담관암에서 성장인자-매개 암 진행과 전이
Growth Factor-mediated Tumor Progression and Metastasis in Cholangiocarcinoma
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Trans Abstract
Different ligands can lead to the activation of epidermal growth factor receptor, and the subsequent signal transduction leads to an increase in cellular motility, proliferation, invasion, and blocking of apoptosis, and all of this contributes to the development and progression of cancer. Our studies clarified; 1) The EGFR expression level is reduced during tumor progression or during ligand-induced EGFR activation. 2) ANXA8 and its regulatory pathway may well be alternative, strategic targets for inhibiting tumor metastasis. 3) The molecular pathway of EphA2 signaling and provide insight into the mechanisms that control cancer progression and metastatic spread in cholangiocarcinoma (CC). Therefore, therapeutic strategies that target EphA2 and its downstream effectors may be useful to control CC.
INTRODUCTION
Human cholangiocarcinoma (CC) is a highly malignant, generally fatal neoplasm originating in the bile duct epithelial cells or cholangiocytes of the intra- and extrahepatic biliary system. The prognosis for CC is quite poor because of difficulties in early diagnosis, and relative resistance of the tumors to chemotherapy [1,2]. At the time of diagnosis, approximately 70% of CC patients have an occult metastasis or advanced local disease that precludes curative resection [3]. Mortality rates have risen sharply in recent years and because CC has no proven adjuvant therapy, only surgical resection with tumor- free margins is associated with improvement in survival, with 5-year survival rates in the range of 20-40% [4]. However, resection is precluded by distant metastases, extensive regional lymph node metastasis, and vascular encasement or invasion.
Abnormal epidermal growth factor (EGF) and EGF receptor (EGFR) signaling can also participate in the genesis and progression of CC [5]. There is accumulating evidence implicating EGF and EGFR signaling pathways that regulate or mediate the epithelial-to-mesenchymal transition (EMT) in the development of CC [6,7]. However, reduced EGFR expression and activation was shown to correlate with a lack of differentiation in CC, even in CC with an altered EMT [8]. The molecular mechanism underlying tumor progression, by which tumor cells acquire the phenotypes of invasiveness and metastasis, is therefore still unclear in CC.
The current chemotherapy regimen that is used to treat CC has very limited benefit in terms of patient survival. Sarcomatoid (SC) CC is predominantly composed of spindle cells, giant cells, and bizarre cells and is believed to have an epithelial origin with transformation to the sarcomatous type. The sarcomatoid change in CC, referred to as the EMT, contributes to a more aggressive intrahepatic spread and widespread metastasis, and CC with a sarcomatoid change has a poorer prognosis than ordinary CC [9,10].
GENOME-WIDE EXPRESSION PATTERNS ASSOCIATED WITH ONCOGENESIS AND SARCOMATOUS TRANSDIFFERENTATION OF CHOLANGIOCARCINOMA
1. Gene expression patterns distinguish CC cells from cultured normal biliary epithelial (NBE) cells
Using BeadChip microarray analysis, we compared the gene expression profiles of nine CC cell lines, an immortalized biliary epithelial cell line, and four types of NBE cells. We selected 828 unique genes with a 2-fold or greater expression difference from the mean, with a p < 0.01 by t-test. Unsupervised hierarchical clustering analysis of all samples was based on the similarity in the expression pattern of all genes (Fig. 1). Cell samples were separated into two main groups, the NBE cluster, and the transformed and immortalized biliary epithelial cells (cholangiocarcinoma cells, CCC cluster). Each distinctive gene cluster was identified by delineation using a hierarchical clustering dendrogram. Cluster I consisted of genes upregulated in CC cells, which included tumor-related genes such as LGR4, AGR2, PCAF, TMEM97, FRAT2, EFNB2 and ZIC2. Cluster II included genes underexpressed in CC cells. These were mainly tumor suppressor genes such as GREM1, THY1, STC2, SERPINE1, SPARC and TAGLN. Cluster III was genes upregulated in NBE cells, and contained the PDGFRA, CD248, and BDKRB1 genes.
