cstrong\u003eE \u003c/strong\u003eThe situation of wound-healing at 24h and 48h of cells in 4 groups: si-NC, si-TAK1, si-PDZD2 and co-inhibition of TAK1 and PDZD2 and the area of the wound was calculated. \u003cstrong\u003eF \u003c/strong\u003eTranswell assay detected the ability of migration and invasion in 4 groups and the number of cells crossed to the lower chamber in 4 groups were calculated. *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4325473/v1/4c105321f2f33ae83ed9c914.jpg"},{"id":56021152,"identity":"cf7e88ee-af8e-4dff-b38a-bc73e3634ba1","added_by":"auto","created_at":"2024-05-07 16:12:08","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":491476,"visible":true,"origin":"","legend":"\u003cp\u003eDown-regulation of TAK1 suppressed the activity of JNK signaling pathway and silence of PDZD2 rescued its activity. \u003cstrong\u003eA \u003c/strong\u003eThe expression of related proteins of MAPK signaling pathways after TAK1 was inhibited: phosphorylated ERK, total-ERK, phosphorylated P38, total-P38, phosphorylated JNK and total-JNK. \u003cstrong\u003eB\u003c/strong\u003e The wound-healing area was detected at 24h, 48h among si-NC, si-TAK1 and si-TAK1+Anisomycin groups. \u003cstrong\u003eC\u003c/strong\u003e Migratory and invasive ability was detected by transwell among si-NC, si-TAK1 and si-TAK1+Anisomycin groups. The number of cells crossed to the lower chamber was calculated. \u003cstrong\u003eD \u003c/strong\u003eThe expression of p-JNK and total-JNK was detected after BON-1 was transfected with si-TAK1 alone, si-TAK1 and si-PDZD2 simultaneously. *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4325473/v1/4bafbf08cedbe97fceaf753b.jpg"},{"id":56488987,"identity":"8ab5b95c-198c-427a-abe8-c0f61339f39c","added_by":"auto","created_at":"2024-05-14 21:38:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1188839,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4325473/v1/c684e66f-65dd-4a31-98ba-c16754d934d9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eDown-regulation of TAK1 inhibits migration and invasion of p-NENs by regulating JNK signaling pathway through interaction with PDZD2\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePancreatic neuroendocrine neoplasms(p-NENs) are a kind of heterogeneous neoplasms derived from pluripotent stem cells of the neuroendocrine system, which is very rare. In recent decades, its incidence rate has gradually increasing, which has ranked the second most common tumor of the pancreas, accounting for about 2%-5% of pancreatic tumors[2]. According to SEER database, the incidence of p-NENs reached 1.0/100000 until 2016[3]. Atypical symptoms of p-NENs, which are easily overlooked by patients, often lead to actual autopsy rates much higher than the 0.8%-10% reported[4].\u003c/p\u003e\n\u003cp\u003eAt present, surgery is still the preferred treatment for p-NENs. When the primary lesion is removed, patients' clinical symptoms can be reduced, their survival time is significantly extended, and even the disease is cured. In spite of this，a total of 49.2% of patients already had lymph node metastasis and 30% had liver metastasis when diagnosed[5]. For patients with extensive metastasis, or patients who lose surgical indications, drug treatment can also be selected. Commonly used drugs include somatostatin analogs (SSAs), cytotoxic drugs, inhibitor of mammalian target of rapamycin(mTOR) and tyrosine kinase inhibitors (TKIs), interferonα-2b(IFNα-2b) and so on. The efficacy of drugs depends on location, size, stage, and grade of the tumor[6-8]. Due to the heterogeneity of p-NENs, the medical curative effect is very limited. Once the disease progresses, there will be no more medicine could be selected. Due to a lack of cellular and biological research on p-NENs, the pathogenesis of its progression is obscure, resulting in an inability to diagnose or treat it effectively. Therefore, it's urging to find an effective target for p-NENs.\u0026nbsp;Our study aims to provide a theoretical basis for finding new therapeutic and diagnostic strategies for p-NENs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTGF-β-activated kinase-1(TAK1) was originally identified as a mitogen-activated kinase kinase kinase (MAPKKK) activated by transforming growth factor-β(TGF-β). It is a key regulator of inflammatory and immune signaling pathways[9]. The role of TAK1 in tumorigenesis is becoming more and more apparent in recent studies.\u0026nbsp;As a result of TAK1 inhibition, the intrinsic chemoresistance of pancreatic cancer may be reversed[10]. Oihana iriondo found that TAK1 participated in mediating tumor microenvironment and promoted lung\u0026nbsp;migration in triple-negative breast cancer[11]. On the other hand, it has also been reported that TAK1 could suppress the development and progression of some tumors. The higher the Gleason grade, which corresponds to the degree of malignancy, the lower the expression of TAK1 in prostate cancer patients. And inhibition of TAK1 promoted invasive carcinoma and cell phenotype both in vivo and in vitro[12]. The tumor suppressor function of TAK1 in prostate cancer has been validated.