The Mechanism Of FBXW7 Suppressing Epithelial-mesenchymal Transition, Stemness And Metastatic Potential In Cholangiocarcinoma Cells

Cholangiocarcinoma (CCA), the second most common primary hepatobiliary malignancy, is an epithelial cell malignancy originating from the bile ducts. The most contemporary classification of CCA based on anatomical location includes intrahepatic (IHCC), perihilar (PHCC) and distal (DCC) CCA. The incidence and mortility rates of CCA are increasing worldwide. Because of its invasive characteristic, more than two thirds CCA patients are diagnosed with advanced stage and have no access to surgical resection. Besides, the effectiveness of adjuvant therapeutic strategies needs further investigation till now. Overall, the median survival time of CCA patients is only 15 months. Therefore, it is essential to further explore the tumorigenic and developmental mechanisms and find new diagnostic markers and effective molecular targets for improving the prognosis of CCA patients.FBXW7 (F-box and WD repeat domain containing 7), a member of the F-box family of proteins, is a substrate recogniton component of the Skp 1-Cul 1-F box (SCF) protein ubiquitin ligase complex. It has been shown to mediate the ubiquitin-dependent proteolysis of several well-known oncoproteins, including Notch, cyclin E1, mammalian target of rapamycin (mTOR), c-Myc and c-Jun. Furthermore, FBXW7 governs diverse cellular processes, including cell-cycle progression, cell proliferation, differentiation, DNA damage response and maintenance of genomic stability. Generally, FBXW7 is regarded as a tumor suppressor and a potential therapeutic target in human cancers. However, the function of FBXW7 in tumor metastasis is rarely reported, and whether it plays a role in CCA metastasis remains unknown.CCA is characterized by its invasive potential and metastasis in early stage, which is one of the most important reasons influencing patient’s prognosis. Increasing evidence has revealed tumor metastasis was correlated with epithelial-mesenchymal transition (EMT). The process of EMT invovles profound phenotypic changes that include loss of cell-cell adhesion, cell polarity and acquisiton of migratory and invasive properties. The cellular mesenchymal state is believed to be associated with the capacity of cells migrating to distant organs and maintaining sternness, which allows their subsequent differentiation into multiple cell types during initiation and development of metastasis. However, few studies have investigated EMT mechanisms in CCA. Thus, uncovering the regulatory mechanisms of EMT should provide greater insight into the signaling programs that govern metastasis in CCA.With respect to these notions, the present study investigated the functional role of FBXW7 regulating EMT, sternness and metastatic potential of CCA cells, and corresponding molecular mechanisms were also explored. Our results not only further verified the metastatic mechanisms of CCA, but also identified FBXW7 pathway may serve as a potential target for CCA diagnosis and treatment.Part I The correlation of E3 ubiquitin ligase FBXW7 and CCA clinicopathologic characteristics[Objective]To verify the functional role of FBXW7 in CCA, the expression of FBXW7 in CCA cells and tissues were detected. Furthermore, the correlation of FBXW7 and CCA clinicopathologic characteristics was analyzed.[Materials and Methods]1. The mRNA and protein levels of FBXW7 were detected by qRT-PCR and Western blot in human intrahepatic bile duct epithelial cell (HIBEpic), IHCC and PHCC cells.2. Collecting 7 paired CCA and adjacent nontumorous bile duct fresh tissues obtained from CCA patients undergoing curative surgical resention of the primary tumor at Qilu Hospital of Shandong University. The expression of FBXW7 in tumor adjacent nontumorous bile duct tissues and primary tumor tissues was analyzed by Western blot.3. Collecting the paraffine-embedded IHCC tissues and adjacent nontumorous bile duct tissues obtained from IHCC patients undergoing curative surgical resection of the primary tumor at Qilu Hospital of Shandong University, and paraffin section was made. The expression of FBXW7 in tumor adjacent nontumorous bile duct tissues, primary tumor tissues without metastasis and primary tumor tissues with metastasis was analyzed by immunohistochemistry (IHC), and the correlation of FBXW7 expression level with tumor size, metastasis, differentiation and TNM stage was statistically analyzed.4. Collecting the paraffine-embedded PHCC tissues and adjacent nontumorous bile duct tissues obtained from PHCC patients undergoing curative surgical resection of the primary tumor at Qilu Hospital of Shandong