Notch signalling pathway in development of cholangiocarcinoma

Table 1 List of main representatives of Notch Signalling pathway and their current findings in development of cholangiocarcinoma

Receptors, ligands and downstream signaling molecules	Type of model	Findings	Ref.
Notch 1	Mice model	Overexpression of NICD1 in mice liver leads to the formation of cystoadenomas and cystoadenocarcinomas. IH-CCA cell migration is enhanced by overexpression of Notch 1. It further enhances the growth of IH-CCA as result of Rac1 activation. The atypical expression by the up-regulation of Vimentin and α-SMA at the expense of E-cadherin expression leads to an EMT	[32,63]
	Human cell lines	Overexpression of Notch 1 receptor along with JAG-1 has been perceived in four human CCA cell lines (MzChA1, TFK1, SZ1and EgI1)	[61]
Notch 2	Wild-type and Notch2 flox/floxmice model	Notch2 is basically involved in regulating the hepatocyte derived CCA. Wild-type and Notch2 flox/floxmice models have been utilized for studying the Notch Signalling pathway in AKT/Yap-driven IH-CCA development and has been found that AKT/Yap-induced IH-CCA development is hepatocyte dependent and based on the canonical Notch signalling cascade in in vivo models	[14]
	Human cell lines	HCC and ICC cell lines reveals the down-regulation in expression of Sox9 and EpCAM, typical biliary markers after Notch 2 silencing	[14]
Notch 3	Mice model	IH-CCA development and survival has been found to be associated with activation of PI3K-AKT cascade by overexpression of the Notch 3 atypical receptor rather than canonical Notch-RBPJ mechanism. Hepatocarcinogen Thioacetamide induces cancer in CK19CreERTeYFPp53 mouse model owing constitutive Notch3 deletion that establishes the IH-CCA inhibition after loss of single copy of Notch 3 gene	[66,62]
Notch 4	Human samples	The overexpression of Notch 4 is associated with raised serum levels of CA125 that suggests the poor prognosis of IH-CCA	[59]
Jagged-1	Human cell lines	The suppression of the Jagged-1 results in the reduction of growth of the human CCA cell lines (HUCCT1 and KKU-156) with consequent down-regulation of downstream signalling molecules (Hes1 and Hes4)	[16]
RBPJ	Mice model	The down-regulation of RBPJ protein in AKT/JAG-1 mouse model results in total inhibition of Notch signalling pathway following the development of IH-CCA. Notch 1 plays its role in advancement of CCA by enhancing the cell survival and proliferation through the regulation of 14-3-3 theta in RBP-J-associated module. Hence, it concludes that that RBPJ is a significant element in Notch signalling pathway in CCA	[16,70]
Hes-1	Mice model	The tamoxifen-inducible Alb CreERT2; R26R Notch/+ mice expresses mouse NICD1 and delivers the constitutive Notch activity in hepatic cells. In Alb-CreERT2;Hes1fl/fl mice, these hepatocytes that lack the Hes1 gene have revealed that on treatment with TAA, CK10 positive cells significantly increases in lobules of Alb CreERT2; R26RNotch/+ and has been found to be reduced in Alb-CreERT2; Hes1fl/fl mice. Besides that, the rapid nodule formation has been found to be obvious in the livers of Alb CreERT2; R26RNotch/+ in contrast to those of Alb-CreERT2; Hes1fl/fl.. This observation confirms the fact that Hes1 is greatly involved in pathogenicity of IH-CCA. Further, this carcinogenesis can be blocked by inhibition of Hes1	[73,74]

CCA: Cholangiocarcinoma; TCC: Thioacetamide; IH-CCA: Intrahepatic cholangiocarcinoma; NICD: Notch intracellular domain; EMT: Epithelial-mesenchymal transition; RBP-J: Recombination signal Binding Protein for immunoglobulin kappa J; JAG: Jagged.

Table 2 Anti-Notch therapeutic targets examined in vivo and/or in vitro for the treatment of cholangiocarcinoma

