CD271 mRNA/hnRNPA2B1 complex promotes proliferation and stemness in oral and head and neck squamous cell carcinoma.

RNAs, such as noncoding RNA, microRNA, and recently mRNA, have been recognized as signal transduction molecules. CD271, also known as nerve growth factor receptor, has a critical role in cancer, although the precise mechanism is still unclear. Here, we show that CD271 mRNA, but not CD271 protein, facilitates spheroid cell proliferation. We established CD271-/- cells lacking both mRNA and protein of CD271, as well as CD271 protein knockout cells lacking only CD271 protein, from hypopharyngeal and oral squamous cell carcinoma lines. Sphere formation was reduced in CD271-/- cells but not in CD271 protein knockout cells. Mutated CD271 mRNA, which is not translated to a protein, promoted sphere formation. CD271 mRNA bound to hnRNPA2B1 protein at the 3'-UTR region, and the inhibition of this interaction reduced sphere formation. In surgical specimens, the CD271 mRNA/protein expression ratio was higher in the cancerous area than in the noncancerous area. These data suggest CD271 mRNA has dual functions, encompassing protein-coding and noncoding roles, with its noncoding RNA function being predominant in oral and head and neck squamous cell carcinoma.

FAXC interacts with ANXA2 and SRC in mitochondria and promotes tumorigenesis in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is one of the most difficult malignancies to treat as the therapeutic options are limited. Although several driver genes have been identified, most remain unknown. In this study, we identified a failed axon connection homolog (FAXC), whose function is unknown in mammals, by analyzing serially passaged CCA xenograft models. Knockdown of FAXC reduced subcutaneous tumorigenicity in mice. FAXC was bound to annexin A2 (ANXA2) and c-SRC, which are tumor-promoting genes. The FAXC/ANXA2/c-SRC complex forms in the mitochondria. FAXC enhances SRC-dependent ANXA2 phosphorylation at tyrosine-24, and the C-terminal amino acid residues (351-375) of FAXC are required for ANXA2 phosphorylation. Transcriptome data from a xenografted CCA cell line revealed that FAXC correlated with epithelial-mesenchymal transition, hypoxia, and KRAS signaling genes. Collectively, these findings advance our understanding of CCA tumorigenesis and provide candidate therapeutic targets.

CD271 promotes proliferation and migration in bladder cancer.

Bladder cancer is a urothelial cancer and effective therapeutic strategies for its advanced stages are limited. Here, we report that CD271, a neurotrophin receptor, promotes the proliferation and migration of bladder cancer cells. CD271 knockdown decreased proliferation in both adherent and spheroid cultures, and vice versa when CD271 was overexpressed in bladder cancer cell lines. CD271 depletion impaired tumorigenicity in vivo. Migration activity was reduced by CD271 knockdown and TAT-Pep5, a known CD271-Rho GDI-binding inhibitor. Apoptosis was induced by CD271 knockdown. Comprehensive gene expression analysis revealed alterations in E2F- and Myc-related pathways upon CD271 expression. In clinical cases, patients with high CD271 expression showed significantly shortened overall survival. In surgically resected specimens, pERK, a known player in proliferation signaling, colocalizes with CD271. These data indicate that CD271 is involved in bladder cancer malignancy by promoting cell proliferation and migration, resulting in poor prognosis.

Multi-gene Liquid Biopsy to Detect Resistance to First-line Osimertinib in Patients With EGFR-mutated Lung Adenocarcinoma.

BACKGROUND/AIM: Osimertinib is currently used as a first-line treatment for EGFR-mutated non-small cell lung cancer, and the emergence of drug resistance poses a substantial challenge. Liquid biopsy with a multi-gene panel can examine both the molecular mechanisms and possibility of early resistance diagnosis. PATIENTS AND METHODS: We used a molecular barcode library construction kit (Archer® LiquidPlex™) that allowed the analysis of multiple cancer-related genes using cell-free DNA from the plasma samples of patients. We collected plasma from 17 consecutive patients with lung adenocarcinoma at our hospital at various time points and cell-free DNA was extracted and subjected to LiquidPlex analysis. RESULTS: Plasma DNA concentration was not associated with the presence or absence of resistance to osimertinib. The pathological mutations detected using next-generation sequencing in the resistant specimens were in MAP2K1, PIK3CA, TP53, BRAF, and EGFR. Among the recurrent cases, EGFR mutations identified at the initial diagnosis were detected within 6 months before relapse confirmation in four cases (average 88 days). Many of the recurrent cases without detection of known EGFR mutations in the liquid biopsy showed a longer interval between the detection of relapse and the last blood draw for the liquid biopsy (average 255 days). CONCLUSION: Frequent liquid biopsies are useful for identifying known EGFR mutations as markers for early detection of relapse. Several cancer driver mutations were observed, suggesting a variety of mechanisms of resistance in first-line osimertinib-treated lung adenocarcinoma.