2. Gene expression patterns distinguish CC tissues from cultured NBE cells
Using BeadChip microarrays, gene expression profiles of 19 CC tissues and 4 types of NBE cells were compared. We selected 1,798 unique genes with 2-fold or greater differences from the mean difference with a p < 0.01 by t-test. Unsupervised hierarchical clustering analysis was as described above (Fig. 2A). All samples separated into two main groups, NBE and CC tissues (CCT). Each distinctive gene cluster was identified using a hierarchical clustering dendrogram as above. Intriguingly, the CC sample cluster was divided into two subclasses by tumor differentiation: differentiated (Df) and undifferentiated (Udf). Clustering data for the CC group revealed three clusters. Cluster I had genes upregulated in NBE and downregulated in CCT including SERPINB2, PAPPA, LRRC17, and GREM1. Cluster II contained genes upregulated in the Df CCT and downregulated in NBE. Cluster III included genes upregulated in poorly differentiated or Udf CCT, and downregulated in NBE. A supervised hierarchical clustering analysis was performed between the NBE class, and the Df and the Udf subclasses based on the similarity of expression pattern of all genes (Fig. 2B, C).
3. Differential expression and verification of CC-related genes
We compared the gene lists from the cell-based and tissue-based databases, and selected 342 commonly regulated genes, including 53 commonly upregulated genes and 289 commonly downregulated genes (Fig. 3A). To verify the microarray data, we examined the mRNA levels of the identified genes using real-time reverse transcription polymerase chain reaction (RT-PCR) in human CC tissues. We selected five up-regulated genes from the commonly upregulated genes of both the cell and tissue sample classes (Fig. 3B). We also chose the IRX3, PTTG1, and PPARγ genes, which were highly upregulated in only the cell sample class. These genes were preferentially expressed in CC cells and tissues. We also examined the expression of the commonly downregulated KRT17 and UCHL1 genes, as well as the cellular downregulated IGFBP7 and SPARC genes using real-time RT-PCR in human CC. The human NBE showed substantial expression of CK-17, UCHL1, IGFBP7 and SPARC, which were barely detected in CC tissues (Fig. 3C).
4. Gene expression patterns distinguish the sck cell line from three CC cell lines
Previously, we established four human CC cell lines and characterized one with a typical sarcomatoid phenotype of SCK. We classified the other cell lines according to tumor cell differentition, as a poorly differentiated JCK, a moderately differentiated Cho-CK, and a well-differentiated Choi-CK cell line [11]. Two-way unsupervised hierarchical clustering analysis of quadruplicate samples for each cell line was conducted, based on the similarity of expression patterns of all genes (Fig. 4A). We selected 559 unique genes whose expression differed from the mean by four-fold or more with p < 0.005 by t-test. Cell samples were separated into two main groups, SC and ordinary or adenocarcinomatous CC (AC), by the gene axis. Genes in cluster I appeared to be downregulated in the SCK cells, compared to ordinary CC cells. In contrast, the genes in cluster II were upregulated in the SCK cells and downregulated in the ordinary CC cells. Cluster I contained the GSTT1, TACSTD, BST2, RAB25, and MAL2 genes. Cluster II contained genes associated with tumor progression and metastasis, including HOXA9, MUC13, and members of the GAGE and CT-45 families. Expression of methylation-silenced genes, such as LDHB, BNIP3, UCHL1, and NPTX2, was barely detectable in the AC group, but appeared in this cluster.
From 559 genes that were differentially regulated between SCK cells and the three ordinary CC lines, we selected six upregulated genes and six downregulated genes, and examined their mRNA expression using real-time RT-PCR (Fig. 4B), which verified the differential expression. We examined protein expression by Western blot analysis of the four CC lines. LDHB, Bnip3, HO-1, and UCHL1 were overexpressed exclusively in SCK cells. The expression of VIM and TWIST1 increased according to tumor dedifferentiation and was highest in SCK cells (Fig. 4C, left). In contrast, LCN2, S100P, KRT7, KRT19, GPX1, and EFNA1 were preferentially expressed in Choi-CK, Cho-CK and JCK cells, but minimally expressed in SCK cells (Fig. 4C, right).