University, and paraffin section was made. The expression of FBXW7 in tumor adjacent nontumorous bile duct tissues, primary tumor tissues without metastasis and primary tumor tissues with metastasis was analyzed by IHC, and the correlation of FBXW7 expression level with tumor size, metastasis, differentiation and TNM stage was statistically analyzed.5. Collecting the paraffine-embedded DCC tissues and adjacent nontumorous bile duct tissues obtained from DCC patients undergoing curative surgical resection of the primary tumor at Qilu Hospital of Shandong University, and paraffin section was made. The expression of FBXW7 in tumor adjacent nontumorous bile duct tissues, primary tumor tissues without metastasis and primary tumor tissues with metastasis was analyzed by IHC, and the correlation of FBXW7 expression level with tumor size, metastasis, differentiation and TNM stage was statistically analyzed.[Results]1. The mRNA and protein levels of FBXW7 in HIBEpic were revealed to be higher than in IHCC cells (HuCCT1 and RBE) and PHCC cells (QBC939 and FRH0201) by qRT-PCR and Western blot, respectively.2. Seven paired tumor adjacent nontumorous tissues and corresponding tumor tissues were collected, and tumor tissues exhibited higher FBXW7 expression level than tumor adjacent nontumorous tissues in 6 cases on Western blot assay.3. IHC staining confirmed the downregulation of FBXW7 in cancer tissues compared with tumor adjacent nontumorous tissues in IHCC. Furthermore, correlation analysis of FBXW7 protein level with clinicopathologic features revealed significant association between FBXW7 deficiency and tumor size, metastasis, TNM stage and differentiation in IHCC.4. IHC staining confirmed the downregulation of FBXW7 in cancer tissues compared with tumor adjacent nontumorous tissues in PHCC. Furthermore, correlation analysis of FBXW7 protein level with clinicopathologic features revealed significant association between FBXW7 deficiency and metastasis, TNM stage and differentiation in PHCC.5. Correlation analysis of FBXW7 protein level with the clinicopathologic features of DCC revealed no significant association between FBXW7 deficiency and tumor size, metastasis, TNM stage and differentiation.[Conclusions]1. The expression of FBXW7 in HIBEpic was much higher than in the IHCC and PHCC cells, and its expression level in tumor adjacent bile duct tissues was also higher than in CCA tissues, which indicated that FBXW7 may be a tumor suppressor in CCA.2. FBXW7 could inhibit the metastasis of IHCC and PHCC, morever, it is correlated with tumor differentiation and TNM stage.Part II Role of FBXW7 in epithelial-mesenchymal transition, stemness and metastatic potential of CCA cells[Objective]Previous studies have demonstrted that FBXW7 could inhibit CCA metastasis and was correlated with tumor differentiation and TNM stage in IHCC and PHCC. In this section, the function of FBXW7 on CCA EMT and metastasis will be further investigated both in vivo and in vitro.[Materials and Methods]1. Two shRNAs against human FBXW7 (pBabe-shFBXW7.1 and pBabe-shFBXW7.2) expressed in pBabe.puro vector were constructed. FBXW7 stable silencing cells and their control vector cells were established retrovirally. To verify whether the stable transfected cells were successfully established, qRT-PCR and Western blot were used to examine the level of FBXW7.2. pBabe.puro retroviral construct containing human FBXW7 cDNA was prepared. FBXW7 stable overexpressed cells and their control vector cells were established retrovirally. To verify whether the stable transfected cells were successfully established, qRT-PCR and Western blot were used to examine the level of FBXW7.3. To investigate the function of FBXW7 on CCA EMT, the morphological changes of FBXW7 silencing cells and overexpressed cells were detected with light microscrope. Furthermore, the expression of epithelial markers (E-cadherin and a-catenin) and mesenchymal markers (Fibronectin and Vimentin) were analyzed by Western blot.4. To detect the role of FBXW7 on CCA cancer stem cells (CSCs), the expression of CSC related markers (Nanog and OCT4) were examined in FBXW7 silencing cells and overexpressed cells. Besides, the self-renewal ability of these cells was also investigated with primary sphere formation and secondary sphere formation assay.5. To explore the funtion of FBXW7 on CCA motility, wound healing assay, Transwell assay and Matrigel assay were carried out in FBXW7 deficient and overexpressed cells.6. Collecting the paraffine-embedded IHCC tissues and adjacent nontumorous bile duct tissues obtained from IHCC patients undergoing curative surgical resection of the primary tumor at Qilu Hospital of Shandong