Molecules/Targets and Functions	Experiments	Results and findings
GSI IX (Gamma Secretase Inhibitor)	In resected specimens of EH-CCA	In all of CC cell lines, treatment of GSI IX considerably reduces the subpopulation of CD24+ +CD44++ cells (Cancer Stem Cells, CScs)[60]
RO4929097 (Gamma Secretase Inhibitor)	Pancreatic adenocarcinoma patients	25% treated patients attained the stable disease, while the 6-month survival rate was found to be 27.8%. Additionally, decrease in the expression of HeyL was observed following the drug administration[82]
DAPT (Gamma Secretase Inhibitor)	Cell lines (SZ1, MzChA1, EgI1 and TFK1)	Cause the decrease of the expression of the cleaved form of the Notch receptor and Hes1, with the subsequent overexpression of the cyclin kinase inhibitors (p21, p27 and p53)[30]
FLI-06 (Gamma Secretase Inhibitor)	IH-CCA cells	Found to be completely diminished the MFAP5- dependent Notch activation in CCA[84]
Brontictuzumab, OMP-52M51 (Anti-Notch1 antibody)	Phase 1 Clinical Trial	The treatment study was done on 07 CCA patients. It showed single-agent efficacy. While patients had a partial response, specifically in the NICD high population[87]
Tarextumab, OMP-59R5 (Anti-Notch2/3 antibody)	Phase 2 Clinical Trial	Phase1b/2 study conducted on SCLC (Small Cell Lung Cancer) and pancreatic cancer. Phase1b results showed a greater tumor reduction at high dose of TRXT[87]
Demcizumab, OMP- 21M18 [Anti-Delta-like ligand 4 (Dll4)]	Phase1/2 clinical trial	In NSCLC (Non-Squamous Non-small Cell Lung Cancer) patients treated with Demcizumab, 3% of patients had a valuable response, 48% had a partial response and 38% showed a stable disease. In pancreatic cancer patients, 50% of valuable patients who received Demcizumab showed a partial response[88,89]
RNA interfering antisense oligonucleotide (AS-ODN, a specific Notch2 Inhibitor)	B-CLL cells	AS-ODNs results in down-regulation of Notch 2 activity in pathogenicity of B-CLL[91]
miR-34a (Anti-tumorgenic effect)	Human CCA cells (CCLP1, TFK1, SG231 and HUCCT1)	miR-34a expression is silenced epigenetically by EZH2 that tends to result in progression of CCA cell growth via Notch cascade activation, so it’s activation via inhibition of EZH2 inhibitor results in the significant decrease of tumor burden[94]
Niclosamide (An antheliminthic drug)	Colon cancer cell lines (HCT116, LoVo and SW620 cells)	Niclosamide results in inhibition of the progression of colon cancer by up-regulating the miR-200 family members and by down-regulating the Notch signaling pathway[95]
Hydrocarbon-stapled α-helical peptide (SAHM1)	T-cell acute lymphoblastic leukaemia	It prevents the formation of Notch trans-activation complex and subsequently suppresses the development of in vivo and in vitro T- Cell acute leukemia[96]
Inhibitor of mastermind Recruitment-1 (IMR-1)	Notch-dependent cell lines and patient-derived tumor xenografts	It disrupts the recruitment of Mastermind-like to the Notch transcriptional activation complex and, thus attenuates the Notch target gene transcription[97]
MO-I-1100 (SMI, Small Molecule inhibitor of β-hydroxylase)	FOCUS, HCC cell line, BNLT3, Huh7, Hep3B and HepG2	It reduces the ASPH enzymatic activity and inhibits the proliferation, invasion and growth of HCC cell lines and animal models. The mechanism of effect is based on the antitumor effect with the reduced activation of Notch Signalling cascade, both in vivo and in vitro[98]
MO-I-1151 (Second Generation Small Molecule Inhibitor, SMI of β-hydroxylase)	Human CCA cell lines (ETK1, RBE, SSP25 and BDE-Neu CL24 cells)	It exhibits the inhibition on CCA cell migration and proliferation and also significantly suppresses tumor growth and progression of CCA. The treatment also results in suppression of overexpression of Notch1 and Hey1 in the tumors[99]
Corilagin (A natural plant derivative, Polyphenol Tannic Acid)	Human CCA cell lines (MZ-Cha-1 and QBC9939)	It inhibits the CCA cell proliferation and invasion and increases the CCA cell apoptosis. It inhibits the AKT phosphorylation and counteracts the AKT phosphorylation that is induced by NICD1 and consequently inhibits the Notch1 and Hes1 mRNA expression[100]
Curcumin (A phytochemical derived from turmeric, Curcuma longa)	Human IH-CCA cell lines (SG-231 and CCLP-1)	It reduces the viability and colony-forming ability of CCA cells. Even at low concentration, it effectively reduces the growth of the cells and stimulates the apoptosis with subsequent suppression of Notch1 and Hes1 mRNA expression[101]
Cinobufagin (Chansu, Toad Venom)	Human prostate carcinoma cell lines (DU145, LNCaP and PC3 cells), Normal breast epithelial cell lines, Human CCA cell lines RBE (IH-CCA) and QBC939 (EX-CCA)	Cinobufagin causes and cell apoptosis and anti-proliferative effects in cancer cells[103]. Cinobufagin-derived inactivation of Notch signalling pathway results in the stimulation of apoptosis in CCA cells[104]
Xanthohumol (A Prenylated Chalcone)	SG-231and CCLP-1 derived mouse xenografts	Xanthohumol causes the cell cycle arrest via an increase in pro-apoptotic markers and induces the apoptosis. It also results in reduction of AKT and Notch1 expression level in a time dependent fashion[105]

CCA: Cholangiocarcinoma; IH-CCA: Intrahepatic cholangiocarcinoma; NICD: Notch intracellular domain; IMR-1: Inhibitor of Mastermind Recruitment-1; SMI: Small-molecule inhibitor; GSI: Gamma-secretase inhibitors.