PRB inhibited cell proliferation through let-7b-E2F1 in breast cancer.

The presence of progesterone receptor (PR) and PR isoform B (PRB) in breast cancer is generally correlated with better clinical outcomes. Additionally, the significance of hormone-independent effects of PR/PRB correlated with better prognosis has been reported in non-small cell lung cancer (NSCLC). However, the detailed mechanism of that still remains unclear. In this study, we examined how microRNAs (miRNAs) could contribute to tumor inhibition via PR/PRB expression, in order to find miRNAs that have tumor-agnostic effects between breast cancer and NSCLC. We obtained miRNA data using human tissues of breast cancer and NSCLC from The Cancer Genome Atlas (TCGA) database and PCR array from NSCLC patients of our cohort. Subsequently, we examined the function of the miRNA through in vitro study using breast cancer cell lines. As a result, only let-7b expression was significantly correlated with PR expression in both cancers. Additionally, the expression of let-7b significantly inhibited cell proliferation by inducing PR and PRB expression in breast cancer cell lines. However, the positive correlation of let-7b and PRB required a mediated factor, E2 promoter binding factor 1 (E2F1), obtained from TCGA database analysis. In vitro experiments showed that let-7b significantly inhibited E2F1, and E2F1 significantly inhibited PRB. This study revealed that PRB inhibits the proliferation of breast cancer cells by the let-7b-E2F1 interaction. In addition, the immunohistochemical analysis in NSCLC was also consistent with these in vitro data. Our results could contribute to developing novel therapeutic strategies for patients with PR/PRB-positive cancer by targeting let-7b or PRB expression in breast cancer and possibly NSCLC.

Morphometric analysis of nuclear shape irregularity as a novel predictor of programmed death-ligand 1 expression in lung squamous cell carcinoma.

Immune checkpoint inhibitor (ICI) therapy has been established as one of the key treatment strategies for lung squamous cell carcinoma (LUSQ). The status of programmed death-ligand 1 (PD-L1) in tumor cells and/or immune cells using immunohistochemistry has been primarily used as a surrogate marker for determining ICI treatment; however, when the tissues to be examined are small, false-negative results could be unavoidable due to the heterogeneity of PD-L1 immunoreactivity. To overcome this practical limitation, we attempted to explore the status of nuclear atypia evaluated using morphometry as a potential predictor of PD-L1 status in LUSQ. We correlated the parameters related to nuclear atypia with PD-L1 status using two different cohorts of LUSQ patients (95 cases from The Cancer Genome Atlas database and 30 cases from the Miyagi Cancer Center). Furthermore, we studied the gene mutation status to elucidate the genetic profile of PD-L1 predictable cases. The results revealed that nuclear atypia, especially morphometric parameters related to nuclear shape irregularity, including aspect ratio, circularity, roundness, and solidity, were all significantly associated with PD-L1 status. Additionally, LUSQ cases with high PD-L1 expression and pronounced nuclear atypia were significantly associated with C10orf71 and COL14A1 mutations compared with those with low PD-L1 expression and mild nuclear atypia. We demonstrated for the first time that nuclear shape irregularity could represent a novel predictor of PD-L1 expression in LUSQ. Including the morphometric parameters related to nuclear atypia in conjunction with PD-L1 status could help determine an effective ICI therapeutic strategy; however, further investigation is required.

Clinical and genomic features of non-small cell lung cancer occurring in families.