In this study, our experimental design primarily investigated the gene expression profiles of 10 cell lines and 19 CC tissues, and compared these profiles to those from four cultured NBE cell line using genome-wide BeadChip microarray analysis. Gene expression profiling appears to be a useful diagnostic tool, especially for differentiating CC from other liver masses, as well as for the subclassification of intrahepatic CC compared to histopathological findings [12].
ANXA8 DOWN-REGULATION BY EGF-FOXO4 SIGNALING IS INVOLVED IN CELL SCATTERING AND TUMOR METASTASIS OF CHOLANGIOCARCINOMA
1. Down-regulation of ANXA8 and EGFR in CC cells
We previously established four distinct CC cell lines (SCK, JCK1, Cho-CK, and Choi-CK) and we analyzed their chromosomal aberrations by cross-species color banding [11,13]. The SCK, JCK and Cho-CK cells were originally derived from a peripheral type of intrahepatic CC. The Choi-CK cells were cultured from a hilar type of infiltrative CC. One of them (SCK) was derived from the sarcomatoid CC.
We assessed ANXA8 and EGFR protein expression according to tumor dedifferentiation in 3 CC cell lines and found that the expression was abruptly down-regulated with poor differentiation and sarcomatoid changes in JCK and SCK cells, respectively, as compared with the well and moderate differentiation in Choi-CK and Cho-CK cells, respectively (Fig. 5A). The expression of phosphorylated EGFR was proportional to the expression of EGFR. We next investigated whether the growth factors could induce cell phenotypic changes in the well-differentiated Choi-CK cells. EGF, but not hepatocyte growth factor or TGF-β, effectively induced rounding, refractile morphologic changes, and cell detachment (Fig. 5B). The down-regulation of EGFR and ANXA8 protein expression by exogenous EGF was time and dose dependent (Fig. 5C). To confirm the finding of transcriptional regulation of EGFR and ANXA8 by EGF, the ER1-luc and ANXA8-luc reporter plasmids were transiently transfected and assayed in Choi-CK cells treated with EGF. EGF effectively reduced the levels of EGFR and ANXA8 promoter activity to 61% and 43% of vehicle controls, respectively (p = .005) (Fig. 5D).
2. Transcriptional Regulation of ANXA8 by FOXO4
Insulin or growth factor inhibits the transcriptional activity of FOXO4 by phosphorylation via phosphatidylinositol-3-kinase (PI3K)/Akt signaling. Therefore, we checked several downstream targets of EGF mediated ANXA8 regulation using an inhibition assay. Inhibiting PI3K and Akt resulted in higher levels of ANXA8 expression than inhibiting c-Jun N-terminal kinase (JNK), p38, and mitogen-activated protein kinases (MAPK)/extracellular signal-regulated kinases (ERK) kinase in Choi-CK cells, and the ANXA8 expression levels were abolished by the addition of EGF (Fig. 6A). PI3K/Akt, and not Ras/Ral, seems to be the major signal downstream of EGF-mediated ANXA8 regulation. The nuclear translocation of FOXO4 is reported to be negatively regulated through phosphorylation by Akt. Our data consistently show that FLAG-FOXO4 are recovered as phosphoproteins from transfected H-69 cells that are metabolically labeled with [32p]orthophosphate in the presence of the wild-type (WT)-Akt, activated (Act)-Akt or dominant negative (Dn)-Akt. FLAG-the triple mutant (TM)-FOXO4 was also processed in the presence of the activated Akt. The results show that FOXO4 is efficiently phosphorylated by WT-Akt and Act-Akt, whereas TMFOXO4, with 3 mutated phosphorylation sites, ie, T28A, S193A, and S258A, did not serve as a substrate (Fig. 6B). EGF increased the PI3K/Akt-induced phosphorylation of FOXO4 in the cytoplasm and simultaneously decreased the nuclear translocation of FOXO4 and subsequent down-regulation of ANXA8 expression, which was inhibited by the PI3K inhibitor wortmannin (Fig. 6C). The TM-FOXO4 increased the luciferase activity up to 5.3-fold, and the mutant of FOXO4, which lacks the DNA-binding domain, decreased the luciferase activity by half, as compared with WT-Akt (Fig. 6D). These findings suggest that FOXO4 and its phosphorylation can positively and negatively regulate ANXA8 promoter activity, respectively. The immunofluorescence data confirm that the ectopically expressed wild-type FOXO4 can translocalize to the nucleus with or without ANXA8 induction, whereas the TM-FOXO4 easily translocates to the nucleus and always induces the expression of ANXA8 (Fig. 6E).
