ΔNp63-restricted viral mimicry response impedes cancer cell viability and remodels tumor microenvironment in esophageal squamous cell carcinoma.

Tumor protein p63 isoform ΔNp63 plays roles in the squamous epithelium and squamous cell carcinomas (SCCs), including esophageal SCC (ESCC). By integrating data from cell lines and our latest patient-derived organoid cultures, derived xenograft models, and clinical sample transcriptomic analyses, we identified a novel and robust oncogenic role of ΔNp63 in ESCC. We showed that ΔNp63 maintains the repression of cancer cell endogenous retrotransposon expression and cellular double-stranded RNA sensing. These subsequently lead to a restricted cancer cell viral mimicry response and suppressed induction of tumor-suppressive type I interferon (IFN-I) signaling through the regulations of Signal transducer and activator of transcription 1, Interferon regulatory factor 1, and cGAS-STING pathway. The cancer cell ΔNp63-IFN-I signaling axis affects both the cancer cell and tumor-infiltrating immune cell (TIIC) compartments. In cancer cells, depletion of ΔNp63 resulted in reduced cell viability. ΔNp63 expression is negatively associated with the anticancer responses to viral mimicry booster treatments targeting cancer cells. In the tumor microenvironment, cancer cell TP63 expression negatively correlates with multiple TIIC signatures in ESCC clinical samples. ΔNp63 depletion leads to increased cancer cell antigen presentation molecule expression and enhanced recruitment and reprogramming of tumor-infiltrating myeloid cells. Similar IFN-I signaling and TIIC signature association with ΔNp63 were also observed in lung SCC. These results support the potential application of ΔNp63 as a therapeutic target and a biomarker to guide candidate anticancer treatments exploring viral mimicry responses.

Multigene Profiling of Circulating Tumor Cells in Esophageal Squamous Cell Carcinoma Identifies Prognostic Cancer Driver Genes Associated with Epithelial-Mesenchymal-Transition Progression and Chemoresistance.

We investigated the clinical significance of CTCs in cancer progression by detecting multiple cancer driver genes associated with epithelial-to-mesenchymal transition (EMT) at the transcript level. The 10-gene panel, comprising CCND1, ECT2, EpCAM, FSCN1, KRT5, KRT18, MET, TFRC, TWIST1, and VEGFC, was established for characterizing CTCs from mouse ESCC xenograft models and clinical ESCC peripheral blood (PB) samples. Correlations between gene expression in CTCs from PB samples (n = 77) and clinicopathological features in ESCC patients (n = 55) were examined. The presence of CTCs at baseline was significantly correlated with tumor size (p = 0.031). The CTC-high patients were significantly correlated with advanced cancer stages (p = 0.013) and distant metastasis (p = 0.029). High mRNA levels of TWIST1 (Hazard Ratio (HR) = 5.44, p = 0.007), VEGFC (HR = 6.67, p < 0.001), TFRC (HR = 2.63, p = 0.034), and EpCAM (HR = 2.53, p = 0.041) at baseline were significantly associated with a shorter overall survival (OS) in ESCC patients. This study also revealed that TWIST1 facilitates EMT and enhances malignant potential by promoting tumor migration, invasion, and cisplatin chemoresistance through the TWIST1-TGFBI-ZEB1 axis in ESCC, highlighting the prognostic and therapeutic potential of TWIST1 in clinical ESCC treatment.

Depletion of DNA Polymerase Theta Inhibits Tumor Growth and Promotes Genome Instability through the cGAS-STING-ISG Pathway in Esophageal Squamous Cell Carcinoma.

