The novel epithelial-mesenchymal transition-related proteins and their therapeutic targets in cholangiocarcinoma: a narrative review

Introduction

Cholangiocarcinoma (CCA), a liver cancer caused by bile duct epithelial cells, is a severe health issue in northeastern Thailand (1). The liver fluke Opisthorchis viverrini (O. viverrini) causes chronic inflammation and progressive periductal fibrosis, which increases the risk of CCA, especially in Thailand. Otherwise, periductal fibrosis appears to be the main cause of CCA (2). The main issue with this malignancy is that it has a very poor prognosis and is difficult to identify until the disease has progressed to an advanced stage (3). There is an immediate need for the discovery and validation of biomarkers that can be employed in the screening, prognosis, and diagnosis of CCA in clinical settings.

Epithelial-mesenchymal transition (EMT), a biological process that loses the epithelial phenotype and becomes mesenchymal, is a critical factor in many cancer types. Briefly, mesenchymal cells improve extracellular matrix (ECM) formation, invasiveness, and apoptosis resistance (4). EMT leads to metastatic cancer cells by acquiring mesenchymal markers and losing epithelial cell adhesion molecules (5). According to several studies, these protein expressions in human tissues or sera can be biomarkers for EMT in many diseases, including cancer (6,7).

This review summarized the 5-year updated literature on PubMed (https://www.ncbi.nlm.nih.gov/pubmed) using the search terms “cholangiocarcinoma AND epithelial-mesenchymal transition AND biomarker AND diagnosis AND prognosis”. All these studies have examined the pathways and possible targeted therapies of the EMT mechanism caused by EMT-related molecules on CCA. Furthermore, the common findings as well as the controversial conclusion regarding the effects of EMT-related factors induced on CCA progression were presented and evaluated comprehensively. We present this article in accordance with the Narrative Review reporting checklist (available at https://jgo.amegroups.com/article/view/10.21037/jgo-22-1126/rc).

Methods

Following discussions among the authors, we conducted a search of the PubMed database for articles on EMT-related proteins and molecular markers of CCA published in the last five years. We then selected, categorized, and summarized the relevant articles. The specific search method is described in Table 1.

Effects of the novel EMT-related proteins on CCA progression

Many EMT-related molecules have been studied for their potential roles in driving or restraining cancer growth, but few studies have looked at their prognostic activities in CCA, which may help establish a medical approach to cancer diagnosis.

Alpha7 nicotinic acetylcholine receptor (α7-nAChR)

In normal physiological conditions, α7-nAChR modulates synaptic transmission in every portion of the brain and affects learning and memory (8). Pathophysiological conditions, including immunological and oncological disorders, decrease α7-nAChR regulation, making it a therapeutic target. One study recently found the mechanism of α7-nAChR in EMT, stimulating CCA generation (9). In that study, 50 human CCA tissue samples showed that most patients had high α7-nAChR expression, which was associated with poorly differentiated histological grade, severe tumor stage, lymphatic and distant metastasis, a shorter survival time, and a poor prognosis. In RBE cells, shRNA-α7-nAChR decreased cancer cell growth, migratory and invasive effects, and early apoptosis. After α7-nAChR knockdown, E-cadherin was down-regulated and vimentin, mesenchymal protein, and snail transcription factor were up-regulated. These findings show that α7-nAChR activates EMT to advance CCA. In addition, α7-nAChR inhibition was found to be an effective anti-CCA strategy in vitro. In vivo studies showed that α7-nAChR gene knockdown reduced tumor volume in BALB/c nude mice with CCA xenografts (9). This is the first study to reveal that α7-nAChR inhibits apoptosis and promotes EMT, causing CCA progression. It may be a useful molecular diagnostic and prognostic factor for CCA.

Annexin A10 (ANXA10)

The biggest class of calcium and phospholipid-binding eukaryotic proteins, the annexin family, is crucial for many physiological activities, including cell division and proliferation (10). ANXA10, a novel member of the Annexin family, is involved in cancer formation but needs more investigation regarding its expression and role in CCA. Sun and colleagues found that elevated ANXA10 expression was an independent biomarker of perihilar CCA (pCCA) and distal CCA (dCCA) using exome and transcriptome sequencing (11). This study found that ANXA10 regulated PLA2G4A, an enzyme that cleaves phospholipids to liberate free fatty acids (FFAs), and that it was necessary for CCA proliferation, invasion, and EMT. EMT, which gives cancer cells increased aggressivity, resistance to apoptosis, and stem-like traits, is what ANXA10-mediated extrahepatic CCA (eCCA) metastasis depends on. EMT and PLA2G4A/COX-2/PGE2 may work together in eCCA, indicating that the inflammation-EMT axis is important for CCA metastasis. This discovery might identify high-risk pCCA and dCCA patients and offer personalised treatment. Finally, inhibiting the ANXA10/PLA2G4A/PGE2 pathway could alleviate pCCA and dCCA patients’ symptoms.