3. ANXA8 Inhibits the Migratory and Metastatic Characteristics of CC Cells
The parental SCK cells and vector control cells (VC1 and VC3) readily penetrated the matrix and colonized the bottom surface of the Matrigel-coated membrane (a modified Boyden chamber assay), whereas the stable transfectants that expressed ANXA8 rarely showed this behavior (Fig. 7A). Next, the role of ANXA8 expression in the metastatic phenotype of the SCK cells was examined by implanting stable transfectants (200 µL of 5 × 106 cells/mL) into the peritoneal cavity and monitoring the lethality over a 120-day period. The parental (SCK) and vector control cells that were inoculated intraperitoneally into the nude mice produced a distention of the abdomen, cachexia, and protruding abdominal masses. The mice bearing these tumors invariably died within 80 days. The stable ANXA8 transfectants produced no mortality until 80 days after intraperitoneal inoculation (Fig. 7B). A necropsy revealed that the parental cells or the vector control cells extensively colonized the visceral organs and formed multiple metastatic masses with bloody ascites. In contrast, the stable ANXA8 transfectants first colonized and then continued growing into the peritoneum with only rare metastatic nodules (Fig. 7C).
In conclusion, ANXA8 is transcriptionally down-regulated by EGF-mediated FOXO4 phosphorylation and that the subsequent deregulation of FAK and F-actin dunamics is responsible for the morphology of EMT. Therefore, ANXA8 is functionally related to tumorigenesis, whereas its down-regulation is essential for tumor progression and metastasis.
ACTIVATION OF MAMMALIAN TARGET OF RAPAMYCIN COMPLEX 1 (MTORC1) AND RAF/PYK2 BY GROWTH FACTOR-MEDIATED EPH RECEPTOR 2 (EPHA2) IS REQUIRED FOR CHOLANGIOCARCINOMA GROWTH AND METASTASIS
1. EphA2 Expression According to the Level of Differentiation in CC
We previously described four CC cells with varying levels of differentiation, including well differentiated Choi-CK, moderately differentiated Cho-CK, poorly differentiated JCK, and sarcomatoid SCK cells. EphA2 expression was more prominent in less-differentiated CC cells, such as JCK and SCK cells (Fig. 8A). However, both of these cell types showed lower expression levels of the known EphA2 ligand, EFNA1, as well as EGFR, compared to the better-differentiated Choi-CK and Cho-CK cells. Accordingly, EphA2 transcript levels were elevated in poorly differentiated JCK and SCK cells. IHC examination of EphA2 expression in CC tissues revealed that normal bile duct epithelial cells were negative or barely positive for EphA2 immunoreactivity. As expected, the poorly differentiated CC tissues were more strongly positive for EphA2 than the better-differentiated CC tissues (Fig. 8B). These results suggest that EphA2 expression correlates with poorer differentiation and more progression in CCs. Among the various growth factors tested, EGF induced EphA2 expression and reciprocal inhibition of EFNA1 in CC cells (Fig. 8C, upper panels). Therefore, EGF seems to transcriptionally regulate EphA2 and EFNA1. The EGF-mediated morphological changes were consistent with EMT changes (Fig. 8D), as we previously described. Among the cells treated with various growth factors, the promoter activity of EphA2 was highest in the EGF-treated cells (Fig. 8E). EGF dose-dependently induced EphA2 overexpression and phosphorylation on S897, but not on Y594 (Fig. 8F). These results suggest that aberrant expression of EphA2 resulting from abnormal growth factor signaling is crucial for tumor promotion and metastasis and this abnormal growth factor signaling contributes to the down-regulation of EFNA1 and EGFR.
