J Hepatobiliary Pancreat Sci (2014) 21:371–379 DOI: 10.1002/jhbp.66
TOPIC
Molecular aspects of cholangiocarcinoma Kaoru Kiguchi
Published online: 13 January 2014 © 2014 Japanese Society of Hepato-Biliary-Pancreatic Surgery
Abstract Novel targets for therapeutic or chemopreventive approaches against cholangiocarcinoma (CCA) are urgently needed. In this review article, we discuss the molecular aspects of CCA including the role of erbB receptor tyrosine kinases (RTKs), downstream signaling pathways of these erbB RTKs, inflammatory mediators during gallbladder carcinogenesis and bile acids based on our study using a mouse model for human CCA (BK5.erbB2 mice) as well as additional information in the literature. Keywords Cholangiocarcinoma · COX-2 and bile acid · Epidermal growth factor receptor · erbB2 Introduction Cholangiocarcinoma (CCA), which includes gallbladder carcinomas (GBCs) and cancers of the intra- and extrahepatic biliary tree, are relatively infrequent but highly lethal malignancies [1]. Although there have been advances in the diagnosis and management of CCA, these cancers still prove challenging to treat due to their insensitivity to conventional therapies and the inability to detect early tumor formation. These factors render GBC nearly incurable with a 5-year survival rate of only 5–21% [2–6]. Novel targets for therapeutic or chemopreventive approaches are urgently needed. To date, very few studies have addressed the molecular and cellular mechanisms underlying the development of CCA. We previously generated transgenic mice that K. Kiguchi (*) Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin, 1400 Barbara Jordan Blvd., Austin, TX 78723, USA e-mail: [email protected] This article is summarized based on the presentation for International Symposium on Cholangiocarcinoma held in Tokyo, 2013. It is based mostly on our previously published studies using BK5.erbB2 mice as well as additional information in the literature. Some of the text and figures/table are adopted from our previous studies as indicated.
overexpress wild type rat erbB2 in epithelial tissues under the control of the bovine keratin 5 (BK5) promoter (BK5.erbB2 mice) [7]. Overexpression of the erbB2 in basal epithelial cells of the gallbladder led to the development of adenocarcinoma of the gallbladder and cystic duct in 90% of these transgenic mice. This was the first direct demonstration that erbB2 overexpression could lead to the development of CCA [7]. We have shown that BK5.erbB2 mice are a valid model for investigating mechanisms underlying development of GBCs and other CCAs. In this article, we discuss the molecular aspect of CCA including the role of erbB receptor tyrosine kinases (RTKs), downstream signaling pathways of these erbB RTKs, inflammatory mediators during gallbladder carcinogenesis and bile acids based on our study using BK5.erbB2 mice as well as additional information in the literature (review in [8, 9]). Understanding the signaling pathways altered in CCA will provide critical clues for novel therapeutic and chemopreventive strategies using drugs and/or agents that selectively target these specific pathways.