Cell migration inducing hyaluronidase 1 (KIAA1199)

The transmembrane protein KIAA1199 is expressed in many non-cancerous and malignant cells. KIAA1199 overexpression increases cancer metastasis and proliferation. Gastric cancer, breast cancer, and hepatocellular carcinoma (HCC) studies have supported similar effects (12-14). Zhai et al. revealed that CCA overexpresses KIAA1199 in cancer databases (15). The testing serum and validation cohorts showed decreased overall and disease-free survival with higher KIAA1199 expression. KIAA1199 and E-cadherin had a very adverse correlation in CCA patients. KIAA1199 also correlated positively with N-cadherin, vimentin, tumor-node-metastasis (TNM) stages, histopathological grade, and lymph node metastases. KIAA1199 increases HuCCT1 and QBC-939 CCA cell proliferation, migration, and invasion. TGF-β-PI3K-AKT signaling via KIAA119 induces EMT. Overexpression and silencing of KIAA1199 influenced downstream target expression in the EMT-related TGF-β pathway. KIAA1199 enhances subcutaneous tumor xenograft CCA cell proliferation in naked mice. This study is the first to reveal that KIAA1199 is linked to EMT and maybe a new CCA biomarker.

Cluster of differentiation 90 (CD90)

Many normal cells contain CD90, a 25–37 kDa glycosylphosphatidylinositol-anchored glycoprotein that interacts with other cells. Many malignancies have a poor prognosis and are affected by CD90 expression (16). CD90 promotes HCC tumor development and metastasis (17). Yamaoka and colleagues examined CD90 expression in intrahepatic CCA (iCCA) clinically (18). CD90 expression has been associated to clinicopathological features and prognosis in human iCCA surgical tissues. In 25 iCCA patients, CD90 expression was strong positive, related with lymph node metastases, and an independent prognostic factor. In vitro, CD90⁺ cells were more migratory and expressed more EMT-related genes like CXCR4 and MMP7 than CD90⁻ cells. CD90⁺ cells enhanced EMT-related Wnt/β-catenin signaling. Using these findings, Wnt/β-catenin pathway activation in CD90⁺ cells induce EMT via overexpressing MMP7 and CXCR4. Hence, CD90 expression may predict iCCA prognosis.

Farnesoid X receptor (FXR)

FXR, a nuclear receptor family member and ligand-activated transcription factor encoded by the Nr1h4 gene, can initiate EMT in CCA. It is extensively expressed in the kidney, stomach, duodenum, colon, and liver (19). Bile acids (BAs) can activate FXR gene transcription and enhance BA production, transmission, and metabolism (20). Recent research found that most iCCA tumor tissues had reduced FXR expression, which correlated with aggressive tumor characteristics and poor prognosis (21). Moreover, FXR demonstrated predictive relevance in carbohydrate antigen 19-9 (CA19-9)-negative iCCA patients and FXR deficiency increased IL-6 level in iCCA patients. After studying FXR in CCA genesis, they investigated CCA cell lines (RBE, HCCC-9810, HuCCT1, and CCLP1) with obeticholic acid (OCA), an agonist-mediated FXR activation, and FXR-shRNA in vitro. FXR suppressed IL-6, elevated E-cadherin, and ZO-1, decreased N-cadherin, snail, and vimentin, and inhibited β-catenin to inhibit CCA cell proliferation, migration, invasion, and EMT. In vivo, non-obese diabetic severe combined immunodeficiency (NOD-SCID) mice tumor xenograft model received RBE-cells and OCA and showed that RBE-cell-derived tumors had larger tumor volume, size, and lung metastasis than OCA-fed tumors. In vivo, OCA reduced tumor development and lung metastasis. In conclusion, FXR suppresses IL-6 and reduces iCCA tumor development and metastasis, making it a promising biomarker for iCCA prognosis, especially in CA19-9-negative patients.