2. Morphological Changes Caused by EphA2
Overexpression of EphA2 is correlated with disease progression and highly malignant cellular phenotypes. We therefore investigated the morphological changes induced by EphA2 overexpression in Cho-CK cells. EphA2 expression was examined in stably transfected Cho-CK cells by immunoblot analysis (Fig. 9A). The stable transfectants expressing EphA2 (A2-20 and A2-22) showed a more migratory and less adhesive phenotype compared to the vector control cells (VC4 and VC10) (Fig. 9B). To assess the ligand-mediated EphA2 expression, we treated the SCK cells with recombinant mouse ephrin-A1 Fc ligand and found that it efficiently down-regulated EphA2 expression in a dose-dependent manner compared to treatment with recombinant human IgG1-Fc. EGF treatment resulted in an increase in EphA2 expression and phosphorylation at S897, but not Y594. In contrast, the ephrin-A1 Fc ligand decreased EphA2 expression, but increased phosphorylation of EphA2 at Y594 (Fig. 9C). The cell morphology changed from fibroblastoid type to epitheloid type, i.e., reverse EMT (mesenchymal-eipthelial trasition) (Fig. 9D). Alternatively, we examined the F-actin staining of stress fibers with fluorescent phalloidin in the EphA2-expressing cells treated with either recombinant IgG1-Fc or recombinant ephrin-A1 Fc ligand. Treatment with recombinant ephrin-A1 Fc ligand resulted in repressed expression of EphA2 and the formation of F-actin stress fibers (Fig. 9E).
3. Enhanced Tumorigenicity of EphA2
In order to determine if human EphA2 has pro-oncogenic properties, the capacity of EphA2 to anchor independent cell growth was compared to vector control cells by determining the level of colony growth on soft agar. The stable EphA2 transfectants formed a greater number of larger colonies on soft agar (Fig. 10A). Furthermore, tumor growth was orthotopically or subcutaneously faster in the mouse tumor model with the stable transfectants than with vector control cells (Fig. 10B). These results suggest that EphA2 plays a crucial role in tumor growth in vitro and in vivo. In addition, intrahepatic tumor metastases were more enhanced in orthotopic xenoplants from the stable EphA2 transfectants than from the vector control cells.
4. Migratory and Metastatic Abilities Induced by EphA2
The EMT of epithelial cancers is correlated with aggressive tumors that are believed to play a critical role in metastatic cancer. We therefore examined whether EphA2 overexpression contributed to tumor invasiveness and metastasis by affecting the EMT phenomena in CC cells. We found that adhesion molecules such as E-cadherin and desmoplakin I/II were down-regulated, and locomotion molecules, including fibronectin, vimentin (VIM), N-cadherin, and alpha smooth muscle actin, were up-regulated in the cells stably expressing EphA2 (Fig. 11A). Wound repair assays were performed on Cho-CK cells stably expressing EphA2, and the stable transfectants demonstrated a visibly enhanced capacity to migrate into the wounded area compared to the vector control cells (Fig. 11B). In a modified Boyden chamber assay, the stable transfectants readily penetrated the matrix and colonized the bottom surface of the Matrigelcoated membrane, while the vector control cells did not (Fig. 11C). Therefore, EphA2 expression appeared to enhance the invasiveness of these cells. The role of EphA2 expression in the metastatic phenotype of the Cho-CK cells was examined by first injecting stable transfectants (100 µL of 5 × 105 cells/mL) into the tail vein of nude mice and subsequently monitoring multiple metastatic nodules in the lungs. The stable EphA2 transfectants first colonized and then continued growing into the lungs with many more metastatic nodules (black arrowhead) than were achieved with the vector control cells (Fig. 11D). A histocytological examination confirmed these results (Fig. 11E). Many metastatic nodules strongly expressed EphA2 in the lungs.
5. Downstream Signaling of EphA2
Next, we sought to identify the phosphorylation of downstream molecules using Human Phospho-Kinase Arrays (Fig. 12A). EphA2 overexpression led to phosphorylation of Akt and Pyk2, as confirmed by immunoblot and immunoprecipitation, respectively (Fig. 12B). Akt phosphorylation occurred at T308 and Pyk2 at Y402 in both JCK and SCK cells. However, other phosphorylation sites, S473 of Akt and Y881 of Pyk2, were not affected by EphA2. Mammalian target of rapamycin (mTOR) is known to function downstream of Akt as part of the mTOR complex 1 (mTORC1) protein complex.28,29 We therefore investigated the mTORC1 pathway in stable EphA2 transfectants of the four CC cell lines. EphA2 overexpression resulted in phosphorylation of mTORC1, 4EBP1, and p70S6K in JCK and SCK cells (Fig. 12C). EphA2 overexpression also led to c-Src phosphorylation (Fig. 12D). The knockdown experiments revealed that EphA2 knockdown itself resulted in EFNA1 induction and was linked to the inhibition of canonical upstream activators of ERK signaling, including b/c-Raf and MEK (Fig. 12E).