Role of ErbB RTKs and their downstream signaling pathways in the development of CCA EGFR and erbB2 in CCA Upregulation of the RTK, erbB2, contributes to tumorigenesis in a number of tissues, however, little is known about its role in CCA including GBC. ErbB2 overexpression has been reported in a significant percentage of human CCA [6, 10–20]. Similarly, overexpression and activation of epidermal growth factor receptor (EGFR) has been reported in 30–60% of CCA samples [11, 12, 21, 22]. We previously reported BK5.erbB2 transgenic mice, in which erbB2 was targeted to overexpress in epithelial tissues, developed spontaneous GBC and intrahepatic CCA with incidences of approximately 90 and 30%, respectively, by 2 months of age (Fig. 1b) [7]. Figure 1a shows the cDNA construct of BK5.erbB2 mouse. The majority of the
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A
B
C
Wild-type D
E
G
H
BK5.erbB2 F
Fig. 1 (a) DNA construct used to generate BK5.erbB2 mice. (b) Gross appearance and histological evaluations of cholangiocarcinoma (CCA) in BK5.erbB2 mice at 3 months of age. (c) H&E staining of gallbladder in wild type mouse and (left) BK5.erbB2 mouse (right)). (d) The junction of the pancreatico-biliary duct (JPBD) in a 3-month-old BK5.erbB2 mouse. (e) H&E staining of the ampulla of Vater. (f) Intrahepatic CCA from a 3-month-old BK5.erbB2 mouse. (g and h) Expression of erbB2: Immunostaining for erbB2 in gallbladder from BK5.erbB2 (g) and wild type (h) of 3-month-old mouse. Figures are adopted from Kiguchi et al. 2001 [7]
gallbladder tumors completely filled the lumen (Fig. 1c) [7]. Most of the tumors were diagnosed as well-differentiated adenocarcinomas. Carcinoma cells frequently invaded into the surrounding connective tissues. Microscopic analysis of H&E-stained serial sections showed that tumor cells of the common bile duct had invaded into the pancreatic duct (Fig. 1d) [7]. In addition, the ampulla of Vater was dilated, and hyperplasia of the epithelium was observed in transgenic mice (Fig. 1e) [7]. Pronounced congestion of bile,
inflammation, necrosis, hyperplasia of biliary duct cells, and/or tumor development (CCA) was also observed frequently in intrahepatic biliary ducts of transgenic mice (Fig. 1f) [7]. The localization of transgene expression in the biliary tract was determined by indirect immunofluorescence staining. Persistent expression of the transgene was observed in both the epithelium and tumor lesion from gallbladder (Fig. 1g) and intrahepatic biliary ducts (data not shown) of BK5.erbB2 mice. Endogenous erbB2 expression
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WT GB
BK5 GB tumor
373
WT GB
p-ErbB2
total-ErbB2
p-EGFR
total-EGFR
p-MAPK
total-MAPK
p-Akt
total-Akt
p-mTOR
total-mTOR
BK5 GB tumor
COX-2 TGR5 Tubulin Fig. 2 Analysis of protein expression and kinase activation in gallbladder from wild type and BK5.erbB2 mice. Whole tissue lysates from wild type and BK5.erbB2 mice were analyzed via Western blot with antibodies against indicated molecules. Tubulin was used as an internal control. Figures are adopted from Kiguchi and DiGiovanni 2014 [9]
was only weakly detectable in both the intrahepatic biliary duct (data not shown) and gall bladder from nontransgenic mice (Fig. 1h). Western blot analysis of gallbladder tissue lysates showed that the level of erbB2 protein was significantly elevated in BK5.erbB2 mice compared to that of wild type mice, as expected (Fig. 2) [7, 9]. ErbB2 was also hyperphosphorylated (p-erbB2) after adjustment for total erbB2 protein level (Fig. 2). Interestingly, the level of EGFR protein (but not erbB3 or erbB4 protein) was elevated and hyperphosphorylated on tyrosine residues in gallbladder tissue from BK5.erbB2 mice [7]. Additional analyses by immunoprecipitation of EGFR and erbB2 followed by Western blot analysis for erbB2 and EGFR, respectively, confirmed elevated heterodimer formation between erbB2 and EGFR in gallbladder tissue of BK5.erbB2 mice [7]. Administration of the selective erbB2/EGFR inhibitor, GW2974, showed a potent chemopreventive and therapeutic effect on GBC in this model [23]. Figure 3 shows the therapeutic effect of GW2974 [24]. Treatment with GW2974 resulted in a significant decrease in the incidence of GBC to 3% (Fig. 3a) [23]. These reductions corresponded to a 95% decrease in tumor incidence compared with BK5.erbB2 mice receiving the control diet, which had a GBC incidence of 72% as determined by histopathological examination. The impact of treatment is evident in the ultrasound images