BACKGROUND: Exposure to environmental carcinogens, such as through smoking, is a major factor in the carcinogenesis of non-small cell lung cancer (NSCLC). However, genetic factors may also contribute. METHODS: To identify candidate tumor suppressor genes for NSCLC, we included 23 patients (10 related pairs and 3 individuals) with NSCLC who had other NSCLC-affected first-degree relatives in a local hospital. Exome analyses for both germline and somatic (NSCLC specimens) DNA were performed for 17 cases. Germline exome data of these 17 cases revealed that most of the short variants were identical to the variants in 14KJPN (a Japanese reference genome panel of more than 14 000 individuals) and only a nonsynonymous variant in the DHODH gene, p.A347T, was shared between a pair of NSCLC patients in the same family. This variant is a known pathogenic variant of the gene for Miller syndrome. RESULTS: Somatic genetic alterations in the exome data of our samples showed frequent mutations in the EGFR and TP53 genes. Principal component analysis of the patterns of 96 types of single nucleotide variants (SNVs) suggested the existence of unique mechanisms inducing somatic SNVs in each family. Delineation of mutational signatures of the somatic SNVs with deconstructSigs for the pair of germline pathogenic DHODH variant-positive cases showed that the mutational signatures of these cases included SBS3 (homologous recombination repair defect), SBS6, 15 (DNA mismatch repair), and SBS7 (ultraviolet exposure), suggesting that disordered pyrimidine production causes increased errors in DNA repair systems in these cases. CONCLUSION: Our results suggest the importance of the detailed collection of data on environmental exposure along with genetic information on NSCLC patients to identify the unique combinations that cause lung tumorigenesis in a particular family.

BEX2 is poor prognostic factor and required for cancer stemness in gastric cancer.

Gastric cancer is the fifth most common malignancy worldwide. However, targeted therapy for advanced gastric cancer is still limited. Here, we report BEX2 (Brain expressed X-linked 2) as a poor prognostic factor in two gastric cancer cohorts. BEX2 expression was increased in spheroid cells, and its knockdown decreased aldefluor activity and cisplatin resistance. BEX2 was found to upregulate CHRNB2 (Cholinergic Receptor Nicotinic Beta 2 Subunit) expression, a cancer stemness-related gene, in a transcriptional manner, and the knockdown of which also decreases aldefluor activity. Collectively, these data are suggestive of the role of BEX2 in the malignant process of gastric cancer, and as a promising therapeutic target.

RELA is required for CD271 expression and stem-like characteristics in hypopharyngeal cancer.

CD271 (also referred to as nerve growth factor receptor or p75NTR) is expressed on cancer stem cells in hypopharyngeal cancer (HPC) and regulates cell proliferation. Because elevated expression of CD271 increases cancer malignancy and correlates with poor prognosis, CD271 could be a promising therapeutic target; however, little is known about the induction of CD271 expression and especially its promoter activity. In this study, we screened transcription factors and found that RELA (p65), a subunit of nuclear factor kappaB (NF-κB), is critical for CD271 transcription in cancer cells. Specifically, we found that RELA promoted CD271 transcription in squamous cell carcinoma cell lines but not in normal epithelium and neuroblastoma cell lines. Within the CD271 promoter sequence, region + 957 to + 1138 was important for RELA binding, and cells harboring deletions in proximity to the + 1045 region decreased CD271 expression and sphere-formation activity. Additionally, we found that clinical tissue samples showing elevated CD271 expression were enriched in RELA-binding sites and that HPC tissues showed elevated levels of both CD271 and phosphorylated RELA. These data suggested that RELA increases CD271 expression and that inhibition of RELA binding to the CD271 promoter could be an effective therapeutic target.

PP6 deficiency in mice with KRAS mutation and Trp53 loss promotes early death by PDAC with cachexia-like features.

To examine effects of PP6 gene (Ppp6c) deficiency on pancreatic tumor development, we developed pancreas-specific, tamoxifen-inducible Cre-mediated KP (KRAS(G12D) plus Trp53-deficient) mice (cKP mice) and crossed them with Ppp6cflox / flox mice. cKP mice with the homozygous Ppp6c deletion developed pancreatic tumors, became emaciated and required euthanasia within 150 days of mutation induction, phenotypes that were not seen in heterozygous or wild-type (WT) mice. At 30 days, a comparative analysis of genes commonly altered in homozygous versus WT Ppp6c cKP mice revealed enhanced activation of Erk and NFκB pathways in homozygotes. By 80 days, the number and size of tumors and number of precancerous lesions had significantly increased in the pancreas of Ppp6c homozygous relative to heterozygous or WT cKP mice. Ppp6c-/- tumors were pathologically diagnosed as pancreatic ductal adenocarcinoma (PDAC) undergoing the epithelial-mesenchymal transition (EMT), and cancer cells had invaded surrounding tissues in three out of six cases. Transcriptome and metabolome analyses indicated an enhanced cancer-specific glycolytic metabolism in Ppp6c-deficient cKP mice and the increased expression of inflammatory cytokines. Individual Ppp6c-/- cKP mice showed weight loss, decreased skeletal muscle and adipose tissue, and increased circulating tumor necrosis factor (TNF)-α and IL-6 levels, suggestive of systemic inflammation. Overall, Ppp6c deficiency in the presence of K-ras mutations and Trp53 gene deficiency promoted pancreatic tumorigenesis with generalized cachexia and early death. This study provided the first evidence that Ppp6c suppresses mouse pancreatic carcinogenesis and supports the use of Ppp6c-deficient cKP mice as a model for developing treatments for cachexia associated with pancreatic cancer.

