In this study, we revealed that CSPG4 played an important role to make peritoneal disseminations through migration/invasion ability and cell-cell interactions in EOC. We first confirmed that CSPG4 was expressed in EOC clinical samples and CSPG4 expression was associated with the proliferation and migration of EOC cells. Specifically, the proteomic analysis suggested that the downregulation of CSPG4 was related to the expression of proteins involved in cell movement. Additionally, CSPG4 downregulation remarkably decreased the ability of EOC cells to form spheroids in both 3D and in vivo models. Further, we found that sh-CSPG4 treatment significantly decreased peritoneal dissemination and tumorigenesis. Moreover, our results suggested that LEF1 was essential in regulating CSPG4; however, the expression of PAX3, which has been reported to be a regulator of CSPG4, was not associated with CSPG4 expression in EOC cells. Therefore, CSPG4 could be a novel therapeutic target, especially for preventing peritoneal dissemination in EOC.
It is well-known that CSPG4 plays an important role in cell adhesion, motility, and invasion in various types of malignant tumors (11, 15). Many reports have revealed a correlation between CSPG4 expression and the risk of metastasis or cancer prognosis (10, 16). However, the evidence regarding the role of CSPG4 in EOC is limited (27). In this study, we revealed that CSPG4 was expressed in nearly half of the clinical EOC samples and related to poor prognosis. We also observed CSPG4 expression in aggressive and mesenchymal EOC cell lines. CSPG4 knockdown in EOC cells changed the cell’s morphology to a round shape. Some studies have shown that CSPG4 expression in malignant melanoma is related to the morphological changes associated with the epithelial-to-mesenchymal transition (37). Similarly, previous studies have revealed that cell lines with expression of CSPG4 exhibit mesenchymal markers and high invasion properties (34, 35). These morphological changes may affect migration and invasion abilities of cancer cells. Actually, downregulation of CSPG4 using siRNA or shRNA decreased the migration, invasion, and wound-healing abilities of EOC cell lines. These results are in accordance with previous reports on other malignant tumors (11, 15). We also revealed the changes in the expression of proteins, especially those involved in cell movement pathways related to the actin cytoskeleton and muscle contraction, in EOC cells with CSPG4-targeted siRNA. Therefore, CSPG4 plays an important role in aggressive features of EOC.
EOC has a unique metastatic root that uses ascites (4). To induce peritoneal metastasis, EOC cells need to invade the mesothelial layer, which consists of mesothelial cells. Therefore, the interaction between EOC and mesothelial cells is critical for the formation of peritoneal metastasis (35). We first found that primary mesothelial cells expressed FN1, which binds to the extracellular domain of CSPG4 (38). Previous studies have revealed that TGF-β secreted by EOC cells in malignant ascites plays an important role in the peritoneal metastasis of EOC cells by altering the conditions of mesothelial cells (32, 39). Interestingly, we found that FN1 expression was significantly higher when mesothelial cells were stimulated with TGF-β. Therefore, TGF-β secretion further promoted the interaction between EOC and mesothelial cells, leading to an enhanced invasion of EOC cells into the mesothelial layer to form peritoneal dissemination. Moreover, we found that EOC cells with CSPG4 knockdown had restricted movement on the surface of mesothelial cells. These movements can be beneficial for EOC cells to find the proper metastatic area, including milky-spots of omenum, on the peritoneal surface by interacting with mesothelial cells (40, 41). These results suggest that blocking CSPG4 in EOC cells decreased their interaction with mesothelial cells, leading to decreased peritoneal metastasis.
Accumulating ascites is one of the unique characteristics of EOC. In ascites, EOC cells do not exist as a single cell, but form aggregated spheroids. Previous reports have revealed that spheroid formation has several merits for EOC, including resistance to anoikis and invasion into the mesothelial layer (4). Therefore, assessing the ability to form spheroids is essential for understanding the metastatic ability of EOC. Previous studies revealed that the expression of CSPG4 in human melanoma cells promoted their resistance to anoikis (42, 43). Interestingly, we found that CSPG4 knockdown remarkably decreased the spheroid formation of EOC cells. In addition, our in vivo model study showed that sh-CSPG4 transfection significantly decreased the spheroid formation of ES2 cells in ascites. Together, CSPG4 strongly affects the formation of spheroids in EOC cells, and these characteristics can strengthen the aggressiveness of EOC in surviving and forming peritoneal metastases in ascites.
Although many studies have investigated the function of CSPG4 in various tumors, little is known regarding how CSPG4 expression is regulated (15). Previous studies have revealed that PAX3 expression are related to CSPG4 expression (6, 14). Although PAX3-targeted siRNA decreased the expression of PAX3, the expression of CSPG4 did not change. From the upstream sequence of the CSPG4 coding area, we found that many LEF1-binding motifs existed. Indeed, LEF1 was found to regulate the CSPG4 promoter. In colorectal cancer, LEF1 is well known to have critical role for cancer stem-like cells survival and self-renewal activity (44). And disruption of the LEF1 pathway can be reduce recurrence (45). In addition, a previous study revealed that LEF1 was consistently expressed in the serous tubal intraepithelial carcinoma, precursor region of ovarian cancer (46). To our knowledge, this is the first reports on the relationship between CSPG4 expression and LEF1 regulation. Therefore, LEF1could be an important transcriptional factor related to aggressive features of EOC with the expression of CSPG4.
CSPG4 is expressed in several normal tissues throughout development but is only expressed in a few cell types in adults (12, 20). Therefore, CSPG4 is considered a suitable target for immune-based therapies (8, 47). Moreover, previous reports have shown no obvious deleterious side effects when using immunotherapy against CSPG4 (15). Many studies revealed that anti-CSPG4 monoclonal antibody or lentivirus encoding a CSPG4-targeted shRNA in tumors restricted tumor growth and metastasis (48, 49). In this study, the deletion of CSPG4 reduced spheroid formation and tumor burden in peritoneal metastasis. CSPG4 is proposed to be an essential protein for EOC cell growth in ascites and metastasis to the peritoneal cavity. These abilities can be related to cell-cell interactions. Therefore, CSPG4 may be a suitable target for blocking peritoneal metastasis and drug resistance in EOC patients.
This study has some limitations. First, we did not reveal the molecular mechanisms underlying CSPG4 expression and spheroid formation. Moreover, some studies have reported that CSPG4 is expressed in endothelial cells and affects vascular formation in the tumor microenvironment (8, 50). In this study, we did not examine the construction of blood vessels in the tumors. Further studies are needed to address these limitations.
In conclusion, this study showed that CSPG4 was expressed in EOC and related with tumor aggressiveness, including proliferation, invasion, and sphere formation. Specifically, we revealed that CSPG4 was related to spheroid formation in EOC in both 3D and in vivo models. Downregulation of CSPG4 was significantly associated with decreased tumor burden and peritoneal metastasis. This ability may be related to the development of peritoneal metastasis. In EOC, CSPG4 was regulated by LEF1 but not by PAX3. CSPG4 has restricted expression in normal tissues and the ability to control peritoneal dissemination; therefore, CSPG4 can be a suitable treatment target for EOC by controlling peritoneal metastasis, which is the most important strategy to improve prognosis in EOC patients.