Metastasis-associated In Colon Cancer-1 Promotes Vasculogenic Mimicry In Gastric Cancer By Upregulating TWIST1/2

Cancer requires an adequate blood supply to sustain its rapid growth. It was long believed that only endothelial cells could form blood vessels. However, when endothelium-dependent vessel growth is insufficient to support the rapid proliferation of tumor tissues, nonendothelial vascular networks also originate from tumors through a process called vasculogenic mimicry (VM). VM is the dominant method that provides blood supply in the early stages of cancer and is also significant for tumor proliferation and metastasis. Although a vascular endothelial growth factor receptor (VEGFR)-2 antagonist, has been reported that increasing overall survival for advanced gastric cancer (GC), clinical studies have shown that anti-angiogenesis drugs such as bevacizumab that targets vascular endothelium could not thoroughly meet the need of cancer treatment. A recent study showed that VM was still markedly increased in the tumors of patients receiving anti-angiogenesis treatment for ovarian carcinoma, indicating that VM is a potential alternative providing blood supply to maintain tumor growth when endothelium-dependent angiogenesis is inhibited. Many authors have conducted investigations of VM in a variety of tumors,but very few studies focused on GC.It was reported that VM occurs in poorly differentiated human GC, and tumor suppressor genes could inhibit VM formation,which could be promoted by oncogenes. Therefore, targeting the oncogene that promotes VM could effectively inhibit the carcinogenensis and tumor progression.Metastasis-associated in colon cancer-1 (MACC1) is an important regulatory factor for the activation of HGF/c-Met (Hepatocyte growth factor/Met tyrosine kinase receptor) signaling pathway. We previously reported that upregulation of MACC1 predicts poor clinical outcomes for patients with GC, and that MACC1 promotes GC cell proliferation and invasion by inducing the epithelial-mesenchymal transition (EMT) through activation of HGF/c-Met signaling pathway. Previous studies showed that EMT played a pivotal role in tumorigenesis and in VM formation. HGF could exert its biological function by promoting MACC1 nuclear translocation. Therefore, we hypothesized that MACC1 has a critical role in the process of VM in GC.It is known that EMT regulatory proteins include TWIST1 and TWIST2, and TWIST1 could induce EMT meanwhile promoting VM genesis in hepatocellular carcinoma. TWIST2 is a member of the basic helix-loop-helix (bHLH) transcription factor family, and it shares more than 90% sequence homology and structural similarity with TWIST1. The ability of TWIST2 to mediate the EMT in several types of cancer suggests that it may also be active in VM, but whether it is particularly involved in VM formation is not yet reported. We hypothesized that nuclear translocation of MACC1 promotes VM in GC by activating the TWIST1/2 signaling pathway.VE-cadherin is the first found VM-related protein, which is essentially classified as the endothelial cell adhesion molecule, but also has the kinase activity or participates in some important signal transduction process. Studies have reported that silencing of VE-cadherin could significantly inhibit the formation of VM cord-like structure.E-cadherin is one of the important phenotypes of epithelial cells, and lack of its expression is considered to be an important symbol of EMT. Normal cells or transformed E-cadherin mesenchymal cells can facilitate the connection and adhesion between cells through expressing E-cadherin. We have confirmed that MACC1 downregulated the expression of E-cadherin. Sun et al. also confirmed that TWIST1 promoted VM in HCC by upregulating VE-cadherin expression and downregualtion of E-cadherin. Therefore, TWIST1/2-VE-cadherin signaling pathway might be involved in MACC1 inducing VM formation. It is well-known that VEGFR2 (also called Flkl/KDR) is the the main factor modulating the mitogenic and angiogenesis functions of VEGF, and is involved in VM formation.TWIST1 unregulated the expression of VE-cadherin, meanwhile increasing the expression of VEGFR2. The above information indicated that MACC1 might promote the expression of endothelial cell associated markers in tumor cells through EMT-related signaling pathway, namely "TWIST1/2-VE-cadherin/ VEGFR2" axis, finally facilitate the formation of VM.In this study, we for the first time investigated whether