University, and paraffin section was made. The expression of E-cadherin in tumor adjacent nontumorous bile duct tissues, primary tumor tissues without metastasis and primary tumor tissues with metastasis was analyzed by IHC, and its correlation with FBXW7 expression was statistically analyzed.7. Collecting the paraffine-embedded PHCC tissues and adjacent nontumorous bile duct tissues obtained from PHCC patients undergoing curative surgical resection of the primary tumor at Qilu Hospital of Shandong University, and paraffin section was made. The expression of E-cadherin in tumor adjacent nontumorous bile duct tissues, primary tumor tissues without metastasis and primary tumor tissues with metastasis was analyzed by IHC, and its correlation with FBXW7 expression was analyzed.8. To investigate the function of FBXW7 on tumor metastasis in vivo, distant metastatic models of nude mice were establisded with FBXW7 deficient and overexpressed cells, and tumor metastasis of the nude mice was detected.9. To further verify whether FBXW7 could inhibit CCA EMT in vivo, IHC was performed to detect the expression of FBXW7 and E-cadherin in the metastic tumors of the nude mice, and their relationship was analyzed.[Results]1. Two shRNAs against human FBXW7 (pBabe-shFBXW7.1 and pBabe-shFBXW7.2) expressed in pBabe.puro vector were constructed. Then, FBXW7 stable silencing cells and their control vector cells (named as HuCCT1-Vector, HuCCT1-shFBXW7.1、 HuCCT1-shFBXW7.2; RBE-Vector、RBE-shFBXW7.1、RBE-shFBXW7.2) were established retrovirally. qRT-PCR and Western blot verified the successful establishment of the stable transfected cells by examining the mRNA and protein levels of FBXW7.2. pBabe.puro retroviral vector containing human FBXW7 cDNA was successfully constructed. FBXW7 stable overexpressed cells and their control vector cells (named as QBC939-Vector、QBC939-FBXW7; FRH0201-Vector、FRH0201-FBXW7) were established retrovirally. qRT-PCR and Western blot verified the successful establishment of the stable transfected cells by examining the mRNA and protein levels of FBXW7.3. It was obviously observed FBXW7 inhibited EMT by comparing the morphalogical type of FBXW7 deficient and overexpressed CCA cells with corresponding control cells with light microscrope. Moreover, Western blot revealed that FBXW7 could increase the expression epithelial markers (E-cadherin and a-catenin) and reduce the expression of mesenchymal markers (Fibronectin and Vimentin) in CCA cells.4. The expression of CSC markers (OCT4 and Nanog) were significantly upregulated in FBXW7 downregulated cells on Western blot assay, accompanied with increased self-renewal ability as shown on primary sphere formation assay and secondary sphere formation assay. Meanwhile, FBXW7 overexpressed cells showed significant reduction of CSC markers (OCT4 and Nanog) on Western blot assay, and their self-renewal abilities were also decreased simultaneously.5. It was also been demonstrated that FBXW7 could inhibit CCA migration and invasion in vitro by wound healing assay, Transwell assay and Matrigel assay.6. Statistical analysis revealed that the expression of E-cadherin was statistically correlated with tumor metastasis, and has a signigficantly positive correlation with FBXW7 expression in IHCC.7. Statistical analysis revealed that the expression of E-cadherin was statistically correlated with tumor metastasis, and has a signigficantly positive correlation with FBXW7 expression in PHCC.8. FBXW7 could inhibit the metaststic potential of CCA cells in vivo.9. FBXW7 could suppress EMT of CCA cells in vivo.[Conclusions]1. FBXW7 could inhibit EMT of CCA cells both in vitro and in vivo.2. FBXW7 could reduce the stem-like capacity of CCA cells in vitro.3. FBXW7 could suppress the metastatic potential of CCA cells both in vitro and inPart Ⅲ The molecular mechanism of FBXW7 suppressing EMT, stemness and metastatic potential of CCA cells[Objective]It has been confirmed that FBXW7 could inhibit EMT and metastasis of CCA cells. However, the molecular mechanism of FBXW7 regualting EMT in CCA has not been identified, which will be explored in following studies.