\u003c/p\u003e\n\u003cp\u003eTAK1 is involved in the activation of NF-κB and MAPK pathways，which are related to tumorigenesis and the progression of cancers. C-Jun N-terminal kinase (JNK) is a subfamily protein of the MAPK family, which can be activated by cytokines, pathogens and other stimuli. The activated JNK signaling pathway can regulate cell growth, immune response and is closely related to the occurrence and progression of tumors[13]. Additionally, Wu Min's team discovered that tumor tissues formed by TAK1 deletion exhibited reduced expression of p-JNK, suggesting that TAK1 may cause prostate cancer growth by controlling MAPK signaling[12].\u003c/p\u003e\n\u003cp\u003ePDZ domain containing 2(PDZD2)\u0026nbsp;is a protein with multiple PDZ domains. The PDZ domain is a protein structure that is related to protein-protein interactions. It can specifically recognize and bind to the special amino acid sequence at the C-terminal of protein, regulate the interaction between protein and protein, and participate in the regulation of cellular signal transduction pathways[14].\u0026nbsp;Several studies have revealed that PDZD2 contributes to tumorigenesis in some ways. Such as, PDZD2 is down-regulated in small intestinal neuroendocrine tumors[15]. In addition, PDZD2 deficiency led to an increase in basal insulin secretion[16]. Tumor progression was also associated with it. It was confirmed that overexpression of circ_0000009 suppressed lung adenocarcinoma progression by upregulating PDZD2 expression[17].\u003c/p\u003e\n\u003cp\u003eIn our study, we identified a link between TAK1, PDZD2 and JNK signaling pathways in p-NENs progression, providing theoretical groundwork for the possibility that TAK1-PDZD2-JNK may represent an important therapeutic target for the treatment of p-NENs in the future.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Cell culture\u003c/h2\u003e \u003cp\u003eHuman pancreatic neuroendocrine neoplasm cell line(BON-1) 、human normal pancreatic duct epithelial cell line(hTERT-HPNE) and human pancreatic cancer cell line(CFPAC-1) were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences(Shanghai, China). BON-1 was cultured in F12 medium containing 10% fetal bovine serum (Gibco, NY, USA). CFPAC-1 and hTERT-HPNE were cultured in DMEM medium with 10% fetal bovine serum. All cells were cultured at a constant temperature of 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Cell transfection\u003c/h2\u003e \u003cp\u003eCells were transfected when cell density reached 80\u0026ndash;90%. In order to increase the expression of TAK1, the cells were transfected with the plasmid using Lipofectamine 2000 reagent (Life Technologies, New York, USA). pcDNA3.1 plasmid was used as scamble control. Small interfering RNAs (siRNAs) specifically targeting corresponding gene synthesized by Ribobio (Guangzhou, China) were transfected with Lipofectamine RNAimax reagent (Life Technologies, NY, USA) into cells to down-regulate the expression of corresponding gene. Si-control were used as negative control. The sequences of siRNAs were as follows, si-TAK1: 5\u0026rsquo;-GGAGTTGTTTGCAAAGCTA-3\u0026rsquo;.si-PDZD2-1:5\u0026rsquo;-GTGTCCCTAGGCTCATCTGC-3\u0026rsquo;,si-PDZD2-2:5\u0026rsquo;-ACTCACAAGGAGCCTGGAAA-3\u0026rsquo;,si-PDZD2-3:5\u0026rsquo;CTCGTGTGCAGAAGAAACCA-3\u0026rsquo; .\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. RNA extraction and quantitative real-time PCR (qRT-PCR)\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from cultured cells with Trizol reagent (Vazyme, Nanjing, China) according to the manufacturer's instructions. The cDNA was synthesized according to Reverse Transcription Kit (Takara, Dalian, China). And the quantitative Real-time PCR(Roche, Basel, Switzerland) was used to analyze the mRNA levels. GAPDH served as an internal control. The sequences of primers were as follows:GAPDH:5\u0026rsquo;-GAAGGTGAAGGTCGGAGT-3\u0026rsquo;(forward), 5\u0026rsquo;-GAAGATGGTGATGGGATTTC-3\u0026rsquo;(reverse);TAK1:5\u0026rsquo;-ATTGTAGAGCTTCGGCAGTTATC-3\u0026rsquo;(forward),5\u0026rsquo;-CTGTAAACACCAACTCATTGCG-3\u0026rsquo;(reverse);PDZD2:5\u0026rsquo;-TCTGTACTGTGTACCTCACCAA-3\u0026rsquo;(forward),5\u0026rsquo;-CCCTGCGCTTTTCACCATAG-3\u0026rsquo;(reverse).