Fascin (FSCN1)

Recent study has shown fascin, a cytoskeletal protein that promotes cells adhere, plays a mechanical role in CCA progression (22). In that study, fascin affected CCA tumorigenicity in vitro and in vivo. In fascin-shRNA knockdown experiments, QBC-939 cells showed that silenced fascin expression decreased cancer cell proliferation, migration, and invasion. Nude mice were given subcutaneous tumors to determine if fascin affects tumor formation in vivo. After 42 days after inoculation, fascin-shRNA-transfected mice had less tumors than the control group, supporting in vitro findings. E-cadherin was up-regulated while vimentin was down-regulated in fascin-induced EMT. In QBC-939 cells, fascin knockdown activated GSK3β, increased phosphorylated β-catenin, and decreased nuclear localization. Their findings revealed that fascin knockdown greatly suppressed Wnt/β-catenin signaling triggering EMT. Hence, fascin regulates Wnt/β-catenin pathway in CCA to increase invasion and metastasis in EMT.

Free fatty acid receptor 4 (FFAR4)

The next novel EMT-related molecule is connected to cancer lipid metabolism. FFAs provide energy and food as extracellular signaling molecules by attaching to its receptor (FFA receptors; FFARs), a transmembrane receptor that couples with G-protein-26 (GPCRs). Most articles stated that FFAR4, also known as GPR120, is a novel member of the GPCR family that has been observed as a receptor for FFA in diabetes mellitus. FFAR4 is increasingly implicated in carcinogenesis in breast and prostate cancer (23-26). Meng et al. examined FFAR4 expression in 98 human CCA tissues and examined its correlation with clinical characteristics in CCA patients (27). CCA overexpress FFAR4, and statistical analysis showed that severe clinicopathological data corresponded with increased FFAR4 expression. In that retrospective investigation, FFAR4 was adversely correlated with CCA E-cadherin expression. FFAR4 was favorably related with Snail-1, vimentin, CK7, and CK19 in CCA. Overexpression of FFAR4 may highly correlate with EMT-related protein expression. These findings suggest that FFAR4 may participate in EMT via PI3K/Akt signaling pathway to alter CCA invasion and metastasis. Finally, FFAR4, a novel therapeutic and diagnostic target for CCA, appears to be promising.

GATA-binding protein 6 (GATA6)

GATA6 interacts with the GATA motif in the promoter region to affect gene expression (28). It is uncertain what molecular processes GATA6 uses to promote CCA formation. According to recent research, GATA6 induces EMT via a novel mechanism and has the potential to be employed as a predictive biomarker for CCA patients (29). In 91 CCA samples, GATA6 expression was inversely correlated with E-cadherin, negatively correlated with vimentin, and positively correlated with N-cadherin. GATA6 knockdown and overexpression accelerated CCA cell EMT and metastasis in vitro and in vivo. ChIP-sequencing showed that GATA6 targets MUC1 downstream. N-cadherin and vimentin were favorably correlated with MUC1 expression, while E-cadherin was negatively correlated. In CCA cells, GATA6-induced EMT was markedly reduced by MUC1 knockdown. Furthermore, in CCA tissues, nuclear β-catenin expression exhibited a high correlation with MUC1 expression. In CCA cells, MUC1 binds to β-catenin and increases its expression in the nucleus (29). In conclusion, GATA6 plays a crucial role in inducing EMT in CCA via the MUC1/β-catenin pathway, which may have significance for anti-metastatic therapy strategies in CCA clinical trials.

H2A histone family member Z (H2A.Z)

The following notable unique EMT-factor is H2A.Z. This molecule is essential for DNA replication, chromosomal segregation, and heterochromatic state maintenance (30). A recent study found that H2A.Z is overexpressed, and that H2A.Z expression is associated with a worse outcome and shorter overall survival time in iCCA patients (31). Cell proliferation was influenced by H2A.Z/S-phase kinase-associated protein 2/p27/p21 signaling in vitro and in vivo. Moreover, H2A.Z inhibition decreased cell growth and promoted apoptosis in CCLP-1 and HCCC-9810 cell lines. Inhibiting H2A.Z decreased tumor metastasis by inhibiting EMT process and improved the anticancer effects of cisplatin, a chemotherapy drug, in the treatment of iCCA (31). As a whole, H2A.Z increased cell proliferation and EMT in iCCA, suggesting it could be a useful biomarker and therapeutic target for this cancer.

