Study Of TROP2Mediated Tumor Growth And Angiogenesis In Hilar Cholangiocarcinoma

Background and SignificanceHilar cholangiocarcinoma (also known as Klatskin tumor) is a biliary epithelial neoplasm with high mortality and a low rate of early diagnosis. Although it is a rare malignancy, incidence is rising every year. At this time, curative therapeutic options are provided only through surgical techniques, including bile-duct resection, concomitant liver resection and liver transplantation. However, the majority of patients present with locally advanced disease, such that only30%of patients attempt curative resection, and recurrence is still high even after curative resection. Chemotherapy and radiotherapy yield unsatisfactory results, while the benefits of photodynamic therapy have been debatable. Therefore, there is a pressing need to identify novel genes to serve as therapeutic targets in hilar cholangiocarcinoma.Trophoblast cell surface antigen2(TROP2), also known as GA733-1or EGP-1, is a36-KDa transmembrane glycoprotein originally defined on normal and malignant trophoblast cells. The protein consists of323amino acids including four N-linked glycosylation sites. Its encoding gene belongs to the tumor-associated calcium signal transducer (TACSTD) gene family, of which GA733-2or epithelial cell-adhesion molecule (EpCAM) gene is another highly conserved member. TROP2is highly expressed in various types of human epithelial cancers, but rarely expressed in normal epithelial cells. Although the ligand for TROP2is unknown, many studies have indicated that TROP2plays a functional role in cancer and in stem cells. TROP2suppression by RNA interference in colon cancer cells can inhibit their invasiveness, anchorage-independent growth, and tumorigenicity in vitro and in vivo. Clinical studies have also indicated that high TROP2expression correlates with poor disease-free and overall survival in a variety of carcinomas. Furthermore, elevated TROP2expression is significantly associated with lymph node metastasis, tumor differentiation and other clinicopathological features in some digestive cancers. Given its relatively low expression in normal epithelial tissues, TROP2may be a potential novel therapeutic target for malignant carcinomas.Pathological angiogenesis is necessary for tumor growth and metastasis and is of prognostic importance in human solid tumors. Quantification of microvessel density (MVD) is widely used to evaluate the degree of pathological angiogenesis in tumors. Anti-angiogenic drugs targeting different molecules have been efficacious in a variety of human cancers. Previous studies have demonstrated a significant correlation between MVD and disease-free survival and overall survival in extrahepatic cholangiocarcinoma and other digestive tumor, moreover, it has been reported that expression of EpCAM is significantly associated with MVD and vascular endothelial growth factor (VEGF) expression in hepatocellular carcinoma (HCC). Therefore, we propose that TROP2may play a functional role in tumor angiogenesis in hilar cholangiocarcinoma. To our knowledge, the relationship between TROP2expression and angiogenesis in hilar cholangiocarcinoma has not been investigated.The present study was conducted to determine TROP2antigen expression, evaluate the correlation between TROP2and angiogenesis and their prognostic significance in hilar cholangiocarcinoma. Then, we screened stable TROP2knockdown hilar cholangiocarcinoma cell line and found that TROP2could promote tumor cell proliferation, colony formation, sphere formation and angiogenesis. Finally, we figured out that β-catenin signaling pathway played an important role in TROP2mediated biological functions and β-catenin could rescue TROP2effects in hilar cholangiocarcinoma. Part I Expression and Prognostic Significance of TROP2in Hilar CholangiocarcinomaObjectiveTROP2was found to be associated with tumor progression and poor prognosis in a variety of epithelial carcinomas. The aim of the study was to investigate TROP2expression and its prognostic impact in hilar cholangiocarcinoma.MethodsImmuohistochemistry and quantitative real-time PCR were used to determine TROP2expression in surgical specimens from70hilar cholangiocarcinoma patients receiving radical resection. TROP2protein and mRNA level was also measured in HBDC, FRH0201and QBC939three common hilar cholangiocarcinoma cell lines. Standard statistical analysis was used to evaluate correlation between TROP2and clinicopathological characteristics