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Protein extraction and western blotting\u003c/h2\u003e \u003cp\u003eThe total protein was extracted from cells was according to manufacturer\u0026rsquo;s recommending protocol (Vazyme, Nanjng, China) and then detected the concentration of protein by BCA reagent (Beyotime Institute of Biotechnology). Protein lysates were separated by electrophoresis(SDA-PAGE) and then transferred to PVDF membranes, 5% BSA was used to blocked with redundancy protein for 1 hour, the specific primary antibodies were incubated at 4 ℃ overnight, and then washing membrane with Tris buffer saline containing 0.1% Tween-20 (TBST) 3\u0026ndash;4 times, the secondary antibodies were incubated at normal temperature (1:5000 dilution)for 1 h. Membranes were visualized by the ECL assay (Millipore, Sigma, USA) after washing by TBST, the gray value of protein was analyzed by ImageJ(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://imagej.nih.gov/ij/\u003c/span\u003e\u003cspan address=\"https://imagej.nih.gov/ij/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Tubulin and GAPDH were used as the inner control.\u003c/p\u003e \u003cp\u003eAll antibodies were purchased from Abcam (Cambridge, MA, USA) or Cell Signaling Technology (Boston, MA, USA). Vimentin (ab8978), Snail (ab53591), E-cadherin (ab1416), N‐cadherin(ab18203), β‐catenin (ab16051), TAK1(CST5206), Actin(ab8224), and GAPDH (ab8245). Phosphor-JNK(CST#4668), JNK(CST#9252), phospho-ERK1/2(CST#4370), ERK1/2(CST#9102), Phosphor-p38(CST#4511), P38(CST#8690), PDZD2(ab196631).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Cell proliferation assay\u003c/h2\u003e \u003cp\u003e8000 cells were planted in 96-well plates per well, each group had 3 replications. After treatment for 24h, 5-Ethynyl-29-deoxyuridine (EdU) assay was performed according to EdU kit instruction (Beyotime Institute of Biotechnology, Shanghai, China). Photos were taken by Olympus fluorescence microscope, and then analyze the cell proliferation by calculating the EdU positive rate between different groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Wound-healing assay\u003c/h2\u003e \u003cp\u003eCells were cultivated in 6-well plates and transfected for 24 h. When cells achieved 90\u0026ndash;100% confluence, wounds were created by a 200\u0026micro;l plastic pipette tip. Fresh serum-free medium was added, and the wound-closing process was evaluated and recorded with a microscope at 0h,24 h and 48h after wounding. Wound-healing rate was calculated to analyze the migration capability of cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Transwell migration and invasion assay\u003c/h2\u003e \u003cp\u003eAfter cells were treated for 24 hours, transwell assay was performed to detect mingration and invasion abilities. 2*10\u003csup\u003e5\u003c/sup\u003ecells with 200\u0026micro;l of serum-free medium were seeded into upper transwell chamber (Corning, NY, USA). And F12 containing 10% serum was added to the lower chamber. For invasive assay, the upper chamber was coated with Matrigel (BD, Franklin Lakes, NJ, USA) for 2 hours in incubator, and washed twice by PBS before seeding cells. After 24h of incubation, the cells were fixed with 4% PFA for 15min. After washing with PBS, the cells were stained with crystal violet for 30min. After washing with PBS twice, the cells in the upper chamber were gently wiped off with cotton swabs, the cells attached to the lower surface of the upper chamber were taken photos under a microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Immunohistochemistry\u003c/h2\u003e \u003cp\u003eThe tissues from the p-NENs\u0026rsquo; patients were cut during endoscopic surgery in the people's hospital of Jiangsu province, and then fixed in formaldehyde. Tissues were embedded with paraffin and sliced with a thickness of 3 \u0026micro;m. Immunohistochemical staining was performed according to the kit instructions to detect the expression of TAK1 protein. Brown spots were deemed as positive expression of TAK1, different areas were photographed by microscope under 200 times field.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Immunoprecipitation assay\u003c/h2\u003e \u003cp\u003eThe cells were harvested and added an appropriate amount of IP lysate, and then splitting for 30 minutes. Some of lysates were taken for western blot analysis, and the remaining were added with 1\u0026micro;g antibody and 20\u0026micro;l protein A-beads. The lysates were shaken softly and incubated overnight at 4℃. After the immunoprecipitation reaction, proteinA-beads were deposited to the bottom of the tube by centrifugation at 4℃, and then sucked off the supernatant. Protein A-beads was washed with 1ml lysate for 3 times, and then added 15\u0026micro;l SDS and boiled for 10 minutes. Then the samples were performed mass spectrometry in Shanghai Applied Protein Technology.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Statistical analysis\u003c/h2\u003e \u003cp\u003eAll the trials were conducted at least three separate times. Statistical analysis was analyzed with GraphPad Prism software. The results were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, Student's t-test was used for measurement data between two groups, and one-way ANOVA analysis was used for comparison of sample mean between multiple groups. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicated statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1. TAK1 was significantly over expressed compared to normal pancreatic epithelial cells and pancreatic cancer cells\u003c/h2\u003e \u003cp\u003eTo evaluate the original expression of TAK1 in pancreatic neuroendocrine tumor cells, we analyzed the TAK1 mRNA and protein expression of BON-1 and compared it with human normal pancreatic epithelial cells(hTERT-HPNE) and human pancreatic cancer cells (CFPAC-1). We found that TAK1 was highly expressed than HPNE and CFPAC-1(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). IHC were analyzed to detect TAK1 expression in tumor tissue and peri-tumorous normal tissue. Compared with adjacent normal tissue, tumor tissue had a deeper brown degree, and the brown area was larger. The difference between p-NENs tumor tissue and normal tissue was statistically significant, indicating higher TAK1 expression in p-NENs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). According to these, we suggested that pancreatic neuroendocrine tumors may lead to an increase in TAK1 expression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2. No significant phenotype was observed in BON-1 after TAK1 was over-expressed\u003c/h2\u003e \u003cp\u003eIt was reported that disruption of TAK1 contributed to carcinogenesis and cancer development[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. We were curious to know whether TAK1 is involved in the development or progression of p-NENs. We increased the expression of TAK1 by transfecting with a plasmid targeting TAK1 into BON-1, and received a high expression after transfection (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). Next, we examined BON-1 cells' phenotype. Edu assay was conducted to assess cell proliferation ability. Cells that are EDU positive are undergoing proliferation, and the proportion of positive cells did not differ from the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). To examine BON-1's migration ability, we also performed a transwell assay where no significant difference was found between the normal control group and the TAK1-overexpressed group(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). As a consequence of all these observations, overexpression of TAK1 has no impact on BON-1 progression. We speculate that because TAK1 was already highly expressed in p-NENs, it is unobvious to observe significant changes in cell phenotype when increasing TAK1 in BON-1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Down-regulation of TAK1 significantly suppressed migration and invasion of BON-1 and altered EMT-related proteins.\u003c/h2\u003e \u003cp\u003eIn order to detect the effect of TAK1 on tumor growth, we transfected siRNA-3 which was reported had the highest efficiency targeting TAK1 on BON-1 to reduce the expression of TAK1. To verify TAK1 knockdown efficiency, we detected the expression of TAK1 mRNA and protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). And then we investigated the cells\u0026rsquo; vitality by EdU assay. Similarly, TAK1 decreased did not affect cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eThe ability of migration and invasion is an important indicator of distant metastasis of tumors. It was reported that inhibition of TAK1 blocked cancer cell invasion, and metastasis[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], so we were curious about whether dysregulation of TAK1 could influence the growth of p-NENs cells. We performed wound-healing assay to evaluate the migratory ability of BON-1. At 24h or 48h, the wound area compared to the initial area represented the wound-healing rate. According to our findings, wound-healing rates in the TAK1 deficient group were aberrantly higher than in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), indicating that cell migration is inhibited. As a confirmation, we conducted transwell experiments, which showed a significant drop in cell crossing from upper chamber to lower chamber when TAK1 was knocked down. The same results were observed with matrigel coated chambers (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), indicating an inhibition of invasion after decreasing TAK1. Therefore, TAK1 is not capable of suppressing cell proliferation, but it can suppress BON-1 migration and invasion.\u003c/p\u003e \u003cp\u003eDue to the fact that EMT plays a key role in tumor metastasis, we detected proteins that represent EMT. The characteristic protein of epithelial cells, E-cadherin, was significantly increased compared with the control group. And the mesenchymal markers, vimentin and N-cadherin, were significantly reduced after TAK1 was down-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). This result was consistent with our previous observation that TAK1 attenuated the EMT process in cells which resulted in the reduction of metastasis to distant sites.