High mobility group A1 (HMGA1)

Small nuclear protein known as HMGA1 serves as a structural transcription factor (32). It is uncommon to observe HMGA1 in mature differentiated tissues, but oncogenic transcription factors, epigenetic modifications, and chromosomal translocation can up-regulate it (33). HMGA1 was expressed in iCCA and has been shown to increase tumorigenicity (34,35). However, the clinical importance of HMGA1 in pCCA remains unknown. Li and colleagues used transcriptome sequencing to identify putative pCCA biomarkers and assessed the predictive importance of HMGA1 in an extensive pCCA cohort (36). Bioinformatics and in vitro/vivo study revealed that HMGA1 promoted thyroid hormone receptor interactor 13 (TRIP13) transcription, which increased tumor cell stemness, EMT, proliferation, migration, and invasion. TRIP13 was a poor biomarker for pCCA, however double high expression of HMGA1/TRIP13 may better predict prognosis. By decreasing FBXW7 transcription and stabilizing c-Myc, TRIP13 helped pCCA cell lines (QBC-939 and FRH-0201) progress. The HMGA-TRIP13 axis boosted pCCA stemness and EMT in a positive feedback process, as evidenced by the fact that c-Myc improved the transcription and expression of both HMGA1 and TRIP13. Interestingly, the positive feedback from the c-Myc/Wnt-β-catenin pathway enabled the HMGA-TRIP13 axis to increase pCCA stemness and EMT (36). This revealed that disrupting the HMGA1-TRIP13-c-Myc nexus could be a very promising strategy for treating pCCA, and that detecting HMGA1 and TRIP13 after surgery could assist stratify high-risk patients, directing individual treatments and facilitating the development of customized therapeutics.

Kidney‑type glutaminase (GLS1)

Glutaminase initiates glutamine to glutamate conversion. GLS1 and GLS2, kidney and liver glutaminases, were initially identified. GLS1 promotes tumor metabolism (37). Some studies found that GLS1 abnormally expressed and enhanced tumor progression in HCC (38). Nevertheless, the functions of GLS1 in iCCA are mainly unclear. Recently, Cao and coworkers aimed to assess expression and clinical importance of GLS1 in iCCA (39). In many digestive system malignancies, including iCCA, GLS1 expression was higher than in peritumoral tissue. GLS1 overexpression in RBE cells promoted metastasis and invasion. E-cadherin and vimentin were also regulated by GLS1 in iCCA cells. In QBC-939 cells, GLS1 knockdown had the opposite effect. GLS1 expression in iCCA samples was negatively connected with E-cadherin and positively correlated with vimentin in clinical studies. Tumor differentiation and lymphatic metastasis were substantially associated with GLS1 protein expression (P=0.001 and 0.029, respectively). Patients with high GLS1 expression had shorter overall survival and recurrence rates than those with low expression. Independent predictive markers included GLS1 expression (39). Overall, the results of this investigation showed that GLS1 is an independent predictive biomarker of iCCA. GLS1 promotes iCCA development via EMT and might thus be a therapeutic target in iCCA.

Mitochondrial pyruvate carrier 1 (MPC1)

Apparently, cancer cells have a distinct metabolism to help them survive. This metabolic shift increases aggressive cancer cell aggressiveness (40). Consequently, therapeutic targets for cancer metabolism molecules may exist. A recently identified transporter in the mitochondrial inner membrane is called the mitochondrial pyruvate carrier (MPC) (41). MPC consists of two subunits, but MPC1 is linked to poor prognoses in a number of malignancies (42,43). MPC1’s effect on iCCA’s malignancy was investigated in a recent study (44). iCCA tumor invasion and distant metastases were also associated with decreased MPC1 expression. These events are clearly connected to EMT. Hence, they studied the effect of altering MPC1 gene expression on cancer cell aggressiveness in vitro using CCA cell lines (TFK-1 and CCLP-1). MPC1 expression was downregulated in EMT cells treated with TGF-β. Inhibiting MPC1 expression promoted EMT-related cancer cell, but overexpressing it decreased tumor cell migration (44). In conclusion, MPC1 regulates the generation of EMT through the TGF-β signaling pathway and assists to the cancerous potential of iCCA. MPC1 is downregulated in a wide range of solid tumors, these data suggest that MPC1 may be a potential therapeutic target in iCCA.

Mortalin

Mortalin is a heat shock protein (HSP) 70 family member, a highly conserved molecular chaperone protein essential in many pathological and physiological circumstances (45). As reported by Kang et al. (46), 125 iCCA patients had high mortalin expression in cancer tissues, which was associated with shorter overall survival time, increased event to recurrence, metastasis to lymphatic organs, and aggressive tumor differentiation. Knocking down of mortalin mRNA expression on QBC-939 and RBE cell lines suppressed iCCA cell growth, increased apoptosis, reduced cancer cell invasion, and delayed wound closure. Mortalin knockdown cells express more E-cadherin and less snail and vimentin. High mortalin expression linked with snail and vimentin and negatively with E-cadherin. These findings suggest that mortalin may activate EMT and progress iCCA.

