MACC1 Mediates Acetylcholine-induced Invasion And Migration By Human Gastric Cancer Cells

BackgroundGastric cancer is one of the most common cancers worldwide, and it is the fifth most frequently diagnosed cancer worldwide and the third cause of death from cancer. Especially, the incidence of gastric cancer in China is much higher than in any other countries. It is the second most frequently diagnosed cancer in Chinese men and the third in women. Although there is a downtrend incidence in recent years, people diagnosed with of gastric cancer still have a large number in our coutry. In 2015, an estimated 679.1 thousand people are diagnosed with gastric cancer and 498 thousand people die from this disease. Surgery remains to be the major therapy for early gastric cancer. But gastric cancer is often diagnosed in advanced stage when only palliative treatment can be sonsidered, such as chemotherapy, radiotherapy, traditional Chinese medicine and targeted therapies. The therapeutic effect in advanced desease is often poor. Advanced gastric cancer is usually accompolished with extensive invasion and metastasis. Therefore, studying the mechanism of metastasis may help us to better inderstand the malignant biological behavior of gastric cancer and find new therapic strategies.Cancer metastasis is defined as a process that cancer cells detach the primary site to disseminate in the body, and then colonize at another site to form a second cancer. Epithelial-mesenchymal transition (EMT) is considered to be the initial step for malignant tumor metastasis, during which process tumor cells developing from the epithelial cells that are non-motile, polarized and adhered to each other by cell-cell junction convert into motile, non-polarized, and invasive mesenchymal cells. The molecular markers of a cell experiences EMT have changed dramatically. For example, the expression of cell-cell adhesion molecule E-cadherin is down-regulated, whereas the expression of the mesenchymal cell molecular markers is up-regulated, including vimentin, fibronectin and so on. Cancer cells also remodel extracellular matrix (ECM) to make it favorable to invasion and metastasis, and secretion of various matrix metalloproteinase (MMP) plays important role.The importance of microenvironment in tumor development and progression has attracted serious attention in rencent years. Nerve is an important part of the tumor microenvironment. It has been reported that tumor can grow along nerve and then disseminate through the route of nerve, which process is defined as perineural invasion (PNI), but the initiative role of nerve in cancer development and progression has attract little attention. As early as the 1940s, there has been a report about the effect of denervation on tumor, which has not yet received much attention. In 2013, the research published by Magnon et al made reseachers to be attracted by the effect of nerve on tumor again. They verified that automatic nervous system was essential for tumorigenesis and progression in mouse prostate cancer. On one hand, deletion of sympathetic nervous systems was found to mainly inhibit growth in the early phases of tumor development, and on the other hand, deletion of sympathetic nervous systems mainy inhibits dissemination in advanced phases. Another study by Zhao et al in 2014 reported that surgical and pharmacologic denervation of stomach, by vagotomy or local injection of neurotoxic agents, dramatically reduced incidence and progression of gastric cancer in mouse. Vagus nerve terminals can synthesis and release acetylcholine (ACh), and ACh and its M3 muscarinic receptor reportedly participates in promoting growth and metastasis in lung cancer, liver cancer, colon cacner, prostate cancer and so on, but their effect on gastric cancer metastasis and the mechanism remain unclear.Metastasis-associated in colon cancer-1 (MACC1) is an oncogene firstly identified in colon cancer in 2008. Stein et al discovered that MACC1 is higher expressed in colon cancer compared to normal tissues and serves an independent prognostic factor. In 2013, it is verified in our laboratory that MACC1 is also over expressed in gastric cancer and is associated with tumor stage and poor outcome. MACC1 acts on hepatocyte growth factor receptor (c-Met) to induce gastric cancer cell EMT and stimulate MMP2 and MMP9 