Overexpression of the specialized DNA polymerase theta (POLQ) is frequent in breast, colon and lung cancers and has been correlated with unfavorable clinical outcomes. Here, we aimed to determine the importance and functional role of POLQ in esophageal squamous cell carcinoma (ESCC). Integrated analysis of four RNA-seq datasets showed POLQ was predominantly upregulated in ESCC tumors. High expression of POLQ was also observed in a cohort of 25 Hong Kong ESCC patients and negatively correlated with ESCC patient survival. POLQ knockout (KO) ESCC cells were sensitized to multiple genotoxic agents. Both rH2AX foci staining and the comet assay indicated a higher level of genomic instability in POLQ-depleted cells. Double KO of POLQ and FANCD2, known to promote POLQ recruitment at sites of damage, significantly impaired cell proliferation both in vitro and in vivo, as compared to either single POLQ or FANCD2 KOs. A significantly increased number of micronuclei was observed in POLQ and/or FANCD2 KO ESCC cells. Loss of POLQ and/or FANCD2 also resulted in the activation of cGAS and upregulation of interferon-stimulated genes (ISGs). Our results suggest that high abundance of POLQ in ESCC contributes to the malignant phenotype through genome instability and activation of the cGAS pathway.

Differentiation-related zinc finger protein 750 suppresses cell growth in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a deadly squamous cell carcinoma (SCC) of the esophagus. Development of SCCs is associated with the deregulation of the squamous cell lineage program and/or keratinocyte terminal differentiation by genomic and genetic aberrations; thus, these processes must be tightly controlled to maintain normal squamous cell development. Zinc finger protein 750 (ZNF750) is a gene involved in keratinocyte terminal differentiation and is frequently mutated and putatively silenced in ESCC, which implicates its function as a potential differentiation-related suppressor of ESCC. The present study aimed to elucidate the relationship between ZNF750 function to induce keratinocyte differentiation and tumor suppression in ESCC. The results demonstrated that chemical manipulation of esophageal keratinocyte differentiation in mouse normal esophageal epithelial organoids (mNEEO) implicated the involvement of the mouse homologue of ZNF750, Zfp750, in keratinocyte differentiation in premalignant cells. Bioinformatics analyses of data from high ZNF750-expressing ESCC tumors obtained from public databases and ZNF750-overexpressing ESCC cells compared with low ZNF750-expressing ESCC tumors and GFP-expressing ESCC cells, respectively, revealed enrichment of keratinocyte differentiation-related gene sets in these samples. Finally, the induction through to terminal differentiation of the keratinocyte by all-trans retinoic acid on parental ESCC cell lines led to the upregulation of the terminal differentiation marker Involucrin and a decrease in cell viability similar to that observed in ZNF750-overexpressing ESCC cells. The results of the present study demonstrated a functional link between the ability of ZNF750 to induce cell differentiation through to terminal differentiation and its function as a growth suppressor in ESCC. This study provides improved understanding of the role of ZNF750, a frequently mutated differentiation-related gene in ESCC, and its effects in ESCC pathogenesis.

Gain-of-function hot spot mutant p53R248Q regulation of integrin/FAK/ERK signaling in esophageal squamous cell carcinoma.

PURPOSE: TP53, encoding the protein p53, is among the most frequently mutated genes in all cancers. A high frequency of 60 - 90% mutations is seen in esophageal squamous cell carcinoma (ESCC) patients. Certain p53 mutants show gain-of-function (GoF) oncogenic features unrelated to its wild type functions. METHODS: This study functionally characterized a panel of p53 mutants in individual ESCC cell lines and assayed for GoF oncogenic properties. RESULTS: The ESCC cell line with endogenous p53R248Q expression showed suppressed tumor growth in an immunocompromised mouse model and suppressed colony growth in in vitro three-dimensional culture, when depleted of the endogenous p53 protein expression. This suppression is accompanied by suppressed cell cycle progression, along with reduced integrin expression and decreased focal adhesion kinase and extracellular-regulated protein kinase signaling and can be compensated by expression of a constitutively active mitogen-activated protein. P53R248Q enhances cell proliferation upon glutamine deprivation, as compared to other non-GoF mutants. CONCLUSIONS: In summary, study of the functional contributions of endogenous p53 mutants identified a novel GoF mechanism through which a specific p53 mutant exerts oncogenic features and contributes to ESCC tumorigenesis.