in Figure 3b [23]. H&E staining in the right panels clearly show the dramatic regression of the tumor with only hyperplasia remaining (Fig. 3b–d). Treatment with GW2974 resulted in decreased levels of both erbB2 and EGFR. Levels of p-erbB2 and p-EGFR were also markedly reduced [23]. Nearly complete inhibition of tumor development by GW2974 suggests a level of erbB2 dependency during gallbladder tumor development in BK5.erbB2 mice. Furthermore, we recently reported that a histone deacetylase inhibitor (HDAC), PCI-24781 showed a potent therapeutic effect against GBC in these mice by reducing erbB2 expression [25]. In this study, PCI-24781 (50 mg/kg per day, i.p. injection twice a day) was delivered to BK5.erbB2 mice for 4 weeks. Each group of mice was screened via the ultrasound image to ensure they had GBCs when the treatment started. Treatment of BK5.erbB2 mice with PCI-24781 for one month prevented 79% of GBC cases from progression and showed a clinical effect in 47% of cases (Fig. 4a) [25]. We also confirmed the evidence for a potent inhibitory effect on tumor cell growth in human CCA cell lines treated with PCI-24781. In both in vitro and in vivo models, this effect was associated with downregulation of erbB2 mRNA and erbB2 protein/activity and upregulation of acetylated histone and acetylated tubulin (Fig. 4b,c) [25]. These results indicate that the significant therapeutic/ inhibitory effect that this HDAC has on the development of gallbladder tumors is due to its ability to block the activation of erbB2. In addition, erbB2 has been shown to be overexpressed in the neoplastic glandular epithelium of furan- and thioacetamide-induced intestinal-type CCAs in rat liver [26, 27]. It has also been reported that erbB2 transfection transformed cultured rat cholangiocytes. These transformed cholangiocytes were tumorigenic when transplanted into isogenic rats, yielding a 100% incidence of CCAs [28]. Collectively, these data suggest that altered expression and activity of erbB2 and EGFR are major mechanisms underlying human CCA carcinogenesis (reviewed in [8]).
Downstream signaling of EGFR/erbB2 in the GBC from BK5.erbB2 mice The activation status of signaling molecules downstream of erbB2/EGFR in the GBC from BK5.erbB2 mice was also examined. Although total protein levels of MAPK were not changed (Fig. 2), the level of p-MAPK was increased in the gallbladder of transgenic mice. Furthermore, p-Akt, but not total Akt level, was elevated in the gallbladder of BK5.erbB2 mice (Fig. 2). We have reported that mTOR and other signaling molecules both immediately upstream (Akt, MAPK) and downstream (p70S6K) of mTOR are hyperphosphorylated in gallbladder tissues from
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Incidence of gallbladder carcinoma (%)
A 100 90 80
21/29 (72.4%)
* P2.2-fold, P < 0.05) compared to gallbladders in control mice (Table 1). Treatment with PCI-24781
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377 Table 1 Hold change in the gallbladder carcinomas (GBCs) from BK5.erbB2 mice vs. GBs from wild-type mice (>2.2 hold). Table is adopted from Kitamura et al. 2012 [25] Downregulated
Fig. 6 Left panel. Therapeutic effect of CS-706 on gallbladder carcinoma in BK5.erbB-2 mice. Mice were treated with AIN76A control diet (left column), AIN76A diet containing 60 ppm CS-706 (middle column), and AIN76A diet containing 100 ppm CS-706 (right column) for 1 mo. *, P < 0.01. n, total number of mice determined. %, percentage of mice diagnosed as PG. Figures are adopted from Kiguchi et al. 2007 [33] Right panel. Western blot analysis of ErbB-2, p-ErbB-2, EGFR, p-EGFR, Akt, and phosphorylated Akt (p-Akt) levels in the gallbladders of BK5.erbB-2 mice on control diet and BK5.erbB-2 mice treated with 100 ppm CS-706 Epithelial cell lysates were prepared from gallbladder tumors from five BK5.erbB-2 mice, and gallbladders from BK5.erbB-2 mice treated with CS-706 were pooled. Figures are adopted from Kiguchi et al. 2007 [33]