MACC1 was associated with VM formation in GC. By studying the clinical data of 88 GC patients, we analyzed the impact of co-expression of MACC1 and VM on the prognosis of GC patients. Then we investigated the role of MACC1 in the genesis of VM using nude mice with GC xenografts and GC cell lines. As a result, we found that the HGF/c-Met-MACC1-TWIST1/2 signaling pathway was the underlying mechanism for VM formation in GC.Section one Clinicopathological analysis-VM exists in GC tissues, and double positive staining for MACC1 and VM predicts an adverse clinical outcomeObjectivesTo observe whether VM could be related to the clinical outcomes; to discuss the relationship between MACC1 and VM, and analyzed the impact of their co-expression on the clinical outcomes.MethodsTumor specimens of 88 GC patients were obtained from the Tumor Tissue Bank of Nanfang Hospital. All of the patients were diagnosed with stage IV GC and underwent palliative surgery or biopsy in Nanfang Hospital from 2005 to 2010. Formalin-fixed tumor tissues were used for HE and CD31/PAS staining, and we used staining for platelet endothelial cell adhesion molecule-1 (CD31) to identify the endothelium in GC tissue sections, as well as periodic acid-Schiff (PAS) staining to identify extracellular matrix-rich channels between tumor cells. Transmission electron microscopy was used to observe the ultra microstructure of VM. Immunohistochemistry was used to examine the correlation between MACC1 expression and VM density, and data was evaluated using the Kaplan-Meier analysis.Results1) We found VM in 43.0% of the GC patients (38/88 tumor samples);2) VM density was positively correlated with tumor differentiation, metastasis and survival status. Kaplan-Meier analysis showed that patients with a higher VM density in tumor tissues had a lower overall survival rate;3) Kaplan-Meier analysis revealed that patients with high levels of MACC1 expression in their tumors had a significantly lower overall survival rate. Interestingly, the 3-year survival rate was only 8.6% for patients whose tumors showed double positive staining for MACC1 and VM, whereas it was 41.7% for patients with tumors that were negative for both MACC1 and VM;4) Univariate and multivariate analyses both demonstrated that VM was an important prognostic factor for patients with GC.Conclusions1) VM structure mainly exists in poorly differentiated GC, indicating that VM is correlated with the malignant biological behavior of GC;2) VM density is correlated with the number of distant metastasis in stage IV GC patients, indicating that VM is correlated with the GC progression;3) VM density is positively correlated with MACC1 expression, which suggests that oncogenes might promote VM formation.Section two MACC1 upregulates TWIST1/2 to promote VM formation in GCObjectivesWe cultivated GC cell lines to observe the impact of MACC1 on the forming ability of VM, and generated GC animal model to further confirm the relationship between MACC1 and VM formation. We analyzed the modulation of MACC1 on TWIST1/2 expression, and investigated the mechanism of VM formation on the molecular level.MethodsBGC-823 and MKN-28 cells stably expressing MACC1 cDNA or MACCl-specific shRNA were described in our previous study. In this study, BGC-823 and MKN-28 cells expressing TWIST1-specific or TWIST2-specific siRNAs were performed by Lipofectamine 2000, and qRT-PCR and Western blot were performed to examine the efficiency of transfection. We used immunohistochemistry in subcutaneous GC xenografts and lung metastasis models to examine VM density and TWIST1/2 protein expression under different MACC1 expression. In 3D culture with Matrigel gel, we observed the formation of typical tube-like structures of VM induced by MACC1, and the change of their amount after silencing of TWIST1 and TWIST2.Results1) We successfully established nude mice models with subcutaneous and metastatic GC xenografts as described previously. In this study, we found that oxMACCl significantly promoted tumor proliferation and the formation of tube-like structures of VM, as well as upregulated TWIST1/2 protein expression; shMACC1 inhibited tumor proliferation and VM formation, as well as downregulated TWIST1/2 protein expression; in the metastatic GC model, VM density is positively correlated with the number of lung metastasis;2) In BGC-823 and MKN-28 cell lines, overexpression of MACC1 significantly upregulated TWIST1/2 expression, while silencing of