Nardilysin (NRDC)

NRDC, a metalloendopeptidase of the M16 family (N-arginine dibasic convertase, NRD-convertase), has been shown to trigger EMT and play important roles in many cancers and inflammation (47). NRDC is highly expressed in many malignancies and promotes tumor growth and poor prognosis proving its importance in cancer biology. Yoh et al. first investigated whether NRDC can cause EMT and CCA progression (48). Ninety-eight iCCA patients had elevated serum NRDC levels, which correlated with shorter overall and disease-free survival and tumor severity. The diagnostic capability of NRDC showed that serum NRDC levels [area under the curve (AUC) =0.689] were comparable to carcinoembryonic antigen (CEA) and CA19-9 (AUC =0.569 and 0.671, respectively). NRDC and CA19-9 had the highest AUC value in the three-marker combination diagnostic analysis (0.756). In surgical iCCA specimens, serum NRDC levels associated positively with EMT-inducer (SNAI1 and ZEB1) mRNA levels. NRDC knockdown in HuCCT-1 and SSP-25 cell lines inhibited iCCA cell growth and migration in vitro. NRDC knockdown-iCCA cells down-regulated EMT-related genes (vimentin, ZEB1, SNAI1, and TWIST) for EMT-induced NRDC mechanisms. Furthermore down-regulated were cancer stem cell marker SOX2 and EMT pathway trigger hypoxia-inducible factor-1 (HIF-1). In vivo, male nude mice were subcutaneously injected with HuCCT-1 negative control and NRDC-knockdown cells for tumor-xenograft. NRDC-knockdown mice had significantly less tumor growth than controls. These findings suggest that NRDC knockdown can inhibit CCA progression, including cancer proliferation and migration.

Phospholipase C beta 1 (PLCB1)

PLCB1, a phospholipase, hydrolyzes phospholipids and is elevated in colorectal cancer, breast cancer, and small cell lung carcinoma (49,50). The biological relevance of PLCB1 in CCA is unknown. PLCB1 promotes human CCA, and Liang et al. studied its role in CCA progression (51). PLCB1 was abundant in human CCA tissues and cell lines. E-cadherin levels were higher in CCA samples with low PLCB1 expression than high expression. PLCB1 and EMT characteristics were related with inverted N-cadherin and vimentin expression patterns. PLCB1 increased tumor growth in CCA animal models, including transposons-based carcinogenesis models. PLCB1 also induced CCA cell EMT via PI3K/AKT signaling. PABPC1, a functional polyadenylate-binding protein conserved gene member, increased PLCB1-mediated EMT via PI3K/AKT/GSK3b/Snail signaling. The AKT inhibitor MK2206 can reverse gemcitabine plus cisplatin resistance caused by ectopic PLCB1. This study also showed for the first time that miR-26b-5p may suppress CCA by targeting PLCB1. These data suggest that a PLCB1-PI3K-AKT signaling axis is crucial for CCA growth and EMT, suggesting that AKT may be a therapeutic target for overcoming chemotherapy resistance in CCA patients with elevated PLCB1. Importance PLCB1, an oncogenic driver of EMT-related CCA, makes AKT inhibition therapeutic.

Protein tyrosine phosphatase type IVA 1 (PTP4A1)

EMT in CCA can also be driven on by PTP4A1, a member of a small class of protein tyrosine phosphatases (PTPs) that removes phosphate groups from phosphorylated tyrosine residues on proteins. PTP4A1’s biological significance in CCA was first explored by Liu and colleagues (52). Three hundred and twenty-two paraffin-embedded tumor samples from iCCA patients showed that PTP4A1 was often overexpressed and correlated with aggressive and severe cancer characteristics like lymph node metastasis, advanced TNM stages, poorer survival, and higher recurrence rates. HCCC-9810, HuCCT-1, and RBE cells were knocked down and overexpressed to show that PTP4A1 encourages the growth, colonization, and invasion of cancer cells. In PTP4A1-silenced iCCA cells, E-cadherin was increased whereas N-cadherin, Zeb1, and Snail were downregulated. E-cadherin downregulation and Zeb1, Snail, and N-cadherin upregulation occurred later than PTP4A1 overexpression in iCCA cells. PTP4A1 may trigger EMT in iCCA, according to their findings. PI3K/AKT signaling pathway enhances metastatic and aggressive tumor microenvironment through EMT process (53). They then used PTP4A1-overexpressed HCCC-9810 cells, PTP4A1-knockdown RBE cells, and their controls to create subcutaneous xenograft tumor animal models. After two weeks of injection, all animals except those injected with PTP4A1-knockdown tumor cells had solid tumors, and the overexpressed mice had faster tumor growth than the silenced mice. In vivo data showed that PTP4A1 promotes iCCA development.

