NGF from pancreatic stellate cells induces pancreatic cancer proliferation and invasion by PI3K/AKT/GSK signal pathway

Abstract Pancreatic cancer (PC) is a continuously high lethal disease, and the tumour microenvironment plays a pivotal role during PC progression. Herein, we focus on that the Nerve growth factor (NGF)/Tropomyosin‐related kinase A (TrkA), in pancreatic stellate cells‐pancreatic cancer cells (PSCs‐PC cells) co‐culture system, influences PC proliferation and invasion. The model of PC cells and PSCs was directly co‐cultured in a no‐touch manner, using the Transwell as the co‐culture system. NGF and TrkA expression was measured in cultured system by real‐time PCR, immunofluorescence, Western blotting analysis or ELISA. Small interfering RNA transfection was used to regulate the expression of TrkA in PC cells. The promotion of cancer invasion was investigated using Matrigel Transwell assay. In our study, NGF/TrkA is overexpressed in PSCs‐PC cells co‐culture system and promotes the invasion and proliferation of PC cells. And the epithelial‐mesenchymal transition‐related genes are influenced by si‐TrkA. What's more, NGF/TrkA regulates the PC cell proliferation and invasion via activation of PI3K/AKT/GSK signalling. The present study demonstrated NGF/TrkA promoted the PC cell proliferation and invasion in the co‐culture system by the activation of the PI3K/AKT/GSK signal cascade, providing a potential therapeutic target for PC patients.

treatments for PC are less effective and easier resisted. PSCs are lipid-storing cells existing in normal pancreatic tissue before activated by cytokines and other factors. PSCs can be activated by a variety of factors, including pro-inflammatory factors, oxidative stress and factors in tumour microenvironment. Activated PSCs can be transformed into myofibroblast-like cells which produce collagenous stroma. 10 Also, some evidence shows that PSCs interact with PC cells through various active factors which promote tumour growth and progression. 5 The interaction between PC cells and PSCs plays their roles through various active factors. 5,11 Nerve growth factor (NGF) is a member of the neurotrophin family. Numerous studies reveal that the high level of NGF in PC is closely correlated with tumour proliferation, tumour cell apoptosis and perineural invasion, especially. 12,13 Tropomyosin-related kinase A (TrkA) is the high-affinity receptor for NGF, in contrast with the low-affinity p75 neurotrophin receptor (p75NTR). 14 Phosphoinositide 3-kinase (PI3K)/protein kinase B(AKT) signalling pathway, which is the downstream of NGF/TrkA, promotes cancer survival and proliferation. 15,16 Herein, we focus on that the high level of NGF in pancreatic stellate cells-PC cells (PSCs-PC cells) co-culture system influences PC proliferation and epithelial-mesenchymal transition (EMT). We built an indirect PSCs-PC cells co-culture system in vitro and used the model to explore the NGF/TrkA works in this system. The data showed that NGF/TrkA promotes PC proliferation and EMT by PI3K/Akt/GSK signal pathway.

| Cell culture and reagents
The human PC cell lines AsPc-1 and Panc-1 were obtained from the American Type Culture Collection and cultured in DMEM supplemented with 10% foetal bovine serum (FBS) and 1% antibiotics/antimycotics in a humidified 5% CO 2 atmosphere at 37°C. Antibodies against MMP-9, vimentin, E-cadherin, NGF, TrkA, p-AKT, AKT, GSK and p-GSK were purchased from Abcam. Recombinant NGF was obtained from R&D Systems.

| Western blotting analysis
Cells were lysed using a lysis buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 1% NP 40, 0.5% sodium deoxycholate, 1 mM EDTA and 0.1% SDS) containing a protease inhibitor cocktail (Sigma-Aldrich), and protein concentrations were measured with the DC Protein Assay (Bio-Rad Laboratories, Inc). After separation on 7.5% SDS-polyacrylamide gels, proteins were transferred to nitrocellulose membranes (Amersham Bioscience), which were then incubated with primary antibodies at 4°C overnight. After being washed 3 times with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour. Immunoreactive bands were visualized using an enhanced chemiluminescence kit (Millipore). Quantitative analysis was performed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc).
The relative protein expression levels were normalized to GAPDH.