significantly decreased the expression of some of these miRNAs, including miR-21, miR-142-3p, miR-142-5p, and miR-223, which were upregulated in GBC (Tables 1,2). PCI-24781 also induced a significant upregulation in the expression of miR-122, which was downregulated in GBC (Tables 1,2). Since miR-21 has been characterized as an antiapoptotic miRNA and is upregulated in many cancer types including CCA cells [40, 41], miR-21 may also play an important role in the development of GBC in BK5.erbB2 mice. Our results indicate that downregulation of miR-122 may also be a marker of GBC. Interestingly, we found miR-101 was significantly upregulated in GBC treated with PCI-24781. It has been reported that both the expression and function of the enhancer of Zeste homolog 2 (EZH2) in cancer cell lines is inhibited by miR-101 [42]. These studies suggest PCI-24781-induced miR-101 may regulate the expression of both EZH2 in GBC. We postulate that the posttranscriptional effect of HDAC inhibition on these miRNAs may play an important role in the anticancer activity of PCI-24781. Conclusion and future direction The dismal outcomes that generally result from gallbladder carcinoma and other CCAs explain the pessimism that sur-
miR-665 miR-714 miR-763 miR-466f-3p miR-145 miR-193 miR-467e* miR-143 miR-881* miR-720 miR-706 miR-122 miR-378
Upregulated −8.233 −3.882 −2.880 −2.750 −2.612 −2.588 −2.511 −2.337 −2.323 −2.311 −2.303 −2.255 −2.241
miR-106a miR-96 miR-223 miR-27a miR-17 miR-15b miR-142-5p miR-142-3p miR-21
2.414 2.499 2.612 2.627 2.700 2.793 2.914 3.582 5.361
Table 2 Hold change in the gallbladder carcinomas (GBCs) treated with PCI-24781 vs. GBCs untreated from BK5. erbB2 mice (>2.2 hold). Table is adopted from Kitamura et al. 2012 [25] Downregulated
Upregulated
miR-223
−8.642 ± 2.539
mmu-miR-193
2.273 ± 0.214
mmu-miR-21
−4.509 ± 0.554
mmu-miR-101b
2.348 ± 0.221
mmu-miR-142-3p
−4.360 ± 0.920
hsa-miR-576-3p
2.600 ± 0.200
mmu-miR-142-5p
−4.417 ± 0.975
mmu-miR-451
2.652 + 0.251
mmu-miR-205
−3.702 ± 1.195
hsa-miR-620
3.335 ± 0.315
mmu-miR-141
−2.189 ± 0.208
hsa-miR-122*
11.141 ± 1.031
hsa-miRPlus-B1114
−2.219 ± 0.121
mmu-miR-122
29.204 ± 14.189
rounds treatment of these cancers. Nevertheless, more aggressive surgical techniques and advanced oncologic radiation therapy have led many institutions to report an increase in long-term survival rates. Although these treatments are progressive, the efforts directed toward early detection and novel treatment derived from basic research to determine the mechanisms involved in CCA development may play a key role in improvement of patients’ survival. Proposed inhibitory effects of each therapeutic compound on the signaling pathways in GBC of BK5.erbB2 mice are shown in Figure 7. New drugs that selectively target specific augmented molecule(s) such as erbB2, COX-2, mTOR, miRNA, and HDAC in CCA may serve as potentially effective adjunct therapeutic strategies for this cancer, for which there is currently no effective medical treatment. In addition, identification of novel candidate gene(s) or protein(s), which regulate these mechanisms, may provide not only potential therapeutic targets, but also novel tumor markers for this lethal disease. The BK5.erbB2 transgenic mouse model provides a unique opportunity to study the mechanisms involved in the development of this cancer.
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Fig. 7 Proposed pathway in which erbB2/EGFR, COX-2, and bile acid may play a role during cholangiocarcinoma (CCA) carcinogenesis. ErbB2 overexpression/activation may accelerate the transactivation cascade of during the development of CCA
Bile acid
EGF-Ligand
TKI
TACE cleave TGR5 (GPCR)
EGFR
erbB2
PI3K
Ras/Raf
Receptor Pro-EGFR Ligand
Akt
MAPK
Rapamycin HDAC inhibitor
Epigenetic Alteration
Acknowledgments The authors would like to thank Dr Takuya Kitamura (Hoshi General Hospital, Fukushima, Japan), Dr Toru Kawamoto (Tokyo Women’s Medical University, Tokyo, Japan), Tetsuo Ajiki (Kobe University School of Medicine, Kobe, Japan), Dr Kevin Connolly (University Medical Center at Brackenridge, Austin, Texas, USA) and Dr John DiGiovanni (College of Pharmacy, University of Texas, Austin, TX, USA) for their contributions and support.