Establishment of a monoclonal antibody against glycosylated CD271 specific for cancer cells in immunohistochemistry.

Various proteins are highly expressed in cancer (e.g., epidermal growth factor receptor); however, the majority are also expressed in normal cells, although they may differ in expression intensity. Recently, we reported that CD271 (nerve growth factor receptor), a glycosylated protein, increases malignant behavior of cancer, particularly stemlike phenotypes in squamous cell carcinoma (SCC). CD271 is expressed in SCC and in normal epithelial basal cells. Glycosylation alterations generally occur in cancer cells; therefore, we attempted to establish a cancer-specific anti-glycosylated CD271 antibody. We purified recombinant glycosylated CD271 protein, immunized mice with the protein, and screened hybridomas using an ELISA assay with cancer cell lines. We established a clone G4B1 against CD271 which is glycosylated with O-glycan and sialic acid. The G4B1 antibody reacted with the CD271 protein expressed in esophageal cancer, but not in normal esophageal basal cells. This specificity was confirmed in hypopharyngeal and cervical cancers. G4B1 antibody recognized the fetal esophageal epithelium and Barrett's esophagus, which possess stem cell-like characteristics. In conclusion, G4B1 antibody could be useful for precise identification of dysplasia and cancer cells in SCC.

A novel 8.57-kb deletion of the upstream region of PRKAR1A in a family with Carney complex.

Carney complex (CNC) is a rare hereditary syndrome that involves endocrine dysfunction and the development of various types of tumors. Chromosome 2p16 and PRKAR1A on chromosome 17 are known susceptibility loci for CNC. Here we report a mother and son with CNC caused by an 8.57-kb deletion involving the transcription start site and non-coding exon 1 of PRKAR1A. The proband is a 28-year-old male with bilateral large-cell calcified Sertoli cell testicular tumors and pituitary adenoma. Comprehensive genomic profiling for cancer mutations using Foundation One CDx failed to detect any mutations in PRKAR1A in DNA from the testicular tumor. Single-nucleotide polymorphism array analysis of the proband's genomic DNA revealed a large deletion in the 5' region of PRKAR1A. Genomic walking further delineated the region an 8.57-kb deletion. A 1.68-kb DNA fragment encompassed by the deleted region showed strong promoter activity in a NanoLuc luciferase reporter assay. The patient's mother, who is suffering from recurrent cardiac myxoma, a critical sign for CNC, carried an identical deletion. The 8.57-kb deleted region is a novel lesion for CNC and will facilitate molecular diagnosis of the disease.

Generation of a lung squamous cell carcinoma three-dimensional culture model with keratinizing structures.

Tumor nests in lung squamous cell carcinoma (LUSC) have a hierarchical structure resembling squamous epithelium. The nests consist of basal-like cells on the periphery and layers of keratinocyte-like cells that differentiate towards the center of the nest, forming keratin pearls. Reproducing this spatial heterogeneity in in vitro models would be useful for understanding the biology of LUSC. Here, we established a three-dimensional (3D) culture model with a squamous epithelial structure using LUSC cell lines PLR327F-LD41 and MCC001F, established in-house. When PLR327F-LD41 cells were cultured in a mixture of Matrigel and collagen I, they generated 3D colonies (designated cancer organoids, or COs) with involucrin (IVL)-positive keratinizing cells in the center (IVLinner COs). COs with uniform size were generated by seeding PLR327F-LD41 cells in a form of small cell aggregates. Since Notch signaling induces the differentiation of squamous epithelium, we confirmed the effect of γ-secretase inhibitor in inhibiting Notch signaling in IVLinner COs. Surprisingly, γ-secretase inhibitor did not block induction of IVL-positive cells; however, cells residing between the CK5-positive basal-like layer and IVL-positive layer decreased significantly. Thus, our 3D culture model with uniform size and structure promises to be a useful tool for elucidating the biology of LUSC and for screening drug-candidates.