| MTT assay
Cell proliferation rate was measured by MTT assays. The cells were seeded in 96-well plates at a density of 1 × 10 4 cells per well and incubated overnight in medium containing 10% FBS. The DMSO concentration was adjusted to 0.4%. The cells incubated in serumfree medium were used as the control group. Following incubation for 12, 24 and 48 hours at 37°C, 20 μL of MTT solution (5 mg/mL in PBS) was added to each well, and the cells were incubated for an additional 4 hours at 37°C. Subsequently, 100 μL DMSO was added to each well at 37°C. The optical density (OD) value was determined using a spectrophotometer (Bio-Rad Laboratories Inc) at 490 nm. The proliferation rate was defined as OD (cell plate)/OD (blank plate).

| Co-culture system for PC cells and PSCs
The fresh pancreas tissue (from the liver transplant donors in the First Affiliated Hospital of Xi'an Jiaotong University) was made as 1-2 mm 3 , and the adherence of tissue and the morphology and quantity of cell were observed under a phase-contrast microscope. All experimental protocols were approved by the Ethical Committee of the First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China. The PSCs began to grow 3-day encompassment of the pancreas tissue and were isolated from pancreas. PSCs were subsequently seeded on 24-well plates cultured in DMEM supplemented with 10% FBS and 1% antibiotics/antimycotics in a humidified 5% CO 2 atmosphere at 37°C. We propose a model in which the PC cells and PSCs were directly co-cultured in a no-touch manner. Using the Transwell as the co-culture system, the upper chamber was inoculated with 1 mL of human PSC suspension (cell density 2 × 10 5 /mL). PC cells suspended in 1.2 mL (cell density 2 × 10 5 /mL) for the different groups were inoculated into the lower plate and cultured for 24 hours at 37°C in a 5% incubator.

| Migration experiment
The

| ELISA
Conditioned medium obtained from the co-culture system was collected at 24 hours after the treatments, centrifuged (1200 rpm) for 10 min and frozen at 80°C until analysed. The levels of secreted NGF were determined by an enzyme-linked immunosorbent assay (ELISA) (R&D Systems) according to the manufacturer's instructions.

| Transfection
Silencing of gene expression was achieved using siRNA technology.
Tumour cells were transfected with TrkA siRNA. Cells were seeded into small dishes and transfected with 100 nmol/L SiRNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The cells were used for further experiments 6 hours after transfection. Negative control siRNA (Ambion Inc) was used as a negative control.

| Statistics
The analyses of the results were carried out using the spss statistical software package (version 16.0). The significance of the data was determined using Student's t test or ANOVA analysis. A value of P < .05 was considered to indicate a statistically significant difference. Data are representative of at least three independent experiments and are reported as means ± SD.

| NGF is expressed in PSCs isolated from human pancreas
To explore the role of PSCs in the development of PC, primary PSCs were isolated from human pancreas ( Figure 1A). These activated PSCs were characteristic stained by Oil Red O which showed the state with accumulated lipid droplets ( Figure 1B). And immunofluorescence showed that NGF was mainly localized to the cytoplasm of PSCs ( Figure 1C,D,E). In summary, we confirmed that NGF was highly expressed in PSCs, which hinted its role in PC progression.

| PSCs promote PC cell proliferation and invasion in PSCs-PC cells co-culture system
To determine the effects of PSCs on PC cells, we used Transwell chamber with Matrigel coating to build a model which we called PSCs-PC cells co-culture system. PC cell lines Panc-1 or AsPc-1 were placed in the lower chamber with or without PSCs in the upper chambers ( Figure 2A). The results showed that the invasion ability of PC cells (Panc-1 and AsPc-1) was visibly increased when they co-cultured with PSCs ( Figure 2B, C) (P < .05). We observed and calculated cell proliferation rate after 12, 24 and 48 hours, and the proliferation rate of PC cells was significantly increased in the groups with PSCs co-cultured ( Figure 2D,E) (P < .05). And, mannitol was a control for excluding influences of interstitial pressure. These results all showed that PSCs co-cultured with PC cells could promote invasion and proliferation ability for PC cells.