MACC1 could effectively downregulate TWIST1/2 expression;3) In 3D culture, upregulation of MACC1 promoted the formation of tube-like structures of the GC cell lines, while downregulation of MACCl could inhibit its formation; silencing of either TWIST1 or TWIST2 could also reduce the tube formation.Conclusions1) MACC1 promotes the formation of VM and tube-like structure of GC cells;2) MACC1 could promote the formation of tube-like structure of GC cells in 3D culture by upregulation of TWIST1/2;3) We previously reported that upregulation of MACC1 promotes GC cell invasion and metastasis by inducing the epithelial-mesenchymal transition (EMT). In the present study, we further discovered that TWIST1/2 were essential modulating proteins in the process of MACC1 inducing VM formation in GC by EMT.Section three "HGF/c-Met-TWISTl/2-VE-cadherin/VEGFR2" axis participates in MACC1 inducing VM formation ObjectivesTo analyze the modulating effect of MACC1 on TWIST1/2 on the transcriptional level; to discuss the role of HGF/c-Met signaling pathway in MACC1 inducing tube formation of GC cells, and clarify the molecular mechanism for MACC1 inducing VM formation in GC cells. MethodsWe further investigated how MACC1 affected the expression of TWIST1 and TWIST2. The luciferase reporter assay was conducted to examine the luciferase activity driven by the TWIST1 and TWIST2 promoter. Western blotting was used to analyze the nuclear expression of MACC1, TWIST1, and TWIST2 proteins in cells treated with rhHGF and co-treatment with the c-Met inhibitor PF-04217903. In 3D cell culture system, we observed the number of tubes formed by VM after being treated with rhHGF and with co-treatment of PF-04217903. We further examined the expression of VE-cadherin, VEGFR2 and E-cadherin in GC cells with different MACC1 expressions by qRT-PCR, Western blot, flow cytometric analysis; Immunohistochemistry was used to examine the expression of VE-cadherin in subcutaneous GC xenografts and in 88 human GC samples, and statistical analysis was performed to find out its correlation to MACC1.Results1) We found in 5 pairs of tissues that the nuclear expressions of MACC1, TWIST 1, and TWIST2 proteins were significantly higher in the tumor tissues of VM-positive GC patients than in their matched adjacent non-tumor tissues;2) MACC1 could bind to TWIST1/2 promoter region under certain micro-environment and therefore enhance its transcriptional activity;3) 20 μM of rhHGF significantly increased the proliferation of BGC-823 cells by 25% and MKN-28 cells by 15% compared with their corresponding controls at 24-48 hours. In addition, the half maximal inhibitory concentration (IC50) of PF-04217903 was 22 μM for BGC-823 cells and 2 μM for MKN-28 cells at 24-48 hours. Western blot showed that the nuclear expressions of MACC1, TWIST1, and TWIST2 proteins were increased in cells treated with rhHGF, while antagonized this effect;4) The number of tubes formed by VM in 3D cell culture was increased by rhHGF, while c-Met inhibitor antagonized this effect;5) MACC1 could upregulate VE-cadherin and VEGFR2 expressions, as well as downregulate the expression E-cadherin;6) MACC1 expression and VM are both positively correlated with the expression of VE-cadherin.Conclusions1) MACC1 could bind to TWIST1/2 promoter region, and promote their transcription, upregulating their nuclear protein expression;2) RhHGF promotes VM formation through upregulating the nuclear protein expressions of MACC1, TWIST1, and TWIST2, while c-Met inhibitor antagonized this effect;3) MACC1 upregulates VE-cadherin and VEGFR2 expressions, and downregulates E-cadherin expression detected by qRT-PCR, western blot, flow cytometric analysis;4) MACC1 expression and VM formation are both significantly correlated to VE-cadherin expression, indicating that MACC1 might modulate VE-cadherin expression via EMT regulatory factors TWIST1/2, that promotes the transformation from the tumor cells to endothelial cells (namely TAEs), and ultimately formed VM.SummaryThe present study did retrospective study with clinical samples of GC, analyzed the relationship between MACC1 expression and VM density, and found that MACC1 promoted VM formation through upregulating TWIST1/2, demonstrating that "HGF/c-Met-TWIST1/2-VE-cadherin/VEGFR2" axis was the potential mechanism for MACC1 inducing VM, including that MACC1 could bind to TWIST1/2 promoter and multiple analysis concerning tissues and cells on the molecular and functional levels.