Liquid biopsy with droplet digital PCR targeted to specific mutations in plasma cell-free tumor DNA can detect ovarian cancer recurrence earlier than CA125.

OBJECTIVE: Ovarian cancer (OC) is an intractable gynecological tumor, and frequent recurrence is experienced within a few years even after the complete eradication of tumor tissues by radical resection and neo-adjuvant chemotherapies. The conventional recurrence marker, CA125, is widely used for follow-up after resection of OC, but CA125 has a long half-life in blood and lacks dynamic responses to tumor recurrence. Recent developments in liquid biopsy procedures are expected to overcome the difficulties in early diagnosis of OC recurrence after surgery. METHODS: We applied droplet digital PCR (ddPCR) technology to detect circulating tumor-derived DNA in OC patients' plasma during follow-up. Exome sequencing of 11 tumor-normal pairs of genomic DNA from consecutive OC patients identified tumor-specific mutations, and ddPCR probes were selected for each sample. RESULTS: Six of 11 cases showed apparent recurrence during follow-up (mean progression-free survival was 348.3 days) and all six cases were positive in ddPCR analyses. In addition, ddPCR became positive before increased plasma CA125 in five out of six cases. Increased allele frequency of circulating tumor DNA (ctDNA) is associated with increased tumor volume after recurrence. ddPCR detected ctDNA signals significantly earlier than increased CA125 in the detection of OC recurrence by imaging (49 days and 7 days before, respectively: p < 0.05). No ctDNA was detected in the plasma of recurrence-free cases. CONCLUSIONS: Our results demonstrate the potential of identifying ctDNA by ddPCR as an early detection tool for OC recurrence.

BEX2 is required for maintaining dormant cancer stem cell in hepatocellular carcinoma.

Cancer stem cells (CSCs) are responsible for therapy resistance and share several properties with normal stem cells. Here, we show that brain-expressed X-linked gene 2 (BEX2), which is essential for dormant CSCs in cholangiocarcinoma, is highly expressed in human hepatocellular carcinoma (HCC) lesions compared with the adjacent normal lesions and that in 41 HCC cases the BEX2high expression group is correlated with a poor prognosis. BEX2 localizes to Ki67-negative (nonproliferative) cancer cells in HCC tissues and is highly expressed in the dormant fraction of HCC cell lines. Knockdown of BEX2 attenuates CSC phenotypes, including sphere formation ability and aldefluor activity, and BEX2 overexpression enhances these phenotypes. Moreover, BEX2 knockdown increases cisplatin sensitivity, and BEX2 expression is induced by cisplatin treatment. Taken together, these data suggest that BEX2 induces dormant CSC properties and affects the prognosis of patients with HCC.

Ppp6c deficiency accelerates K-rasG12D -induced tongue carcinogenesis.

BACKGROUND: Effective treatments for cancer harboring mutant RAS are lacking. In Drosophila, it was reported that PP6 suppresses tumorigenicity of mutant RAS. However, the information how PP6 regulates oncogenic RAS in mammals is limited. METHODS: We examined the effects of PP6 gene (Ppp6c) deficiency on tongue tumor development in K (K-rasG12D)- and KP (K-rasG12D + Trp53-deficient)-inducible mice. RESULTS: Mice of K and KP genotypes developed squamous cell carcinoma in situ in the tongue approximately 2 weeks after the induction of Ppp6c deficiency and was euthanized due to 20% loss of body weight. Transcriptome analysis revealed significantly different gene expressions between tissues of Ppp6c-deficient tongues and those of Ppp6c wild type, while Trp53 deficiency had a relatively smaller effect. We then analyzed genes commonly altered by Ppp6c deficiency, with or without Trp53 deficiency, and identified a group concentrated in KEGG database pathways defined as 'Pathways in Cancer' and 'Cytokine-cytokine receptor interaction'. We then evaluated signals downstream of oncogenic RAS and those regulated by PP6 substrates and found that in the presence of K-rasG12D, Ppp6c deletion enhanced the activation of the ERK-ELK1-FOS, AKT-4EBP1, and AKT-FOXO-CyclinD1 axes. Ppp6c deletion combined with K-rasG12D also enhanced DNA double-strand break (DSB) accumulation and activated NFκB signaling, upregulating IL-1ß, COX2, and TNF.