6. Proliferation and Metastatic Ability of Akt/mTORC1 and c-Src Activation
In the wound healing assay, 4-Amino-3-(4-chlorophenyl) -1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine (PP2) completely inhibited EphA2-mediated wound closure. Rapamycin, however, caused only a partial inhibition of wound repair (Fig. 13A). The Matrigel-coated transwell invasion assay similarly showed that PP2 completely inhibited invasion of Matrigel pores, while rapamycin provided only partial inhibition (Fig. 13B). These results suggest that the Akt/mTORC1 pathway plays a major role in regulating cell proliferation while the Pyk2/c-Src pathway generally acts to modulate metastatic ability. However, the Akt/mTOR and Pyk2/c-Src pathways also appeared to substantially influence each other. We further examined the morphological changes occurring in the stable transfectants expressing EphA2 after rapamycin or PP2 treatment. Rapamycin partially inhibited the migratory characteristics and stress fiber formation, while PP2 completely suppressed these phenomena (Fig. 13C).
To exclude the cell-specific function of EphA2 we used metastasis-prone CC cells, JCK, to determine whether EphA2 expression is critical for metastasis of CC cells because these cells express high levels of EphA2. EphA2 knockdown cells were established by lentiviral delivery of EphA2 shRNA with stable expression of luciferase (JCK-luc) (Fig. 14A). The metastatic phenotype of the JCK-luc cells was examined by injection into the tail veins (100 µL of 5 × 105 cells/mL) of nude mice. Sufficient bioluminescence data were collected at 4 weeks postinjection (Fig. 14B). The parent and non-target cells first colonized and then continued growing into the lungs with many metastatic nodules (Fig. 14C). A histological examination demonstrated that injection of the parent and nontarget cells resulted in numerous EphA2-positive metastatic lesions, and the target cell injection resulted in EphA2-negative and fewer metastatic nodules. These results suggest that EphA2 expression plays a major role in tumor metastasis and invasion in CC cells.
In addition to the canonical Raf/MEK activation, overexpression of EphA leads to activation of Akt and Pyk2 by phosphorylation. In turn, activated Akt and Pyk2 phosphorylate mTORC1 and c-Src, respectively. The activation of molecules downstream of mTORC1 and ERK contribute to tumor growth and/or metastasis in CCs (Fig. 14D) [14].
THERANOSTICS-BASED IDENTIFICATION OF TARGET MOLECULES FOR DIAGNOSIS AND THERAPY IN CHOLANGIOCARCINOMA
To control cholangiocarcinoma growth and metastasis, we screened EphA2 kinase inhibitor by homogeneous time resolved fluorescence (HTRF). Thirty substances were founded and we validated these substances related to inhibit cell proliferation and cell death. Human cholangiocarcinoma cell lines, Choi-CK, Cho-CK, JCK and SCK were treated various concentration of EKI, EphA2 kinase inhibitor (1, 2.5, 5, 10, 25 µM) for 24h. At 1 and 2.5 µM of EKI, proliferation of these cells was inhibited. At 5 µM of EKI, cell death was induced in these cells. As quantitatively assessed by flow cytometry, apoptosis was induced by EKI in CC cells. We investigated apoptotic signaling by Western blot and observed cleavage or overexpression of pro-caspase-3 and PARP in EKI treated cells compared with vehicle. We investigated EKI up-or down-signal transdection pathways in cholangiocarcinoma to use therapeutic target for CC. Therefore, the EphA2 kinase inhibitor EKI is therapeutic target for cholnagiocarcinoma.
Notes
Conflict of Interest
The author has no conflict to disclose.