Tripartite motif-containing protein 44 (TRIM44)

An essential member of the TRIM family, TRIM44, possessed a zinc-finger domain that was associated with ubiquitin-specific proteases (USPs). TRIM44 is a cancer-promoting gene that has been linked to multiple different types of the disease, including head and neck squamous cell carcinomas and esophagogastric cancer. TRIM44 is responsible for activating the PI3K/AKT/mTOR pathway, which in turn promotes the EMT of cancer cells as well as the initiation and growth of tumors (54,55). TRIM44 expression and function in human iCCA were examined by Peng et al. (56). TRIM44 mRNA and protein expressions in iCCA and corresponding peritumoral tissues were evaluated using the public Oncomine database. Second, TRIM44 interference and cDNA transfection were utilized to study its roles and mechanisms in iCCA cells (QBC-939 and RBE). Finally, TRIM44’s prognostic effect on CCA progression was examined. TRIM44 expression was higher in iCCA tissues, supporting the public cancer database findings. TRIM44 knockdown reduced iCCA cell invasion and migration and increased apoptosis. High TRIM44 levels also trigger EMT in iCCA cells. AZD6244 prevented cell EMT and death caused by TRIM44 overexpression, which increased MAPK signaling. Clinically, TRIM44 expression was significantly associated with substantial tumor development, lymphatic metastasis, and poor tumor differentiation. In comparison to the TRIM44low group, the TRIM44high group exhibited a poorer overall survival rate and a higher cumulative incidence of recurrence. These findings indicate that TRIM44 is a potential predictive biomarker and therapeutic target for iCCA patients because it promotes iCCA formation by causing cancer cell EMT and apoptosis resistance.

Ubiquitin-conjugating enzyme E2T (UBE2T)

A ubiquitin-conjugating enzyme called UBE2T is overexpressed in bladder and lung malignancies and promotes prostate and breast cancer (57,58). Its role in CCA advancement is neglected. UBE2T expression in CCA was examined recently (59). The results demonstrated that UBE2T is critical to CCA genesis. UBE2T was highly expressed in both in vitro and in vivo human CCA models. The research showed that UBE2T overexpression increased mesenchymal markers vimentin and N-cadherin, and decreased epithelial markers β-catenin and E-cadherin (59). Overexpression of UBE2T promoted EMT, invasion, migration, and proliferation of CCA cells, while suppressing it had the reverse effect. The mTOR inhibitor rapamycin inhibits UBE2T activity, indicating that it operates through the selected target of mTOR pathway. According to this research, UBE2T might act as a carcinogenic driver of CCA formation via boosting EMT and mTOR pathway. These discoveries have helped uncover underlying processes of EMT and CCA progression and identify novel therapeutic targets for CCA treatment.

V-set domain containing T-cell activation inhibitor 1 (VTCN1/B7x/B7S1/B7 homolog 4) (B7-H4)

B7-H4, a novel member of the B7 family (also known as VTCN1). B7-H4 boosts the synthesis of cytokines and T cell proliferation, allowing tumors to evade immune detection (60). B7-H4 is generally absent in normal human tissues, apart from lung, kidney, and pancreatic epithelial cells (61). B7-H4 is highly expressed in lung, breast, and pancreatic ductal adenocarcinoma, according to recent studies (61-63). The role and mechanism of B7-H4 in iCCA, however, remain unknown. The study of the B7-H4 expression and its therapeutic importance in iCCA progression were recently explored by Xie and colleagues (64). The findings showed that both at the mRNA and protein levels, B7-H4 expression in iCCA was significantly greater than in peritumoral tissues. The elevated B7-H4 in iCCA cells induced EMT and increased tumor cell invasion and metastasis via ERK1/2 signaling. Tumor samples with high B7-H4 expression had correlated with poorer differentiation, advanced tumor stage, and lymph node metastases. B7-H4-expressing iCCA patients had lower overall and disease-free survival. B7-H4 expression also independently predicted survival and tumor recurrence in iCCA patients after surgery (64). In conclusion, enhanced iCCA tumor aggressiveness via EMT is associated with increased B7-H4 expression, suggesting that this protein may be an interesting target for therapy for iCCA patients.