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.4. TAK1 affected tumor progression by inhibiting the expression of PDZD2\u003c/h2\u003e \u003cp\u003eTo find out which downstream molecule TAK1 interacted with, we performed immunoprecipitation experiments, followed by mass spectrometry, and found that PDZD2 could bind with TAK1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The PDZ domain-containing protein 2 (PDZD2) is a multi-PDZ protein that has been shown to be downregulated in lung adenocarcinoma[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs mentioned above, the expression of TAK1 was highest in BON-1, so we also tested the expression of PDZD2 in these three cells, and found that the expression of TAK1 was the highest in BON-1, but the expression of PDZD2 was the lowest oppositely (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). It seemed that TAK1 and PDZD2 have opposite effects on the progression of p-NENs. Therefore, we speculate whether TAK1 negatively regulates PDZD2. In order to explore the relationship between TAK1 and PDZD2, we detected the expression of PDZD2 after down-regulation of TAK1 at mRNA and protein levels. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, TAK1 knockdown significantly increased expression of PDZD2. To identify PDZD2's role in p-NENs cell progression, we generated three pairs of siRNAs targeting PDZD2 to reduce its expression and analyze its effect on cell phenotype. Among the three pairs of siRNAs, siRNA-1 knocked down PDZD2 most effectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Following siRNA-1 treatment, we conducted wound-healing assays and transwell assays to test BON-1's migratory and invasive abilities. It showed that the wound-healing rate and the number of cells that migrated or invaded to the lower chambers both augmented after PDZD2 was decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, F). We found that the expression trend of PDZD2 in BON-1 was opposite to that of TAK1, and the phenotype of BON-1 was also opposite. We speculated that TAK1 may attenuate PDZD2 expression in p-NENs, therefore promoting the development of p-NENs.\u003c/p\u003e \u003cp\u003eTo validate the above speculation, we conducted co-transfection of PDZD2 and TAK1 siRNAs. Originally, down-regulation of TAK1 alone attenuated cell migration. Cell migration was partially reversed when TAK1 and PDZD2 were reduced together. As a result of co-knockdown of TAK1 and PDZD2, the percentage of cells crossing into the lower chambers was somewhat increased versus TAK1 reduction alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, F). Apparently, it can reverse some of the effects of TAK1 on p-NENs. These studies demonstrated that PDZD2 was a target gene for TAK1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.5 A down-regulation of TAK1 suppressed JNK signaling activity, while PDZD2 rescued this effect.\u003c/h2\u003e \u003cp\u003eAs we all know, TAK1 plays a crucial role in the MAPK signaling pathway which participates in the modulation of cell growth, differentiation, inflammatory response and other processes. Accumulated evidence recovered that the aberrant MAPK pathway was related to tumorigenesis and the development of cancers. It has been reported that down-regulation of TAK1 inhibited the progression of pancreatic ductal adenocarcinoma by inactivating the NF-KB pathway and the MAPK pathway[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. We also wanted to explore whether TAK1 affected tumor development by modulating the activity of the MAPK signaling pathway.\u003c/p\u003e \u003cp\u003eWe detected related proteins of the MAPK pathway after TAK1 was silenced in BON-1. We found no significant difference in p-ERK and p-P38 between the control and knockdown groups. However, the p-JNK notably decreased after TAK1 was silenced (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). We investigated the influence of BON-1 phenotype following anisomycin treatment, which can activate JNK signaling. The results of wound-healing assay and transwell assay both showed that migration and invasion of BON-1 were significantly suppressed after the inactivation of JNK (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C). Based on these findings, it was concluded that depleting TAK1 inhibited migration and invasion of BON-1 by inhibiting the JNK signaling pathway.