Conflict of interest
None declared.
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TOPIC
Molecular aspects of cholangiocarcinoma Kaoru Kiguchi
Published online: 13 January 2014 © 2014 Japanese Society of Hepato-Biliary-Pancreatic Surgery
Abstract Novel targets for therapeutic or chemopreventive approaches against cholangiocarcinoma (CCA) are urgently needed. In this review article, we discuss the molecular aspects of CCA including the role of erbB receptor tyrosine kinases (RTKs), downstream signaling pathways of these erbB RTKs, inflammatory mediators during gallbladder carcinogenesis and bile acids based on our study using a mouse model for human CCA (BK5.erbB2 mice) as well as additional information in the literature. Keywords Cholangiocarcinoma · COX-2 and bile acid · Epidermal growth factor receptor · erbB2 Introduction Cholangiocarcinoma (CCA), which includes gallbladder carcinomas (GBCs) and cancers of the intra- and extrahepatic biliary tree, are relatively infrequent but highly lethal malignancies [1]. Although there have been advances in the diagnosis and management of CCA, these cancers still prove challenging to treat due to their insensitivity to conventional therapies and the inability to detect early tumor formation. These factors render GBC nearly incurable with a 5-year survival rate of only 5–21% [2–6]. Novel targets for therapeutic or chemopreventive approaches are urgently needed. To date, very few studies have addressed the molecular and cellular mechanisms underlying the development of CCA. We previously generated transgenic mice that K. Kiguchi (*) Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin, 1400 Barbara Jordan Blvd., Austin, TX 78723, USA e-mail: [email protected] This article is summarized based on the presentation for International Symposium on Cholangiocarcinoma held in Tokyo, 2013. It is based mostly on our previously published studies using BK5.erbB2 mice as well as additional information in the literature. Some of the text and figures/table are adopted from our previous studies as indicated.
overexpress wild type rat erbB2 in epithelial tissues under the control of the bovine keratin 5 (BK5) promoter (BK5.erbB2 mice) [7]. Overexpression of the erbB2 in basal epithelial cells of the gallbladder led to the development of adenocarcinoma of the gallbladder and cystic duct in 90% of these transgenic mice. This was the first direct demonstration that erbB2 overexpression could lead to the development of CCA [7]. We have shown that BK5.erbB2 mice are a valid model for investigating mechanisms underlying development of GBCs and other CCAs. In this article, we discuss the molecular aspect of CCA including the role of erbB receptor tyrosine kinases (RTKs), downstream signaling pathways of these erbB RTKs, inflammatory mediators during gallbladder carcinogenesis and bile acids based on our study using BK5.erbB2 mice as well as additional information in the literature (review in [8, 9]). Understanding the signaling pathways altered in CCA will provide critical clues for novel therapeutic and chemopreventive strategies using drugs and/or agents that selectively target these specific pathways.
Role of ErbB RTKs and their downstream signaling pathways in the development of CCA EGFR and erbB2 in CCA Upregulation of the RTK, erbB2, contributes to tumorigenesis in a number of tissues, however, little is known about its role in CCA including GBC. ErbB2 overexpression has been reported in a significant percentage of human CCA [6, 10–20]. Similarly, overexpression and activation of epidermal growth factor receptor (EGFR) has been reported in 30–60% of CCA samples [11, 12, 21, 22]. We previously reported BK5.erbB2 transgenic mice, in which erbB2 was targeted to overexpress in epithelial tissues, developed spontaneous GBC and intrahepatic CCA with incidences of approximately 90 and 30%, respectively, by 2 months of age (Fig. 1b) [7]. Figure 1a shows the cDNA construct of BK5.erbB2 mouse. The majority of the
