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.