\u003c/p\u003e \u003cp\u003eAccording to previous studies, TAK1 could bind to PDZD2 and inhibit tumor growth. Furthermore, the silence of TAK1 inhibit the activation of the downstream p-JNK signaling pathway, suppressing the progression of p-NENs. We wondered whether there was any connection between PDZD2 and the JNK signaling pathway. A decrease in p-JNK is seen in BON-1 after TAK1 is downregulated. Interestingly, the decrease of p-JNK reversed after co-transfecting with si-TAK1 and si-PDZD2 on BON-1, suggesting that PDZD2 could rescue the effect of TAK1to p-JNK partly (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe incidence rate of neuroendocrine neoplasms (NENs) has steadily increased over the past forty years, increasing 6.4-fold from 1973 to 2012, mostly in early-stage tumors[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].The gastroenteric tract and pancreas are the most prevalent sites for neuroendocrine tumors. In recent years, with the popularization of endoscopic techniques, the improvement of diagnostic level and the gradual enhancement of people's awareness of physical examination, the incidence of gastroenteropancreatic neuroendocrine tumors ( GEP-NENs ) has gradually increased, which was nearly 3.8 times in 30 years in the United States[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn China, the pancreas is the most common site of GEP-NENs, accounting for 49.8%[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. But the heterogeneity of p-NENs\u0026rsquo; clinical features and vague pathogenesis still result in unsatisfactory therapies. So effective biomarkers are necessary for early diagnosis, prognosis prediction and monitoring therapeutic outcome[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Currently, chromogranin A (CgA), peppeptide (PP) and neuron specific enolase (NSE) have been commonly used in clinic[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], but their sensitivity and specificity are still limited. Our study established a link between molecules and tumor progression, providing a theoretical basis for clinical diagnosis and treatment.\u003c/p\u003e \u003cp\u003eTGF-β is a vital cytokine that regulates the progression of cancer, which may slow tumor progression in early stage by inhibiting cell cycle and apoptosis. And in the late stage of cancer, tumor cells resist the inhibition of TGF-β, which can promote tumor migration and invasion[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. TAK1, which could be activated by TGF-β, is also an essential kinase participates in physical and pathological process. Furthermore, accumulating research have reported that dysregulation of TAK1 was closely associated with the initiation and progression of numerous tumors. TAK1 was demonstrated to be both a tumor promoter and suppressor. In triple-negative breast cancer, TAK1 is highly expressed and aberrantly activated, which contributes to tumor metastasis and progression[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The depletion of TAK1 in tumor endothelial cells suppressed tumor progression by inhibiting blood vessel growth[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Acted as a tumor suppressor, TAK1 also inhibited development of prostate cancer. Despite this, TAK1 plays a complicated role in some tumor cells. Similarly, Sayaka Inokuchi discovered that deletion of TAK1 in hepatocytes resulted in inflammation and fibrosis of liver cells, resulting in hepatocellular carcinoma in vivo[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. However, a recent study found that TAK1 is overexpressed in mice HCC models and tissues of human HCC, and high levels of this gene are associated with poor outcomes[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt is the first time that we revealed the function of TAK1 in p-NENs. Over-expression of TAK1 had no significant difference in cell phenotype. As TAK1 is highly expressed in p-NENs cells already, we hypothesized that up-regulation would not have a discernible effect. According to some of the published studies about TAK1, almost all of them performed knockout or knockdown experiments on TAK1 rather than overexpressing it. Undeniably, the suppressive function of si-TAK1 on BON-1 was significantly observed. Transwell assay and wound-healing assay both demonstrated that migration and invasion was suppressed after TAK1 was decreased. It is thought that EMT was a critical event during tumor metastasis, resulting in the down-regulation of expression of some epithelial proteins, such as E-cadherin. As mesenchymal characteristic proteins, such as vimentin, N-cadherin, and snail, are increased significantly, allowing tumor cells to invade surrounding tissues and migrate to distant organs more effectively[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Our results showed that after TAK1 was inhibited, epithelial marker (E-cadherin) was significantly up-regulated and mesenchymal marker, vimentin and N-cadherin were significantly down-regulated, indicating that the EMT process was suppressed. As a result of downregulated TAK1 expression, BON-1 exhibits less migratory and invasive abilities due to inhibition of EMT, resulting in fewer tumor cells that metastasize to distant organs.