WD repeat domain 5 (WDR5)

The histone methyltransferase complex SET1/MLL, which largely catalyzes histone 3 lysine 4 methylation (H3K4me), contains WDR5 as a vital component (65). It promotes tumors and poor prognoses in colorectal, breast, bladder, and prostate cancers (66). Moreover, the recruitment of c-Myc at particular chromosomal locations is promoted by WDR5, a critical regulatory element (67), although the way WDR5 and c-Myc interact in CCA was not previously known. The clinical significance of WDR5 and c-Myc expression in all CCA subtypes was recently studied by Chen et al. (68). Co-expression of WDR5 and c-Myc was a more accurate prognostic indicator, and WDR5 was highly related with poor CCA prognosis. WDR5’s impact on EMT-specific proteins was also examined. WDR5 overexpression had the opposite impact on the expression of EMT biomarkers as compared to WDR5 knockdown, which boosted E-cadherin while decreasing N-cadherin and vimentin. WDR5 increased Myc-induced HIF1A transcription by interacting with the box IIIb (MBIIIb) motif on c-Myc, which promoted CCA EMT, invasion, and metastasis. WDR5 decreased chromatin opening and PHD2 expression while increasing the accumulation of the HIF-1α and perhaps stabilizing and accumulating HIF-1α (68). These findings imply that stratification of high-risk CCA patients and treatment planning can be aided by WDR5, c-Myc, and HIF-1α. CCA patients may benefit from inhibiting the WDR5-Myc interface and HIF-1α accumulation.

Targeting protein for Xenopus kinesin-like protein 2 (TPX2)

Although TPX2 promotes oncogenesis in numerous malignancies, its role in EMT-induced CCA has been challenging to understand. Recently, Zou et al.’s article found increased TPX2 expression in CCA tissues (69). TPX2 upregulation linked with severe TNM stage, lymph node metastases, shorter survival, and poorer prognosis. TPX2 suppression by siRNA lentivirus-infection in CCA cell lines has a significant effect on tumor cell biology (HCCC-9810 and RBE). After TPX2-silencing transfection, CCA cells apoptosed, proliferated, and invaded less. TPX2 knockdown enhanced E-cadherin and decreased N-cadherin, β-catenin, MMP-2, and MMP-9 in TPX2 induced-EMT. Zou and colleagues revealed that TPX2 inhibition down-regulated Slug and Twist1, EMT transcription factors. TPX2 modulates EMT via an unknown mechanism. The sole investigation on TPX2 function in CCA revealed that it may be a predictive indicator and therapeutic target.

Finally, Tables 2-4 summarize the available experimental evidence on the most recent EMT-related proteins that possess the molecular pathways to initiate the EMT process during CCA development.

Discussion

Organ development and cancer are only two examples of the many processes in which EMT plays a role. By understanding about the key regulatory mechanisms and EMT-mediators involved in this process, novel therapeutics can be established. There is no doubt that EMT plays a vital role in CCA development given the massive amount of data presented in this analysis. EMT must be studied extensively because of its potential to induce advanced tumor metastasis and chemoresistance characteristics. New therapy approaches that can halt this cellular transition during CCA progression must be developed, and it is imperative that researchers focus on identifying and targeting the key EMT pathways involved in this cancer.

This narrative review covers seven proteins involved in EMT, most of which can activate this process through the PI3K/AKT/mTOR signaling cascade. These proteins include α7-nAChR, FFAR4, mortalin, PLCB1, PTP4A1, TRIM44, and UBE2T. The PI3K/AKT/mTOR pathway appears to be the most important mechanism for regulating EMT-induced CCA formation. In addition, the Serine/Threonine kinase AKT plays a major role in regulating a wide variety of cellular functions, including proliferation, survival, glucose metabolism, and neovascularization (70,71). Growth factor receptors activate PI3K, a signal transducer enzyme, which in turn phosphorylates AKT (72). Several studies demonstrate that EMT, angiogenesis, and metastasis can be induced by AKT, which is overexpressed in many human malignancies and promotes cancer cell proliferation, metabolism, and survival (73). Furthermore, CCA cells are resistant to radiation therapy and chemotherapy when the PI3K/AKT pathway is activated, but can be made more sensitive to these treatments when the pathway is inhibited (74). In recent studies, a new dual PI3K/mTOR inhibitor known as NVPBEZ235 was shown to drastically limit CCA cell proliferation and migration by inhibiting AKT. Moreover, it significantly triggered G1 arrest without inducing apoptosis while simultaneously enhancing autophagy response (75).