Nasopharyngeal carcinoma MHC region deep sequencing identifies HLA and novel non-HLA TRIM31 and TRIM39 loci.

Despite pronounced associations of major histocompatibility complex (MHC) regions with nasopharyngeal carcinoma (NPC), causal variants underlying NPC pathogenesis remain elusive. Our large-scale comprehensive MHC region deep sequencing study of 5689 Hong Kong Chinese identifies eight independent NPC-associated signals and provides mechanistic insight for disrupted transcription factor binding, altering target gene transcription. Two novel protective variants, rs2517664 (Trs2517664 = 4.6%, P = 6.38 × 10-21) and rs117495548 (Grs117495548 = 3.0%, P = 4.53 × 10-13), map near TRIM31 and TRIM39/TRIM39-RPP21; multiple independent protective signals map near HLA-B including a previously unreported variant, rs2523589 (P = 1.77 × 10-36). The rare HLA-B*07:05 allele (OR < 0.015, P = 5.83 × 10-21) is absent in NPC, but present in controls. The most prevalent haplotype lacks seven independent protective alleles (OR = 1.56) and the one with additional Asian-specific susceptibility rs9391681 allele (OR = 2.66) significantly increased NPC risk. Importantly, this study provides new evidence implicating two non-human leukocyte antigen (HLA) genes, E3 ubiquitin ligases, TRIM31 and TRIM39, impacting innate immune responses, with NPC risk reduction, independent of classical HLA class I/II alleles.

FANCD2 Confers a Malignant Phenotype in Esophageal Squamous Cell Carcinoma by Regulating Cell Cycle Progression.

Fanconi anemia patients with germline genetic defects in FANCD2 are highly susceptible to cancers. Esophageal squamous cell carcinoma (ESCC) is a deadly cancer. Little is known about the function of FANCD2 in ESCC. For detailed molecular and mechanistic insights on the functional role of FANCD2 in ESCC, in vivo and in vitro assays and RNA sequencing approaches were used. Utilizing Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) technology, FANCD2 knockout models were established to examine the functional impact in mouse models for tumor growth and metastasis and in vitro assays for cell growth, cell cycle, and cellular localization. Our RNA sequence analyses were integrated with public datasets. FANCD2 confers a malignant phenotype in ESCC. FANCD2 is significantly upregulated in ESCC tumors, as compared to normal counterparts. Depletion of FANCD2 protein expression significantly suppresses the cancer cell proliferation and tumor colony formation and metastasis potential, as well as cell cycle progression, by involving cyclin-CDK and ATR/ATM signaling. FANCD2 translocates from the nucleus to the cytoplasm during cell cycle progression. We provide evidence of a novel role of FANCD2 in ESCC tumor progression and its potential usefulness as a biomarker for ESCC disease management.

Clinical Outcome-Related Mutational Signatures Identified by Integrative Genomic Analysis in Nasopharyngeal Carcinoma.

PURPOSE: Investigation of biological mechanisms underlying genetic alterations in cancer can assist the understanding of etiology and identify the potential prognostic biomarkers. EXPERIMENTAL DESIGN: We performed an integrative genomic analysis for a total of 731 nasopharyngeal carcinoma cases from five independent nasopharyngeal carcinoma cohorts to identify the genetic events associated with clinical outcomes. RESULTS: In addition to the known mutational signatures associated with aging, APOBEC and mismatch repair (MMR), a new signature for homologous recombination deficiency (BRCAness) was discovered in 64 of 216 (29.6%) cases in the discovery set including three cohorts. This signature appeared more frequently in the recurrent and metastatic tumors and significantly correlated with shorter overall survival (OS) in the primary tumors. Independent prognostic value of MMR and BRCAness signatures was revealed by multivariable Cox analysis after adjustment for clinical parameters and stratification by studies. The cases with both signatures had much worse clinical outcome than those without these signatures [hazard ratio (HR), 12.4; P = 0.002]. This correlation was confirmed in the validation set (HR, 8.9; P = 0.003). The BRCAness signature is highly associated with BRCA2 pathogenic germline or somatic alterations (7.8% vs. 0%; P = 0.002). Targeted sequencing results from a prospective nasopharyngeal carcinoma cohort (N = 402) showed that the cases carrying BRCA2 germline rare variants are more likely to have poor OS and progression-free survival. CONCLUSIONS: Our study highlights importance of defects of DNA repair machinery in nasopharyngeal carcinoma pathogenesis and their prognostic values for clinical implications. These signatures will be useful for patient stratification to evaluate conventional and new treatment for precision medicine in nasopharyngeal carcinoma.