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A
B
C
Wild-type D
E
G
H
BK5.erbB2 F
Fig. 1 (a) DNA construct used to generate BK5.erbB2 mice. (b) Gross appearance and histological evaluations of cholangiocarcinoma (CCA) in BK5.erbB2 mice at 3 months of age. (c) H&E staining of gallbladder in wild type mouse and (left) BK5.erbB2 mouse (right)). (d) The junction of the pancreatico-biliary duct (JPBD) in a 3-month-old BK5.erbB2 mouse. (e) H&E staining of the ampulla of Vater. (f) Intrahepatic CCA from a 3-month-old BK5.erbB2 mouse. (g and h) Expression of erbB2: Immunostaining for erbB2 in gallbladder from BK5.erbB2 (g) and wild type (h) of 3-month-old mouse. Figures are adopted from Kiguchi et al. 2001 [7]
gallbladder tumors completely filled the lumen (Fig. 1c) [7]. Most of the tumors were diagnosed as well-differentiated adenocarcinomas. Carcinoma cells frequently invaded into the surrounding connective tissues. Microscopic analysis of H&E-stained serial sections showed that tumor cells of the common bile duct had invaded into the pancreatic duct (Fig. 1d) [7]. In addition, the ampulla of Vater was dilated, and hyperplasia of the epithelium was observed in transgenic mice (Fig. 1e) [7]. Pronounced congestion of bile,
inflammation, necrosis, hyperplasia of biliary duct cells, and/or tumor development (CCA) was also observed frequently in intrahepatic biliary ducts of transgenic mice (Fig. 1f) [7]. The localization of transgene expression in the biliary tract was determined by indirect immunofluorescence staining. Persistent expression of the transgene was observed in both the epithelium and tumor lesion from gallbladder (Fig. 1g) and intrahepatic biliary ducts (data not shown) of BK5.erbB2 mice. Endogenous erbB2 expression
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WT GB
BK5 GB tumor
373
WT GB
p-ErbB2
total-ErbB2
p-EGFR
total-EGFR
p-MAPK
total-MAPK
p-Akt
total-Akt
p-mTOR
total-mTOR
BK5 GB tumor
COX-2 TGR5 Tubulin Fig. 2 Analysis of protein expression and kinase activation in gallbladder from wild type and BK5.erbB2 mice. Whole tissue lysates from wild type and BK5.erbB2 mice were analyzed via Western blot with antibodies against indicated molecules. Tubulin was used as an internal control. Figures are adopted from Kiguchi and DiGiovanni 2014 [9]
was only weakly detectable in both the intrahepatic biliary duct (data not shown) and gall bladder from nontransgenic mice (Fig. 1h). Western blot analysis of gallbladder tissue lysates showed that the level of erbB2 protein was significantly elevated in BK5.erbB2 mice compared to that of wild type mice, as expected (Fig. 2) [7, 9]. ErbB2 was also hyperphosphorylated (p-erbB2) after adjustment for total erbB2 protein level (Fig. 2). Interestingly, the level of EGFR protein (but not erbB3 or erbB4 protein) was elevated and hyperphosphorylated on tyrosine residues in gallbladder tissue from BK5.erbB2 mice [7]. Additional analyses by immunoprecipitation of EGFR and erbB2 followed by Western blot analysis for erbB2 and EGFR, respectively, confirmed elevated heterodimer formation between erbB2 and EGFR in gallbladder tissue of BK5.erbB2 mice [7]. Administration of the selective erbB2/EGFR inhibitor, GW2974, showed a potent chemopreventive and therapeutic effect on GBC in this model [23]. Figure 3 shows the therapeutic effect of GW2974 [24]. Treatment with GW2974 resulted in a significant decrease in the incidence of GBC to 3% (Fig. 3a) [23]. These reductions corresponded to a 95% decrease in tumor incidence compared with BK5.erbB2 mice receiving the control diet, which had a GBC incidence of 72% as determined by histopathological examination. The impact of treatment is evident in the ultrasound images