Both in vitro and in vivo investigations have indicated that targeting the cooperation that exists amongst EMT signaling pathways is beneficial (76). For instance, the essential cytokine TGF-β1 is involved in a variety of cellular processes, and it is this cytokine that stimulates the mTOR signaling pathway (77). mTORC2 is a critical downstream effector that is part of the TGF-β signaling cascade. It directly phosphorylates Akt, which in turn promotes EMT (78). In addition, KIAA1199, GLS1, and MPC1 are shown to drive EMT in CCA through the TGF- β1 signaling pathway in this narrative review. Treatment strategies that are based on anti-TGF-β have been examined using preclinical models of CCA. Neutralizing monoclonal antibodies against TGF-β were recently tested in vivo on mice with an induced liver fibrosis model, and the results showed that these animals had lower levels of fibrosis and CCA formation (79). Additionally, M7824 has a dual anti-tumor action in that it boosts immune defenses against the tumor by inhibiting the immunological checkpoint protein programmed cell death ligand-1 (PD-L1). This is accomplished by trapping the TGF-β ligand binding in the tumor microenvironment and preventing it from occurring (80,81). As a result, patients with second-line biliary tract cancer, including CCA, who have locally progressed or metastatic disease, are participating in a recently begun multicenter phase II clinical research (NCT03833661) that is assessing the efficacy of M7824 monotherapy (82).

WNT signaling collaborates with members of the fibroblast growth factor (FGF) and TGF-β families to regulate EMT during gastrulation and neural crest cell delamination (83,84), whereas Notch and TGF-β signaling regulate endocardial cushion formation (85). In cancer cell, there is a synergy between TGF-β signaling and RTK signaling, which is triggered by the epidermal growth factor (EGF)-related TGF-α or FGF. Indeed, TGF-β enhances the epithelial to mesenchymal gene expression shift in cancer cell types by promoting EGF- or FGF-induced EMT (86-88). Similarly, TGF-β induced EMT is promoted by the activation of ERK/MAPK pathway when responding to mutant RAS or growth factors (89). Moreover, CD90, fascin, and HMGA1 in this study can trigger the canonical WNT signaling that binding of WNT ligands to Frizzled receptors inhibits GSK3β, resulting in inhibiting β-catenin phosphorylation, ubiquitylation, and degradation while allowing β-catenin to influence gene expression (90). GSK3β kinase inhibition promotes EMT through increasing SNAIL stability (91). Thus, WNT signaling is important in developmental EMT in cancer. In conclusion, it appears that targeting these signaling pathways might be a promising therapeutic strategy for preventing EMT, metastasis, and invasion in tumor cells.

For CCA, EMT has shown great promise as a therapeutic target. Further work is required to develop combination treatments targeting EMT in CCA due to redundancy and bypasses among the multiple signaling pathways and cell types involved. Even though many studies have failed to find direct evidence that these serum levels predict the prognosis of CCA patients, we believe that the levels of these potential EMT-related proteins may emerge as a novel biomarker in predicting diagnosis and prognosis, as well as a potential therapeutic target for those CCA patients.

Conclusions

This narrative review compiles in vitro and in vivo findings that support the idea that novel EMT-related proteins can trigger CCA progression. In CCA, the expression of epithelial markers can be induced by some of these molecules, while the expression of mesenchymal markers can be reversed by others. This narrative review summarizes how the molecules shown to mediate EMT in CCA formation can generate molecular pathways that ultimately lead to cell proliferation, migration, and invasion. Both pathways ultimately lead to a poorer prognosis in the treatment of CCA, independently and seriously. Hence, future treatment methods to handle anti-tumor-related difficulties in CCA patients may prioritize addressing these EMT-related molecules’ pathways. EMT-related proteins may serve as important molecular markers in the diagnosis, prognosis, and therapy of CCA; however, more study is needed to establish this. Better patient data as well as important and relevant clinical experience are two of these aspects.

Acknowledgments

Funding: This work was supported by National Research Council of Thailand (NRCT).

Footnote

Reporting Checklist: The authors have completed the Narrative Review reporting checklist. Available at https://jgo.amegroups.com/article/view/10.21037/jgo-22-1126/rc

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