Cylindromatosis Lysine 63 Deubiquitinase (CYLD) Regulates NF-kB Signaling Pathway and Modulates Fibroblast and Endothelial Cells Recruitment in Nasopharyngeal Carcinoma.

Nasopharyngeal carcinoma (NPC) is a malignant epithelial carcinoma of the nasopharynx. Cylindromatosis lysine 63 deubiquitinase (CYLD), a NF-kB inhibitor, was reported as one of the top mutated candidate genes in NPC. NF-kB is an inducible transcription factor, contributing to cancer via regulating inflammation, angiogenesis, cell proliferation, and metastasis. In this study, the impact of CYLD on regulating the NF-kB signaling pathway and its contribution to NPC development was studied using in vitro and in vivo functional assays, together with single cell RNA sequencing to understand the NPC tumor microenvironment. CYLD was downregulated in NPC clinical specimens and multiple cell lines. Functional assays revealed CYLD inhibits NPC cell proliferation and migration in vitro and suppresses NPC tumorigenicity and metastasis in vivo by negatively regulating the NF-kB signaling pathway. Additionally, CYLD was able to inhibit fibroblast and endothelial stromal cell infiltration into the NPC tumor microenvironment. These findings suggest that CYLD inhibits NPC development and provides strong evidence supporting a role for CYLD inhibiting fibroblast and endothelial stromal cell infiltration into NPC via suppressing the NF-kB pathway.

Endoplasmic reticulum-localized ECM1b suppresses tumor growth and regulates MYC and MTORC1 through modulating MTORC2 activation in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a deadly disease with dismal 5-year survival. Extracellular matrix protein 1 (ECM1) was identified as one of the most downregulated genes by transcriptomic analysis of normal esophageal/ESCC paired tissue samples. ECM1 plays oncogenic roles in cancer development in various cancer types. However, little is known about its role in ESCC. In vivo and in vitro functional assays coupled with analyses on public datasets and detailed molecular and mechanistic analyses were used to study the gene. We demonstrate that as opposed to the previously identified oncogenic role of ECM1a, ECM1b is a novel tumor suppressor in ESCC. ECM1 is significantly downregulated in ESCC and several other squamous cell carcinomas. ECM1b encodes a cellular protein that suppresses MYC protein expression and MTORC1 signaling activity. MTORC2 inactivation leads to suppressed MYC expression and MTORC1 signaling. ECM1b localizes to the endoplasmic reticulum and suppresses MTORC2 activation by inhibiting MTORC2/ribosome association. By regulating MTORC2/MYC/MTORC1 signaling, ECM1b suppresses general protein translation and enhances chemosensitivity. We provide evidence establishing a novel role of ECM1 in cancer that suggests ECM1b as a biomarker for ESCC disease management.

Chemotherapeutic Treatments Increase PD-L1 Expression in Esophageal Squamous Cell Carcinoma through EGFR/ERK Activation.

