Genomic characterization and risk stratification of esophageal squamous dysplasia.

Objectives: The majority of esophageal squamous dysplasia (ESD) patients progress slowly, while a subset of patients can undergo recurrence rapidly or progress to invasive cancer even after proper treatment. However, the molecular mechanisms underlying these clinical observations are still largely unknown. Methods: By sequencing the genomic data of 160 clinical samples from 49 tumor-free ESD patients and 88 esophageal squamous cell carcinoma (ESCC) patients, we demonstrated lower somatic mutation and copy number alteration (CNA) burden in ESD compared with ESCC. Results: Cross-species screening and functional assays identified ACSM5 as a novel driver gene for ESD progression. Furthermore, we revealed that miR-4292 promoted ESD progression and could serve as a non-invasive diagnostic marker for ESD. Conclusions: These findings largely expanded our understanding of ESD genetics and tumorigenesis, which possessed promising significance for improving early diagnosis, reducing overtreatment, and identifying high-risk ESD patients.

Antitumour effects of artesunate via cell cycle checkpoint controls in human oesophageal squamous carcinoma cells.

Artesunate (ART), an effective antimalarial semisynthetic derivative of artemisinin, exhibits antitumour properties, but the mechanism(s) involved remain elusive. In this study, we investigated the antitumour effects of ART on human oesophageal squamous cell carcinoma (ESCC) cell lines. Treatment of ESCC cell lines with ART resulted in the production of excessive reactive oxygen species (ROS) that induced DNA damage, reduced cell proliferation and inhibited clonogenicity via G1-S cell cycle arrest and/or apoptosis in vitro. The administration of ART to nude mice with ESCC cell xenografts inhibited tumour formation in vivo. However, the cytotoxicity of ART strongly differed among the ESCC cell lines tested. Transcriptomic profiling revealed that although the expression of large numbers of genes in ESCC cell lines was affected by ART treatment, these genes could be functionally clustered into pathways involved in regulating cell cycle progression, DNA metabolism and apoptosis. We revealed that p53 and Cdk4/6-p16-Rb cell cycle checkpoint controls were critical determinants required for mediating ART cytotoxicity in ESCC cell lines. Specifically, KYSE30 cells with p53Mut/p16Mut were the most sensitive to ART, KYSE150 and KYSE180 cells with p53Mut/p16Nor exhibited intermediate responses to ART, and Eca109 cells with p53Nor/p16Nor exhibited the most resistance to ATR. Consistently, perturbation of p53 expression using RNA interference (RNAi) and/or Cdk4/6 activity using the inhibitor palbociclib altered ART cytotoxicity in KYSE30 cells. Given that the p53 and Cdk4/6-cyclin D1-p16-Rb genes are commonly mutated in ESCC, our results potentially shed new light on neoadjuvant chemotherapy strategies for ESCC.

Mammalian esophageal stratified tissue homeostasis is maintained distinctively by the epithelial pluripotent p63+Sox2+ and p63-Sox2+ cell populations.

Self-renewing, damage-repair and differentiation of mammalian stratified squamous epithelia are subject to tissue homeostasis, but the regulation mechanisms remain elusive. Here, we investigate the esophageal squamous epithelial tissue homeostasis in vitro and in vivo. We establish a rat esophageal organoid (rEO) in vitro system and show that the landscapes of rEO formation, development and maturation trajectories can mimic those of rat esophageal epithelia in vivo. Single-cell RNA sequencing (scRNA-seq), snapshot immunostaining and functional analyses of stratified "matured" rEOs define that the epithelial pluripotent stem cell determinants, p63 and Sox2, play crucial but distinctive roles for regulating mammalian esophageal tissue homeostasis. We identify two cell populations, p63+Sox2+ and p63-Sox2+, of which the p63+Sox2+ population presented at the basal layer is the cells of origin required for esophageal epithelial stemness maintenance and proliferation, whereas the p63-Sox2+ population presented at the suprabasal layers is the cells of origin having a dual role for esophageal epithelial differentiation (differentiation-prone fate) and rapid tissue damage-repair responses (proliferation-prone fate). Given the fact that p63 and Sox2 are developmental lineage oncogenes and commonly overexpressed in ESCC tissues, p63-Sox2+ population could not be detected in organoids formed by esophageal squamous cell carcinoma (ESCC) cell lines. Taken together, these findings reveal that the tissue homeostasis is maintained distinctively by p63 and/or Sox2-dependent cell lineage populations required for the tissue renewing, damage-repair and protection of carcinogenesis in mammalian esophagi.

Identification and characterization of stem cells in mammalian esophageal stratified squamous epithelia.