and its prognosis significance in hilar cholangiocarcinoma.ResultsHigh TROP2expression by immunohistochemistry was found in43(61.4%) of the70tumor specimens. Quantitative real-time PCR confirmed that TROP2level in tumor was significantly higher than in adjacent normal biliary tissues (P=0.001). Significant correlations were found between TROP2expression and histological differentiation (P=0.016) and tumor T stage (P=0.031) in hilar cholangiocarcinoma. TROP2expression was found in all three cell lines, but it is the highest in HBDC cell line. High TROP2expression patients had a significantly poorer overall survival rate than those with low TROP2expression (30%vs.68.5%, P=0.001) and multivariate Cox regression analysis indicated TROP2as an independent prognostic factor in hilar cholangiocarcinoma (P=0.004).ConclusionsTROP2protein increased and correlated with histological differentiation (P=0.016) and tumor T stage (P=0.031) and is an independent prognostic factor in human hilar cholangiocarcinoma (P=0.004). Part Ⅱ TROP2Promoted Tumor Growth and Angiogenesis in Hilar CholangiocarcinomaObjectiveThe aim is to investigate the roles of TROP2in tumor growth and angiogenesis in hilar cholangiocarcinoma.MethodsWe constructed TROP2knockdown lentivirus plasmids, infected HBDC hilar cholangiocarcinoma cell line and screened the stable TROP2knockdown HBDC cell line. MTT assay, colony formation assay, cell sphere culture and HUVEC tube formation assay were used to measure the effects of TROP2on proliferation, colony formation, self-renewal and angiogenesis in hilar cholangiocarcinoma respectively. TROP2knockdown stable cells were injected into nude mice subcutaneously and tumor growth was measured every five days.1month later, tumor was removed and MVD was detected, evaluating the correlation between TROP2and angiogenesis in vivo. To confirm the results, test the microvessel endothelial cells using anti-CD34antibody in human hilar cholangiocarcinoma tissue species and evaluate it as mean microvessel density (MVD). Chi-square and Fisher’s exact test was used to evaluate the correlation between TROP2expression and MVD in human hilar cholangiocarcinoma.ResultsTROP2protein was down-regulated significantly in screened stable HBDC cell line. MTT assay demonstrated that TROP2knockdown decreased the cell proliferation. Colony formation assay showed that TROP2knockdown inhibited colony formation in hilar cholangiocarcinoma. Sphere formation assay showed that TROP2knockdown decreased their ability of self-renewal. HUVEC tube formation assay indicated that TROP2knockdown inhibited tube formation of HUVEC on Matrigel. In vivo data showed that TROP2could promote tumor growth and angiogenesis obviously. MVD is about4to71in all the hilar cholangiocarcinoma species. MVD is much higher in TROP2overexpression group than TROP2lower group. Chi-square and Fisher’s exact test proved that TROP2correlated with MVD in hilar cholangiocarcinoma significantly.ConclusionsTROP2knockdown inhibited cell proliferation, colony formation and self-renewal and HUVEC tube formation. In vivo data also showed that TROP2knockdown could inhibit tumor growth and angiogenesis significantly in hilar cholangiocarcinoma. Part III Wnt/beta-catenin Signaling Mediated TROP2Biological Functions in Hilar CholangiocarcinomaObjectiveThe aim is to study the mechanism through which TROP2promotes tumor growth and angiogenesis in hilar cholangiocarcinoma.MethodsWe collected TROP2knockdown HBDC protein and measured (3-catenin level in cells and nucleus. TOP/FOP luciferase reporter was used to make sure the activation of wnt/β-catenin signaling by TROP2. We transfected mutant β-catenin (S37A) into TROP2knockdown HBDC stable cell lines using lentivirus and test the effects on tumor cell proliferation, self-renewal and angiogenesis, testing whether P-catenin mediated TROP2biological functions on tumor growth and angiogenesis in Hilar Cholangiocarcinoma.ResultsWestern results showed that TROP2knockdown decreased the nuclear β-catenin level but not total β-catenin. TOP/FOP luciferase reporter showed that β-catenin/TCF transcription activity was down-regulated after TROP2knockdown in HBDC. Mutant beta-catenin (S37A) overexpression in TROP2knockdown HBDC cells could rescue the related functions of TROP2on proliferation, self-renewal and angiogenesis.ConclusionsTROP2promoted tumor growth and angiogenesis through activating wnt/β-catenin signaling pathway, which shed light on therapeutic treatment for hilar cholangiocarcinoma.