[Materials and Methods]1. The expression of mTOR and p-mTOR were detected in FBXW7 deficient and overexpressed CCA cells to verify the role of FBXW7 in the ubiquitin-dependent degradation of mTOR.2. FBXW7 deficient CCA cells and their control vector cells were treated with rapamycin, a mTOR inhibitor. And then, the expression of EMT related markers (E-cadherin and Vimentin) were examined by Western blot. Furthermore, primary sphere formation assay and secondary sphere formation assay were carried out to detect the self-renewal ability of these cells. Besides, migratory capacity of these cells was confirmed on wound healing assay and Transwell assy, and invasive ability was investigated by Matrigel assay.3. FBXW7 deficient CCA cells and their control vector cells were treated with rapamycin firstly. Then, the mRNA and protein levels of EMT transcriptional regualtors, including Snail, Slug and ZEB1, were analyzed by qRT-PCR and Western blot, respectively.4. The most essential EMT transcriptional factor was chosen for further study. To verify its role in CCA, its expression was detected in HIBEpic and three CCA cells. To further reveal the funtional role of the EMT transcriptional factor in CCA and FBXW7 regulated CCA metastasis, shRNA was used to silence its expression in CCA cells and FBXW7 deficient CCA cells. Furthermore, the expression of EMT related markers (E-cadherin and Vimentin) were examined by Western blot. The migratory capacity was confirmed on wound healing assay and Transwell assay, invasive ability was investigated by Matrigel assay.5. Collecting the paraffine-embedded IHCC tissues and adjacent nontumorous bile duct tissues obtained from IHCC patients undergoing curative surgical resection of the primary tumor at Qilu Hospital of Shandong University, and paraffin section was made. The expression of the EMT transcriptional factor in tumor adjacent nontumorous bile duct tissues, primary tumor tissues without metastasis and primary tumor tissues with metastasis was analyzed by IHC, and its correlation with FBXW7 and E-cadherin expression was statistically analyzed.6. Collecting the paraffine-embedded PHCC tissues and adjacent nontumorous bile duct tissues obtained from PHCC patients undergoing curative surgical resection of the primary tumor at Qilu Hospital of Shandong University, and paraffin section was made. The expression of the EMT transcriptional factor in tumor adjacent nontumorous bile duct tissues, primary tumor tissues without metastasis and primary tumor tissues with metastasis was analyzed by IHC, and its correlation with FBXW7 and E-cadherin expression was statistically analyzed.7. The role of rapamycin on tumor metastasis of the nude mice models established with FBXW7-silenced cells was also examined to investigate whether mTOR could mediate FBXW7 regulated CCA metastasis in vivo.8. To investigate whether mTOR could regulate FBXW7 mediated CCA EMT in vivo, IHC was carried out to detect FBXW7 and E-cadherin expression of the metastatic tissues obtained from the nude mice models established by FBXW7-silenced cells with/without rapamycin treatment.[Results]1. The expression of FBXW7 was negatively correlated with both mTOR and p-mTOR in CCA cells on Western blot assay. Combined with previous litratures, it was confirmed that mTOR could be degraded by FBXW7 mediated ubiquitination in CCA cells.2. Rapamycin could suppress the reduction of E-cadherin and upregulation of Vimentin mediated by FBXW7 silencing. FBXW7 silencing induced functional changes in tumor sphere formation capacities, migratory and invasive behaviors of CCA cells were also eliminated with rapamycin treatment.3. qRT-PCR and Western blot revealed that rapamycin could inhibit the increase of EMT transcriptional factors, including Snail, Slug and ZEB1, caused by FBXW7 deficiency.4. To our known, the role of ZEB1 in CCA has not been identified previously. Therefore, the function of ZEB1 in CCA and FBXW7/mTOR signaling pathway mediated CCA EMT and metastasis was further investigated. It was detected that ZEB1 mRNA and protein levels in HIBEpic were much lower than in CCA cells. Morever, by transiently silencing ZEB1 with pSingle-shZEB1.1, pSingle-shZEB1.2 and pSingle-Vector in FBXW7 deficient cells, the expression reduction of E-cadherin and upregulation of Vimentin mediated by loss of FBXW7 were eliminated. Besides, the increase of the migratory and invasive potentials mediated by FBXW7 silencing were also reduced with ZEB1 deficiency in CCA cells.5. Statistical analysis showed that ZEB1 expression was significantly correlated with tumor metastasis in IHCC. Moreover, its level was negatively correlated with FBXW7 and E-cadherin.6. Statistical analysis showed that ZEB1 expression was significantly correlated with tumor metastasis in PHCC. Moreover, its level was negatively correlated with FBXW7 and E-cadherin.7. The increase of metastasis in nude mice mediated by FBXW7 deficiency was reduced with rapamycin treatment.8. Rapamycin treatment inhibited EMT mediated by FBXW7 silencing in vivo.[Conclusions]1. mTOR could mediate the EMT, stem-like capacity and metastasis of CCA cells regulated by FBXW7.2. ZEB1 could regulate the FBXW7/mTOR signaling pathway mediated EMT and metastasis in CCA cells.