Somatic stem cells are essential for the maintenance of tissue homeostasis. Despite its importance, how the esophageal stratified squamous epithelium executes its self-renewal and maintenance remains elusive. In this study, using 5-bromo-2'-deoxyuridine label-chase in rats in vivo and rat esophageal organoids in vitro together with genome-wide DNA methylation and single-cell RNA sequencing, we identified a slow-cycling/quiescent stem cell population that contained high levels of hemidesmosomes (HDs) and low levels of Wnt signaling localized spatially and randomly at the basal layer of the esophageal epithelium. Pseudotime cell trajectory analysis indicated that tissue cells originated from quiescent basal stem cells in the basal layer. Perturbations of HD component expression and/or Wnt signaling reduced the stem cell population in the basal layer of esophageal keratinocyte organoids, resulting in alterations in the organoid formation rate, size, morphogenesis, and proliferation-differentiation homeostasis. Furthermore, not only high levels of HDs and low levels of Wnt signaling but also an interplay between HD and Wnt signaling defined the stem cells of the basal layer. Hence, HDs and Wnt signaling are critical determinants for defining the stem cells of the basal layer required for tissue homeostasis in mammalian esophagi.

Aspirin enhances the therapeutic efficacy of cisplatin in oesophageal squamous cell carcinoma by inhibition of putative cancer stem cells.

BACKGROUND: Cancer stem cells (CSCs) are related to the patient's prognosis, recurrence and therapy resistance in oesophageal squamous cell carcinoma (ESCC). Although increasing evidence suggests that aspirin (acetylsalicylic acid, ASA) could lower the incidence and improve the prognosis of ESCC, the mechanism(s) remains to be fully understood. METHODS: We investigated the role of ASA in chemotherapy/chemoprevention in human ESCC cell lines and an N-nitrosomethylbenzylamine-induced rat ESCC carcinogenesis model. The effects of combined treatment with ASA/cisplatin on ESCC cell lines were examined in vitro and in vivo. Sphere-forming cells enriched with putative CSCs (pCSCs) were used to investigate the effect of ASA in CSCs. Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) was performed to determine the alterations in chromatin accessibility caused by ASA in ESCC cells. RESULTS: ASA inhibits the CSC properties and enhances cisplatin treatment in human ESCC cells. ATAC-seq indicates that ASA treatment results in remarkable epigenetic alterations on chromatin in ESCC cells, especially their pCSCs, through the modification of histone acetylation levels. The epigenetic changes activate Bim expression and promote cell death in CSCs of ESCC. Furthermore, ASA prevents the carcinogenesis of NMBzA-induced ESCC in the rat model. CONCLUSIONS: ASA could be a potential chemotherapeutic adjuvant and chemopreventive drug for ESCC treatment.

Metformin suppresses the esophageal carcinogenesis in rats treated with NMBzA through inhibiting AMPK/mTOR signaling pathway.

Metformin is a widely used antidiabetic drug for the management of type 2 diabetes mellitus. Recently, epidemiological studies demonstrate that metformin has anticancer effects on esophageal squamous cell carcinoma (ESCC) and other cancers. However, the effects and potential mechanisms of metformin on ESCC remain elusive. In this study, we used N-nitroso-N-methylbenzylamine (NMBzA), a special carcinogen for esophagi, to develop a rat ESCC model, in which the carcinogenesis progression of ESCC in rat was induced and promoted. We investigated the effects of metformin on carcinogenesis of ESCC in this model. Our results revealed that metformin significantly decreased the incidence and precancerous lesions of ESCC and inhibited proliferation and promoted apoptosis of esophageal epithelial cells in rat treated with NMBzA. Moreover, metformin also increased apoptosis and inhibited migration, colony formation and tumor sphere formation of human ESCC cells in vitro. Immunohistochemistry and western blotting showed that without interfering the metabolism of NMBzA, metformin inhibited the inflammation of esophagi via reducing the expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and interleukin-6 (IL-6). Treatment of metformin led to activation of AMP-activated protein kinase (AMPK) and attenuated signaling of the downstream molecules such as p-mTOR, p-p70S6K and cyclin D1 expression both in vivo and in vitro. Taken together, our study demonstrated that metformin suppressed the carcinogenesis of ESCC through inhibiting AMPK/mammalian target of the rapamycin (mTOR) signaling pathway, resulting in its chemopreventive effects on the carcinogenesis of ESCC.

DNMT1 ablation suppresses tumorigenesis by inhibiting the self-renewal of esophageal cancer stem cells.

Cancer stem cells (CSCs) have been isolated from many tumors and considered as the main reason of cancer recurrence and metastasis. DNA methyltransferase 1 (DNMT1) mediates DNA methylation and plays an important role in CSCs maintenance. However, the function of DNMT1 in CSCs of esophageal squamous cell carcinoma (ESCC) remains unclear. In this study, we examined the role of DNMT1 in regulating self-renewal in CSCs of ESCC. We found a high expression of DNMT1 in both side population (SP) cells and sphere formation cells that represented as substitutes for CSCs in KYSE150 and EC109 ESCC cell lines. We performed the knockdown of DNMT1 using lentivirus-mediated RNA interference (RNAi) methods. We revealed that ablation of DNMT1 resulted in the numbers and self-renewal abilities of CSCs refrained significantly in ESCC cells. As a result of the CSCs inhibition, the malignant phenotypes such as cell proliferation, colony formation, migration and drug resistance abilities were dramatically inhibited in ESCC cells. Treatment of 5-aza-2'-deoxycytidine (5-aza-dC), a DNMT inhibitor, also resulted in the inhibition of CSCs and malignant profiles in ESCC cells. Our findings also provided the first evidence that 5-aza-dC inhibited the colony and sphere formation of CSCs. Thus, our results indicated that DNMT1 was important for the self-renewal maintenance of CSCs in ESCC, and 5-aza-dC could be a potential therapy for the CSCs of ESCC.