expression, thus promotes growth, invasion and metastasis. Furthermore, gastric cancer would up-regulate MACC1 via phosphorylated AMP-activated protein kinase (AMPK) in response to glucose deprivation, and MACC1 promotes Warburg effect to protect gastric cancer from metabolic stress.We used bioinformatics analysis to find that phosphorylated AMPK is positively correlated with CHRM3 (the gene encoding M3 muscarinic receptor) mRNA expression in gastric adenocarcinoma. We think that ACh with M3 muscarinic receptor would promote MACC1 expression via phosphorylated AMPK, and MACC1 plays an intermediate role in ACh-induced metastasis of gastric cancer. Therefore, this study aimed to explore the effect of ACh in human gastric cancer cell invasion/migration and EMT and clarify M3 muscarinic receptor/AMPK/MACC1 signal pathway and its role in human gastric cancer cell invasion/migration and EMT.HypothesisACh stimulates invasion/migration and EMT of human gastric cancer cell.M3 muscarinic receptor mediates the effect of ACh on invasion/migration and EMT of human gastric cancer cell.MACC1 is the downstream signal for ACh and play essential role in ACh-induced invasion/migration and EMT of human gastric cancer cell.Research contents1. Choosing gstric cancer cells with high expression of M3 muscarinic receptorTotal RNA of BGC-803, BGC-823, MGC-803, MKN-28, MKN-45, SGC-7901 human gastric cancer cell lines was extracted respectively and reversed to cDNA. And then the mRNA expression of CHRM3 was determined by real-time quantitative PCR (qRT-PCR). The two highest CHRM3 expression cell linses were seleted for the following experiments.2. The effect of ACh on invasion, migration and EMT in gastric cancer10uM ACh was added to the culture medium of MKN-45 and MGC-803 gastric cancer cells, and then they were incubated for 0 h,24 h and 48 h respectively. And then matrigel coated trans well and non-coated transwell assays were carried out to detect the invasion and migration ability respectively. After MKN-45 and MGC-803 cells were incubated with 10uM ACh for 0 h,24 h and 48 h respectively, their mRNA and total protein were extracted. The mRNA was reverse to cDNA. The relative expression of EMT markers (E-cadherin, vimentin, fibronectin, MMP2 and MMP9) was detected by qRT-PCR at mRNA level and by western blot at protein level.3. The effect of M3 muscarinic receptor inhibitor on ACh activityMKN-45 and MGC-803 gastric cancer cells were pretreated with lOuM darifenacin and 10 uM ACh was added after 30 minutes. They were incubated for 48 h. They were designed in four groups:negative control, ACh, darifenacin, ACh+darifenacin. Matrigel coated transwell and non-coated transwell assays were carried out to detect the invasion and migration ability respectively. After MKN-45 and MGC-803 cells were incubated according to the above groups, their mRNA and total protein were extracted. The mRNA was reverse to cDNA. The relative expression of EMT markers (E-cadherin, vimentin, fibronectin, MMP2 and MMP9) was detected by qRT-PCR at mRNA level and by western blot at protein level.4. The effect of ACh stimulation on expression of MACC1At first, we use bioinformatics to analyze the relationship between CHRM3 and phosphorylated AMPK. Since MACC1 is suspected to be downstream signal for MACC1, we treated MKN-45 and MGC-803 cells with 10 uM ACh for 0 h,24 h and 48 h respectively and extracted the total mRNA and protein. The mRNA was reverse to cDNA. The relative expression of MACC1 was detected by qRT-PCR at mRNA level and by western blot at protein level. MKN-45 and MGC-803 cells were incubated for 48 h as four groups:negative control, ACh, darifenacin, ACh+darifenacin, their mRNA and total protein were extracted. The mRNA was reverse to cDNA. The relative expression of MACC1 was detected by qRT-PCR at mRNA level and by western blot at protein level. Immunofluorescence assay was used to detect the nuclear translocation of MACC1 after ACh stimulation.5. The importance of MACC1 in ACh induced invasion/migration and EMT of gastric cancer cellMKN-45 and MGC-803 cells were transfected with MACC1 siRNA (siMACC1) or negative control siRNA (siCtrl), and qRT-PCR and western blot were used to determine transfection efficiency. Transfected cells was incubated with 10 uM ACh. There were four groups in total:siCtrl, siMACC1, siCtrl+ACh, siMACCl+ACh. Matrigel