The current study reveals the clinicopathological association of PD-L1 in Hong Kong esophageal squamous cell carcinoma (ESCC) patients and the differential regulation of PD-L1 by standard first-line chemotherapy in ESCC. Immunohistochemical analysis of tissue microarray data from 84 Hong Kong ESCC patients shows that PD-L1 was expressed in 21% of the tumors. Positive PD-L1 staining was significantly associated with later disease stage (stages III and IV) (P value = .0379) and lymph node metastasis (P value = .0466) in the Hong Kong cohort. Furthermore, PD-L1 expression was significantly induced in ESCC cell lines after standard chemotherapy treatments, along with EGFR and ERK activation in both in vitro studies and the in vivo esophageal orthotopic model. The endogenous expression of PD-L1 was reduced by treatment with an EGFR inhibitor (erlotinib) or by the knockdown of EGFR. Moreover, the upregulation of PD-L1 by chemotherapy was also attenuated by the treatment with erlotinib and a MAPK/MEK inhibitor (AZD6244), suggesting that PD-L1 is regulated by the EGFR/ERK pathway in ESCC. The regulation of PD-L1 by the EGFR pathway was further supported by the correlation of PD-L1 and EGFR expression observed in the commercially available tissue microarray set (P value = .028). Taken together, the current study was the first to demonstrate the upregulation of PD-L1 by chemotherapy in ESCC and its regulation through the EGFR/ERK pathway. The results suggest the potential usefulness of combined conventional chemotherapy together with anti-PD-L1 immunotherapy to achieve better treatment outcome.

Corrigendum to "A Versatile Orthotopic Nude Mouse Model for Study of Esophageal Squamous Cell Carcinoma".

[This corrects the article DOI: 10.1155/2015/910715.].

DESC1, a novel tumor suppressor, sensitizes cells to apoptosis by downregulating the EGFR/AKT pathway in esophageal squamous cell carcinoma.

Esophageal cancer is ranked as the eighth most common cancer and the sixth leading cause of cancer deaths worldwide. To identify candidate tumor suppressor genes related to esophageal squamous cell carcinoma (ESCC) development, a cDNA microarray analysis was performed using paired tumor and nontumor tissue samples from ESCC patients. Differentially expressed in squamous cell carcinoma 1 (DESC1), which belongs to the Type II transmembrane serine protease family, was frequently downregulated in ESCC. This study aims to elucidate the molecular mechanism for the tumor suppressive function of DESC1 in ESCC. We show that DESC1 reduced cell viability and sensitized cells to apoptosis, when cells were under apoptotic stimuli. The proapoptotic effect of DESC1 was mediated through downregulating AKT1 activation and the restoration of AKT activation by the introduction of the constitutively active AKT, myr-AKT, abolished the apoptosis-sensitizing effect of DESC1. DESC1 also reduced EGFR protein level, which was abrogated when the proteolytic function of DESC1 was lost, suggesting that DESC1 cleaved EGFR and downregulated the EGFR/AKT pathway to favor apoptosis. The transmembrane localization and the structural domains provide an opportunity for DESC1 to interact with the extracellular environment. The importance of such interaction was highlighted by the finding that DESC1 reduced cell colony formation ability in three-dimensional culture. In line with this, DESC1 reduced tumor growth kinetics in the in vivo orthotopic tumorigenesis assay. Taken together, our novel findings suggest how DESC1 may suppress ESCC development by sensitizing cells to apoptosis under an apoptotic stimulus through downregulating the EGFR/AKT signaling pathway.

Nuclear Localization of DNAJB6 Is Associated With Survival of Patients With Esophageal Cancer and Reduces AKT Signaling and Proliferation of Cancer Cells.