TM4SF1 promotes the self-renewal of esophageal cancer stem-like cells and is regulated by miR-141.

Cancer stem-like cells have been identified in primary human tumors and cancer cell lines. Previously we found TM4SF1 gene was highly expressed in side population (SP) cells from esophageal squamous cell carcinoma (ESCC) cell lines, but the role and underlying mechanism of TM4SF1 in ESCC remain unclear. In this study, we observed TM4SF1 was up-regulated but miR-141 was down-regulated in SP cells isolated from ESCC cell lines. TM4SF1 could stimulate the self-renewal ability and carcinogenicity of esophageal cancer stem-like cells, and promote cell invasion and migration. In miR-141 overexpression cells, the expression of TM4SF1 was significantly reduced. We also found that overexpression of miR-141 could abolish the self-renewal ability and carcinogenicity of esophageal cancer stem-like cells and decrease cell invasion and migration by suppressing TM4SF1. Consequently, TM4SF1 is a direct target gene of miR-141. The regulation of TM4SF1 by miR-141 may play an important role in controlling self-renewals of esophageal cancer stem-like cells. It may also promote the development of new therapeutic strategies and efficient drugs to target ESCC stem-like cells.

Prognostic impact of mutation profiling in patients with stage II and III colon cancer.

Development of colorectal cancer (CRC) associates with accumulation of genetic mutations include the epidermal growth factor receptor (EGFR) signaling pathway. However, whether mutations in KRAS together with downstream factors BRAF, PIK3CA and NRAS impact prognosis is still unclear for stage II-III colon cancer. In the present study a total of 228 stage II-III colon cancer samples were retrospectively collected, KRAS (codons 12, 13 and 61), BRAF (exon 11 and exon 15), PIK3CA (exon 9 and exon 20) and NRAS (codons 12, 13 and 61) status was detected by Sanger sequencing, 37.89% (86/227) tumors harbored a KRAS mutation, 7.02% (16/228) harbored a BRAF mutation, 13.18% (29/220) harbored a PIK3CA mutation and 0.89% (2/224) harbored a NRAS mutation. NRAS mutations existed only in stage II colon cancer. Older groups harbored a higher KRAS and BRAF mutation (P < 0.05), PIK3CA (exon9) mutations appeared more common in worse differentiation tumors (P = 0.032). Moreover, PIK3CA (E545K) mutation was significantly associated with tumor recurrence (P = 0.031) and acted independently prognostic for poor OS (P = 0.044), while only in stage III colon cancer. KRAS, BRAF and NRAS mutations do not have major prognostic value in stage II and III colon cancer, subtypes of gene mutations should be further investigated for a better understanding in CRC.

Assessment of insulin-like growth factor 1 receptor as an oncogene in esophageal squamous cell carcinoma and its potential implication in chemotherapy.

Insulin-like growth factor-1 receptor (IGF-1R) is a tyrosine kinase receptor implicated in the pathogenesis of multiple cancers. After ligand binding, IGF-1R can initiate the activation of the PI3K/AKT/mTOR and Ras/Raf/MEK/MAPK pathways to modulate cell proliferation, survival, differentiation, motility, invasion and angiogenesis. IGF-1R is a prerequisite for tumor progression and is one of the most attractive targets for therapeutic interventions in several types of cancer. In the present study, we determined the expression of IGF-1R in an esophageal squamous cell carcinoma (ESCC) cohort, investigated the detailed function of IGF-1R and screened the potential application of IGF-1R in the clinic. We verified the higher expression of IGF-1R in ESCC tumor tissues as compared to adjacent normal tissues. We also found that high expression of IGF-1R was associated with advanced tumor progression. We used ESCC cell lines and a mouse xenograft model to detect the function of IGF-1R in vitro and in vivo. Our results suggest the oncogenic function of IGF-1R in regulating cell proliferation, clonogenesis, the cell cycle and apoptosis. In addition, we found that IGF-1R was associated with the response to standard chemotherapy drugs 5-FU and cisplatin in an ESCC cell line. More importantly, we confirmed that the serum concentration of IGF-1/IGFBP3 can be used for predicting response to chemotherapy, and increased serum levels of IGF-1 and IGFBP-3 are associated with significantly higher rates of tumor response. In the present study, we demonstrated that IGF-1R is an important oncogene in ESCC and can be used to detect the chemotherapeutic response.