| Expressions of NGF and TrkA are increased in PSCs-PC cells co-culture system
To assess the expression levels of NGF and TrkA, the RT-PCR, ELISA and Western blot analysis were conducted to compare the mRNA and protein expression ( Figure 3). We found that NGF expression level was increased in PSCs when co-cultured with PC cells

| NGF/TrkA axis plays a role in proliferation and invasion of PC cells in co-culture system
To further investigate the role of NGF/TrkA axis in the interaction between PSCs and PC cells, the synthetic NGF and K252a (inhibitor of TrkA) were used in the co-culture system (Figure 4).
The results demonstrated that the invasion ability in co-culture system was enhanced by NGF (100 ng/mL) compared with control

| NGF/TrkA signalling regulates EMT of PC cells via PI3K/AKT/GSK
To further determine the mechanism of NGF/TrkA for the pro-

| D ISCUSS I ON
PC is one of the leading causes of cancer death among solid cancers. 1 In fact, the high potential metastasis and rapid growth may be the main cause contributing to its high mortality rate. 17 Recent years, tumour microenvironment is the research focus of cancer progression, and PSCs are involved in PC progression as the functional components. 6,7,[18][19][20] NGF and its receptors (P75 and TrkA) were always shown to be the acceleration role for the cancer development. 21 Here, the work presented identifies that NGF and the PI3K/

F I G U R E 2 PSCs promote invasion and increase the proliferation of PC cells. (A)
The PC cells and PSCs were directly co-cultured in a no-touch manner. Pancreatic cancer cell lines Panc-1 or AsPc-1 were placed in the lower chamber with or without PSCs in the upper chambers. (B) The invasion ability of Panc-1 (the above listed) and AsPc-1 (the below listed) cells was visibly increased when they co-cultured with PSCs (the right side) compared with control group (the left side). (C) The migrated cancer cell numbers were visibly increased in the co-cultured with PSCs in Panc-1 and AsPc-1 cells. (D, E) The proliferation rate of PC cells is significantly increased in the groups with PSCs co-cultured, and the mannitol was a control for excluding influences of interstitial pressure. *P < .05 compared with control. All data from three independent experiments were analysed AKT signal pathway were involved in the mechanisms of interaction between PC and PSCs, promoting the proliferation and EMT in PC.
The pancreatic tumour microenvironment contains several cellular components, including the PC cells and PSCs, 18,22 which support tumour growth and progression. To clarify the interaction between both of them, the co-culture system of PC cells and PSCs was used in this study. Based on our previous work, we propose a model in which the PC cells and PSCs were directly co-cultured in a no-touch manner. 23 The advantage of this model is that the system could give consideration to the co-culture which signify the microenvironment and also make it easier to analyse the action of PC cells and PSCs separately.
In this study, we showed that NGF was localized in PSCs We also show in this study that PSC treatment increased the expression of both NGF and its receptor within the co-culture system leading to increased invasiveness and proliferation. This finding suggests that the molecular function of NGF binding TrkA offers partly acceleration, and the high expression and activation of TrkA make contributions in the course of PSCs resulting in cell growth and invasiveness of PC cells. These findings provide the evidence that the pancreatic tumour microenvironment is a heterogeneous ecology and the interaction between cancer cells and stromal elements is complex and hard to make certain. 25,26 So, the deeper mechanism of action should be continued in the further investigation.
The evidence has come out that induction of the EMT programme is required for invasion. 27,28 The expression of cellular EMT marker changes, including MMP-9, vimentin and E-cadherin, has implications for the cancer invasion-metastasis process. In this study, we demon- The E-cadherin expression was increased by si-TrkA or K252a but decreased by NGF in co-culture system. (E, F) The expression of vimentin and MMP-9 was decreased by si-TrkA or K252a but increased obviously by NGF in co-culture system. *P < .05 compared with control. # P < .05 between NGF group and si-TrkA group. All data from three independent experiments were analysed factors or non-canonical pathway may be excluded in the PC process promoted by PSCs. 11,29,30 Further studies will be needed to investigate the mechanisms in animal experiments because of its simulation with the similar tumour microenvironment.
In conclusion, the present study showed that PC cells co-cultured with PSCs can promote invasion and proliferation ability in PC cells. What's more, NGF/TrkA promoted the PC cell proliferation and invasion in the co-culture system of PSCs and PC cells through the activation of the PI3K/AKT/GSK signal cascade. In the balance function of tumour microenvironment, we propose that NGF/TrkA could serve as an effective and potential therapeutic target for PC patients.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

AUTH O R CO NTR I B UTI O N S
LH and ZW designed the study. LH, ZW and JJ wrote the manuscript.
JJ and JB performed analysis. LH, TQ, JB and JJ contributed to methodology. All authors read and approved the final manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets used and analysed during the current study are available from the corresponding author upon reasonable request.