in Figure 3b [23]. H&E staining in the right panels clearly show the dramatic regression of the tumor with only hyperplasia remaining (Fig. 3b–d). Treatment with GW2974 resulted in decreased levels of both erbB2 and EGFR. Levels of p-erbB2 and p-EGFR were also markedly reduced [23]. Nearly complete inhibition of tumor development by GW2974 suggests a level of erbB2 dependency during gallbladder tumor development in BK5.erbB2 mice. Furthermore, we recently reported that a histone deacetylase inhibitor (HDAC), PCI-24781 showed a potent therapeutic effect against GBC in these mice by reducing erbB2 expression [25]. In this study, PCI-24781 (50 mg/kg per day, i.p. injection twice a day) was delivered to BK5.erbB2 mice for 4 weeks. Each group of mice was screened via the ultrasound image to ensure they had GBCs when the treatment started. Treatment of BK5.erbB2 mice with PCI-24781 for one month prevented 79% of GBC cases from progression and showed a clinical effect in 47% of cases (Fig. 4a) [25]. We also confirmed the evidence for a potent inhibitory effect on tumor cell growth in human CCA cell lines treated with PCI-24781. In both in vitro and in vivo models, this effect was associated with downregulation of erbB2 mRNA and erbB2 protein/activity and upregulation of acetylated histone and acetylated tubulin (Fig. 4b,c) [25]. These results indicate that the significant therapeutic/ inhibitory effect that this HDAC has on the development of gallbladder tumors is due to its ability to block the activation of erbB2. In addition, erbB2 has been shown to be overexpressed in the neoplastic glandular epithelium of furan- and thioacetamide-induced intestinal-type CCAs in rat liver [26, 27]. It has also been reported that erbB2 transfection transformed cultured rat cholangiocytes. These transformed cholangiocytes were tumorigenic when transplanted into isogenic rats, yielding a 100% incidence of CCAs [28]. Collectively, these data suggest that altered expression and activity of erbB2 and EGFR are major mechanisms underlying human CCA carcinogenesis (reviewed in [8]).
Downstream signaling of EGFR/erbB2 in the GBC from BK5.erbB2 mice The activation status of signaling molecules downstream of erbB2/EGFR in the GBC from BK5.erbB2 mice was also examined. Although total protein levels of MAPK were not changed (Fig. 2), the level of p-MAPK was increased in the gallbladder of transgenic mice. Furthermore, p-Akt, but not total Akt level, was elevated in the gallbladder of BK5.erbB2 mice (Fig. 2). We have reported that mTOR and other signaling molecules both immediately upstream (Akt, MAPK) and downstream (p70S6K) of mTOR are hyperphosphorylated in gallbladder tissues from
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Incidence of gallbladder carcinoma (%)
A 100 90 80
21/29 (72.4%)
* P2.2-fold, P < 0.05) compared to gallbladders in control mice (Table 1). Treatment with PCI-24781
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377 Table 1 Hold change in the gallbladder carcinomas (GBCs) from BK5.erbB2 mice vs. GBs from wild-type mice (>2.2 hold). Table is adopted from Kitamura et al. 2012 [25] Downregulated
Fig. 6 Left panel. Therapeutic effect of CS-706 on gallbladder carcinoma in BK5.erbB-2 mice. Mice were treated with AIN76A control diet (left column), AIN76A diet containing 60 ppm CS-706 (middle column), and AIN76A diet containing 100 ppm CS-706 (right column) for 1 mo. *, P < 0.01. n, total number of mice determined. %, percentage of mice diagnosed as PG. Figures are adopted from Kiguchi et al. 2007 [33] Right panel. Western blot analysis of ErbB-2, p-ErbB-2, EGFR, p-EGFR, Akt, and phosphorylated Akt (p-Akt) levels in the gallbladders of BK5.erbB-2 mice on control diet and BK5.erbB-2 mice treated with 100 ppm CS-706 Epithelial cell lysates were prepared from gallbladder tumors from five BK5.erbB-2 mice, and gallbladders from BK5.erbB-2 mice treated with CS-706 were pooled. Figures are adopted from Kiguchi et al. 2007 [33]