Robustness of a Cancer Profiling Test Using Formalin-fixed Paraffin Embedded Tumor Specimens.

BACKGROUND/AIM: Cancer profiling tests using formalin-fixed paraffin-embedded (FFPE) specimens with various conditions have become an essential tool for cancer treatment. The robustness of these tests needs to be addressed. MATERIALS AND METHODS: A cancer profiling test, NCC oncopanel, was tested with FFPE specimens from various tissues with different storage conditions and fixation lengths. Next generation sequencing was performed with Miseq and the data were assembled using the human reference genome hg19. RESULTS: Duration of storage and fixation affected the mapping statistics. Prolonged storage increased outward read paring and longer fixation rates caused increased singletons and unmapped reads. CONCLUSION: Our results indicate that a cancer profiling test with target capturing method, NCC oncopanel, shows robustness for FFPE cancer specimens with various storage conditions.

Gilteritinib overcomes lorlatinib resistance in ALK-rearranged cancer.

ALK gene rearrangement was observed in 3%-5% of non-small cell lung cancer patients, and multiple ALK-tyrosine kinase inhibitors (TKIs) have been sequentially used. Multiple ALK-TKI resistance mutations have been identified from the patients, and several compound mutations, such as I1171N + F1174I or I1171N + L1198H are resistant to all the approved ALK-TKIs. In this study, we found that gilteritinib has an inhibitory effect on ALK-TKI-resistant single mutants and I1171N compound mutants in vitro and in vivo. Surprisingly, EML4-ALK I1171N + F1174I compound mutant-expressing tumors were not completely shrunk but regrew within a short period of time after alectinib or lorlatinib treatment. However, the relapsed tumor was markedly shrunk after switching to the gilteritinib in vivo model. In addition, gilteritinib was effective against NTRK-rearranged cancers including entrectinib-resistant NTRK1 G667C-mutant and ROS1 fusion-positive cancer.

Discovery of a chemical compound that suppresses expression of BEX2, a dormant cancer stem cell-related protein.

Cancer stem cells (CSCs) are believed to cause cancer metastasis and recurrence. BEX2 (brain expressed X-linked gene 2) is a CSC-related gene that is expressed in dormant CSCs in cholangiocarcinoma and induces resistance against chemotherapy. The aim of the present study was to identify small compounds that have activity to inhibit BEX2 expression and result in the attenuation of CSC-related phenotypes. We screened 9600 small chemical compounds in high-throughput screening using cholangiocarcinoma cell line HuCCT1 expressing BEX2 protein fused with NanoLuc, and identified a compound, BMPP (1, 3-Benzenediol, [4-(4-methoxyphenyl)-1H-pyrazol-3-yl]). BMPP was found to exert decreasing effects on BEX2 protein expression and G0 phase population of the tumor cells, and increasing effects on ATP levels and chemotherapeutic sensitivity of the cells. These findings indicate that BMPP is a valuable chemical compound for reducing dormant CSC-related phenotypes. Thus, the identification of BMPP as a potential CSC suppressor provides scope for the development of novel therapeutic modalities for the treatment of cancers with BEX2 overexpressing CSCs.

Cancer Stem Cells in Intrahepatic Cholangiocarcinoma; Their Molecular Basis, and Therapeutic Implications.

Cancer tissue consists of heterogenous cell types, and cancer stem cells (CSCs) are a subpopulation of the tissue which possess therapy resistance, tumor reconstruction capability, and are responsible for metastasis. Intrahepatic cholangiocarcinoma (iCCA) is one of the most common type of liver cancer that is highly aggressive with poor prognosis. Since no target therapy is efficient in improving patient outcomes, new therapeutic approaches need to be developed. CSC is thought to be a promising therapeutic target because of its resistance to therapy. Accumulating evidences suggests that there are many factors (surface marker, stemness-related genes, etc.) and mechanisms (epithelial-mesenchymal transition, mitochondria activity, etc.) which are linked to CSC-like phenotypes. Nevertheless, limited studies are reported about the application of therapy using these mechanisms, suggesting that more precise understandings are still needed. In this review, we overview the molecular mechanisms which modulate CSC-like phenotypes, and discuss the future perspective for targeting CSC in iCCA.