BACKGROUND & AIMS: The DnaJ (Hsp40) homolog, subfamily B, member 6 (DNAJB6) is part of a family of proteins that regulates chaperone activities. One of its isoforms, DNAJB6a, contains a nuclear localization signal and regulates ß-catenin signaling during breast cancer development. We investigated the role of DNAJB6 in the pathogenesis of esophageal squamous cell carcinoma (ESCC). METHODS: We performed immunohistochemical analyses of primary ESCC samples and lymph node metastases from a cohort of 160 patients who underwent esophagectomy with no preoperative chemoradiotherapy at Hong Kong Queen Mary Hospital. Data were collected on patient outcomes over a median time of 12.1 ± 2.9 months. Retrospective survival association analyses were performed. Wild-type and mutant forms of DNAJB6a were overexpressed in cancer cell lines (KYSE510, KYSE 30TSI, KYSE140, and KYSE70TS), which were analyzed in proliferation and immunoblot assays, or injected subcutaneously into nude mice. Levels of DNAJB6 were knocked down in ESCC cell lines (KYSE450 and T.Tn), immortalized normal esophageal epithelial cell lines (NE3 and NE083), and other cells with short hairpin RNAs, or by genome engineering. Bimolecular fluorescence complementation was used to study interactions between proteins in living cells. RESULTS: In primary ESCC samples, patients whose tumors had high nuclear levels of DNAJB6 had longer overall survival times (19.2 ± 1.8 months; 95% confidence interval [CI], 15.6-22.8 mo) than patients whose tumors had low nuclear levels of DNAJB6 (12.6 ± 1.4 mo; 95% CI, 9.8-15.4 mo; P = .004, log-rank test). Based on Cox regression analysis, patients whose tumors had high nuclear levels of DNAJB6 had a lower risk of death than patients with low levels (hazard ratio, 0.562; 95% CI, 0.379-0.834; P = .004). Based on log-rank analysis and Cox regression analysis, the combination of the nuclear level of DNAJB6 and the presence of lymph node metastases at diagnosis could be used to stratify patients into groups with good or bad outcomes (P < .0005 for both analyses). There was a negative association between the nuclear level of DNAJB6 and the presence of lymph node metastases (P = .022; Pearson χ(2) test). Cancer cell lines that overexpressed DNAJB6a formed tumors more slowly in nude mice than control cells or cells that expressed a mutant form of DNAJB6a that did not localize to the nucleus. DNAJB6 knockdown in cancer cell lines promoted their growth as xenograft tumors in mice. A motif of histidine, proline, and aspartic acid in the J domain of DNAJB6a was required for its tumor-suppressive effects and signaling via AKT1. Loss of DNAJB6a resulted in up-regulation of AKT signaling in cancer cell lines and immortalized esophageal epithelial cells. Expression of a constitutively active form of AKT1 restored proliferation to tumor cells that overexpressed DNAJB6a, and DNAJB6a formed a complex with AKT1 in living cells. The expression of DNAJB6a reduced the sensitivity of ESCC to AKT inhibitors; the expression level of DNAJB6a affected AKT signaling in multiple cancer cell lines. CONCLUSIONS: Nuclear localization of DNAJB6 is associated with longer survival times of patients with ESCC. DNAJB6a reduces AKT signaling, and DNAJB6 expression in cancer cells reduces their proliferation and growth of xenograft tumors in mice. DNAJB6a might be developed as a biomarker for progression of ESCC.

A versatile orthotopic nude mouse model for study of esophageal squamous cell carcinoma.

Increasing evidence indicates tumor-stromal interactions play a crucial role in cancer. An in vivo esophageal squamous cell carcinoma (ESCC) orthotopic animal model was developed with bioluminescence imaging established with a real-time monitoring platform for functional and signaling investigation of tumor-stromal interactions. The model was produced by injection of luciferase-labelled ESCC cells into the intraesophageal wall of nude mice. Histological examination indicates this orthotopic model is highly reproducible with 100% tumorigenesis among the four ESCC cell lines tested. This new model recapitulates many clinical and pathological properties of human ESCC, including esophageal luminal stricture by squamous cell carcinoma with nodular tumor growth, adventitia invasion, lymphovascular invasion, and perineural infiltration. It was tested using an AKT shRNA knockdown of ESCC cell lines and the in vivo tumor suppressive effects of AKT knockdown were observed. In conclusion, this ESCC orthotopic mouse model allows investigation of gene functions of cancer cells in a more natural tumor microenvironment and has advantages over previous established models. It provides a versatile platform with potential application for metastasis and therapeutic regimen testing.