significantly decreased the expression of some of these miRNAs, including miR-21, miR-142-3p, miR-142-5p, and miR-223, which were upregulated in GBC (Tables 1,2). PCI-24781 also induced a significant upregulation in the expression of miR-122, which was downregulated in GBC (Tables 1,2). Since miR-21 has been characterized as an antiapoptotic miRNA and is upregulated in many cancer types including CCA cells [40, 41], miR-21 may also play an important role in the development of GBC in BK5.erbB2 mice. Our results indicate that downregulation of miR-122 may also be a marker of GBC. Interestingly, we found miR-101 was significantly upregulated in GBC treated with PCI-24781. It has been reported that both the expression and function of the enhancer of Zeste homolog 2 (EZH2) in cancer cell lines is inhibited by miR-101 [42]. These studies suggest PCI-24781-induced miR-101 may regulate the expression of both EZH2 in GBC. We postulate that the posttranscriptional effect of HDAC inhibition on these miRNAs may play an important role in the anticancer activity of PCI-24781. Conclusion and future direction The dismal outcomes that generally result from gallbladder carcinoma and other CCAs explain the pessimism that sur-
miR-665 miR-714 miR-763 miR-466f-3p miR-145 miR-193 miR-467e* miR-143 miR-881* miR-720 miR-706 miR-122 miR-378
Upregulated −8.233 −3.882 −2.880 −2.750 −2.612 −2.588 −2.511 −2.337 −2.323 −2.311 −2.303 −2.255 −2.241
miR-106a miR-96 miR-223 miR-27a miR-17 miR-15b miR-142-5p miR-142-3p miR-21
2.414 2.499 2.612 2.627 2.700 2.793 2.914 3.582 5.361
Table 2 Hold change in the gallbladder carcinomas (GBCs) treated with PCI-24781 vs. GBCs untreated from BK5. erbB2 mice (>2.2 hold). Table is adopted from Kitamura et al. 2012 [25] Downregulated
Upregulated
miR-223
−8.642 ± 2.539
mmu-miR-193
2.273 ± 0.214
mmu-miR-21
−4.509 ± 0.554
mmu-miR-101b
2.348 ± 0.221
mmu-miR-142-3p
−4.360 ± 0.920
hsa-miR-576-3p
2.600 ± 0.200
mmu-miR-142-5p
−4.417 ± 0.975
mmu-miR-451
2.652 + 0.251
mmu-miR-205
−3.702 ± 1.195
hsa-miR-620
3.335 ± 0.315
mmu-miR-141
−2.189 ± 0.208
hsa-miR-122*
11.141 ± 1.031
hsa-miRPlus-B1114
−2.219 ± 0.121
mmu-miR-122
29.204 ± 14.189
rounds treatment of these cancers. Nevertheless, more aggressive surgical techniques and advanced oncologic radiation therapy have led many institutions to report an increase in long-term survival rates. Although these treatments are progressive, the efforts directed toward early detection and novel treatment derived from basic research to determine the mechanisms involved in CCA development may play a key role in improvement of patients’ survival. Proposed inhibitory effects of each therapeutic compound on the signaling pathways in GBC of BK5.erbB2 mice are shown in Figure 7. New drugs that selectively target specific augmented molecule(s) such as erbB2, COX-2, mTOR, miRNA, and HDAC in CCA may serve as potentially effective adjunct therapeutic strategies for this cancer, for which there is currently no effective medical treatment. In addition, identification of novel candidate gene(s) or protein(s), which regulate these mechanisms, may provide not only potential therapeutic targets, but also novel tumor markers for this lethal disease. The BK5.erbB2 transgenic mouse model provides a unique opportunity to study the mechanisms involved in the development of this cancer.
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Fig. 7 Proposed pathway in which erbB2/EGFR, COX-2, and bile acid may play a role during cholangiocarcinoma (CCA) carcinogenesis. ErbB2 overexpression/activation may accelerate the transactivation cascade of during the development of CCA
Bile acid
EGF-Ligand
TKI
TACE cleave TGR5 (GPCR)
EGFR
erbB2
PI3K
Ras/Raf
Receptor Pro-EGFR Ligand
Akt
MAPK
Rapamycin HDAC inhibitor
Epigenetic Alteration
Acknowledgments The authors would like to thank Dr Takuya Kitamura (Hoshi General Hospital, Fukushima, Japan), Dr Toru Kawamoto (Tokyo Women’s Medical University, Tokyo, Japan), Tetsuo Ajiki (Kobe University School of Medicine, Kobe, Japan), Dr Kevin Connolly (University Medical Center at Brackenridge, Austin, Texas, USA) and Dr John DiGiovanni (College of Pharmacy, University of Texas, Austin, TX, USA) for their contributions and support.
Conflict of interest
None declared.
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