coated transwell and non-coated transwell assays were carried out to detect the invasion and migration ability respectively. Their mRNA and total protein were extracted. The mRNA was reverse to cDNA. The relative expression of EMT markers (E-cadherin, vimentin, fibronectin, MMP2 and MMP9) was detected by qRT-PCR at mRNA level and by western blot at protein level.6. Verifying that AMPK is the intermediate signal between ACh/M3 muscarinic receptor and MACC1MKN-45 and MGC-803 cells were incubated for 48 h as four groups:negative control, ACh, darifenacin, ACh+darifenacin. Total protein was extracted for detection of p-AMPK expression by western blot. Then we pretreated the two cell lines with 8 uM dorsomorphin before adding 10 uM ACh in 30 min. There were four groups in total:negative control, ACh, dorsomorphin, ACh+ dorsomorphin. After incubation for 48 h, total protein was extracted for detection of p-AMPK and MACC1 expression by western blot.7. Statistical analysisAll statistical analyses were performed using SPSS software version 20.0. Data are presented as the mean±SEM. Differences between groups were analyzed using one-way ANOVA. Two-sided P values less than 0.05 were considered significant.Results1. Choosing gstric cancer cells with high expression of M3 muscarinic receptorWe used qRT-PCR to determin the expression oCHRM3 mRNA and found that MKN-45 and MGC803 cell lines have the highest mRNA expression of CHRM3, and then they were selected for the following experiments.2. ACh promotes GC cell invasion/migration and induces EMTThe transwell invasion and migration assays show that ACh stimulation increased the number of invading and migrating cells in a time-dependent manner within 48 h. From the result of qRT-PCR and western blot, ACh decreased the mRNA and protein expression of E-cadherin and increased expression of vimentin, fibronectin, MMP2 and MMP9 and, suggesting that ACh gradually promotes EMT progression within 48 h. However, ACh induced no significant morphological changes in gastric cancer cells.3. M3 muscarinic receptors mediate the effect of ACh on GC cell invasion/migration and EMTIn transwell invasion and migration assays, ACh markedly increase the number of invading and migrating cells, but when with darifenacin, ACh has no observable effect on MKN-45 and MGC-803 cells invasion and migration. Form the results of qRT-PCR and western blot, darifenacin also revesed ACh-induced decrease of E-cadherin expression and increase of vimentin, fibronectin, MMP2 and MMP9. What is more, when compared to a negative control, darifenacin also inhibited gastric cancer cell invasion/migration and EMT in the absence of exogenous ACh.4. ACh increases expression of MACC1By analyzing stomach adenocarcinoma data from the TCGA dataset, we discovered that the level of AMPK phosphorylated at Thr 172 is positively correlated with expression of CHRM3 mRNA. Combination with the conclusion from our previous study that MACC1 can be up-regulated by p-AMPK, we next verified the relationship between ACh and MACC1. We found that ACh time-dependently up-regulated MACC1 expression both at mRNA and protein level within 48 h, and this effect was suppressed by darifenacin. However, ACh-induced nuclear translocation of MACC1 was not significant.5. MACC1 is essential for ACh-induced GC cell invasion/migration and EMTAfter MKN-45 and MGC-803 cells were transfected with MACC1 siRNA, the expression of MACC1 was significantly decreased, indicating effective knockdown. The ACh-induced increase in GC cell invasion/migration was restrained after MACC1 knockdown. MACC1 siRNA also also revesed ACh-induced decrease of E-cadherin expression and increase of vimentin, fibronectin, MMP2 and MMP9 both at mRNA and protein level.6. MACC1 is up-regulated by ACh through p-AMPKFrom the results of western blots, p-AMPK levels increased after ACh stimulation, and pretreatment of darifenacin attenuated the ACh-induced increase of p-AMPK. When MKN-45 and MGC-803 cells were pretreated with the p-AMPK inhibitor dorsomorphin, ACh-stimulated up-regulation of MACC1 was greatly inhibited, indicating that p-AMPK is the intermediate signal between ACh/M3 muscarinic receptor and MACC1.ConclusionACh acts on M3 muscarinic receptor/AMPK/MACC1 signal pathway to promote gastric cancer cell invasion/migration and induce EMT.