\u003c/p\u003e \u003cp\u003eTAK1 is involved in the regulation of many signaling pathways, which are involved in processes of cell growth, inflammation, and tumor development, such as NF-κB, MAPK, TGF-β signaling pathways. Nuclear Met promotes HCC metastasis and invasion by phosphorylating TAK1 and activating its downstream NF-κB signaling pathway[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. It has been illuminated that hepatocyte dual-specificity phosphatase 14 (DUSP14) reduced activation of TAK1 and its downstream signaling molecules JNK, p38, and NF-κB, maintaining metabolic homeostasis and preventing inflammation in the liver[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Therefore, we hypothesized that TAK1 regulates p-NENs progression through MAPK signaling. And our results showed that p-JNK was significantly decreased after TAK1 was silenced. Similar to the results of down-regulating TAK1, inhibiting JNK signaling suppressed BON-1's migratory and invasive abilities. These observations suggested that inhibition of TAK1 caused the JNK signaling pathway to be inactivated, which suppressed migration and invasion of p-NENs.\u003c/p\u003e \u003cp\u003eAn important role for the PDZ domain is in protein-protein interactions, which regulate signal transduction in cells. PDZD2 is expressed in multiple tissues such as the heart, brain, lungs, pancreas. Since very few reports are available about PDZD2, its function and detailed mechanism remain unknown. Our research found that TAK1 could blind PDZD2 and significantly repress the expression of PDZD2 in BON-1. In a reduction in PDZD2, the effect of TAK1 on BON-1 might be partially rescued. It is obvious that the two acted antagonistically on p-NENs. As reduction of TAK1 could inhibit the activation of JNK, we also demonstrated that p-JNK activity partially increased when BON-1 was co-transfected with si-TAK1 and si-PDZD2 compared to TAK1 was reduced alone. The simultaneous attenuation of TAK1 and PDZD2 rescued the inhibition of BON-1 migration and invasion mainly by activating the JNK pathway.\u003c/p\u003e \u003cp\u003eTherefore, we speculated that TAK1 could suppress the function of PDZD2 and affect the progression of p-NENs through the JNK signaling pathway. In addition, the down-regulation of TAK1 caused an inhibition of p-JNK activation that could be restored by silencing PDZD2. Thus, it is likely that the TAK1-PDZD2-JNK axis may be a potential therapy target for p-NENs in the future.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eXY and JQ contributed to the experiment design, XY performed all experiments，analyzed the data and prepared the manuscript text. JQ was responsible for revising articles. All authors approved the publication of the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding was received.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data used and analyzed during the present study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declared that there are no conflicts of interest.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePastushenko, I. and C. 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It has been found that TAK1 plays a crucial role in the progression of many tumors. We discovered TAK1 was highly expressed in BON-1 compared to two other pancreatic cell lines（CFPAC-1and hTERT-HPNE). Inhibition of TAK1 could significantly suppress the migration and invasion of p-NENs cell line (BON-1). Furthermore, the down-regulation of TAK1 suppressed the process of epithelial mesenchymal transition[1],which increased the epithelial marker E-cadherin and decreased the mesenchymal marker vimentin and N-cadherin. Through mass spectrometry analysis, we found that TAK1 is potentially blinded to PDZ domain containing 2(PDZD2) and negatively regulates its expression. In addition, down-regulation of TAK1 resulted in decrease of p-JNK.JNK activator (Anisomycin) significantly enhanced invasion and migration in comparison with TAK1 knockdown groups without activator. And silence of PDZD2 could rescue the changes in cancer cells’ phenotype and JNK expression caused by TAK1. All in all, the results of this study indicate that TAK1 regulates the JNK pathway in controlling p-NENs migration and invasion through its interaction with PDZD2, providing us with a new target for the treatment of p-NENs.\u003c/p\u003e","manuscriptTitle":"Down-regulation of TAK1 inhibits migration and invasion of p-NENs by regulating JNK signaling pathway through interaction with PDZD2","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-07 16:12:03","doi":"10.21203/rs.3.rs-4325473/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"70199995-783a-47b4-a1ba-e262d9ab43e4","owner":[],"postedDate":"May 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-23T07:38:11+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-07 16:12:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4325473","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4325473","identity":"rs-4325473","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}