Liver tumour immune microenvironment subtypes and neutrophil heterogeneity.

The heterogeneity of the tumour immune microenvironment (TIME), organized by various immune and stromal cells, is a major contributing factor of tumour metastasis, relapse and drug resistance1-3, but how different TIME subtypes are connected to the clinical relevance in liver cancer remains unclear. Here we performed single-cell RNA-sequencing (scRNA-seq) analysis of 189 samples collected from 124 patients and 8 mice with liver cancer. With more than 1 million cells analysed, we stratified patients into five TIME subtypes, including immune activation, immune suppression mediated by myeloid or stromal cells, immune exclusion and immune residence phenotypes. Different TIME subtypes were spatially organized and associated with chemokine networks and genomic features. Notably, tumour-associated neutrophil (TAN) populations enriched in the myeloid-cell-enriched subtype were associated with an unfavourable prognosis. Through in vitro induction of TANs and ex vivo analyses of patient TANs, we showed that CCL4+ TANs can recruit macrophages and that PD-L1+ TANs can suppress T cell cytotoxicity. Furthermore, scRNA-seq analysis of mouse neutrophil subsets revealed that they are largely conserved with those of humans. In vivo neutrophil depletion in mouse models attenuated tumour progression, confirming the pro-tumour phenotypes of TANs. With this detailed cellular heterogeneity landscape of liver cancer, our study illustrates diverse TIME subtypes, highlights immunosuppressive functions of TANs and sheds light on potential immunotherapies targeting TANs.

Tumor-associated macrophage (TAM)-secreted CCL22 confers cisplatin resistance of esophageal squamous cell carcinoma (ESCC) cells via regulating the activity of diacylglycerol kinase α (DGKα)/NOX4 axis.

Tumor-associated macrophages (TAMs) are often associated with chemoresistance and resultant poor clinical outcome in solid tumors. Here, we demonstrated that TAMs-released chemokine-C-C motif chemokine 22 (CCL22) in esophageal squamous cell carcinoma (ESCC) stroma was tightly correlated with the chemoresistance of ESCC patients. TAMs-secreted CCL22 was able to block the growth inhibitory and apoptosis-promoting effects of cisplatin on ESCC cells. Mechanistically, CCL22 stimulated intratumoral diacylglycerol kinase α (DGKα) to produce phosphatidic acid (PA), which suppressed the activity of NADPH oxidase 4 (NOX4) and then blocked the overproduction of intratumoral reactive species oxygen (ROS) induced by cisplatin. CCL22 activated DGKα/nuclear factor-κB (NF-κB) axis to upregulate the level of several members of ATP binding cassette (ABC) transporter superfamily, including ABC sub-family G member 4 (ABCG4), ABC sub-family A member 3 (ABCA3), and ABC sub-family A member 5 (ABCA5), to lower the intratumoral concentration of cisplatin. Consequently, these processes induced the cisplatin resistance in ESCC cells. In xenografted models, targeting DGKα with 5'-cholesterol-conjugated small-interfering (si) RNA enhanced the chemosensitivity of cisplatin in ESCC treatment, especially in the context of TAMs. Our data establish the correlation between the TAMs-induced intratumoral metabolic product/ROS axis and chemotherapy efficacy in ESCC treatment and reveal relevant molecular mechanisms.

Quiescent cancer cells induced by high-density cultivation reveals cholesterol-mediated survival and lung metastatic traits.

BACKGROUND: The metastatic cascade, a multifaceted and highly aggressive process, is the primary cause of mortality. The survival of quiescent cancer cells in circulatory system during metastasis is crucial, yet our comprehension is constrained by the absence of universally accepted quiescent cancer models. METHOD: We developed a quiescent cancer cell model using high-density cultivation. Based on the scRNA-seq analysis, IP-MS, metabolomics, mouse lung metastasis models, cholesterol assay, PLA and other molecular experiments, we explored the molecular mechanism. Immunofluorescence, atomic force microscope, FluidFM, and shear stress stimulation were used to analyze the cytoskeleton and membrane properties contributing to mechanical force resistance. RESULT: We established a quiescent cancer cell model induced by high-density cultivation. Single-cell RNA sequencing (scRNA-seq) analysis reveals that CDC25A plays a crucial role in the transition to quiescence, with its expression significantly elevated in the quiescent state. Depletion of CDC25A leads to an increased proliferative capacity, and reduced metastasis under high-density conditions. Mechanistically, upregulated CDC25A in quiescent cells enhances cholesterol metabolism via endosome pathways, leading to cell cycle arrest. This increase in cholesterol reinforces the cytoskeleton, alters membrane properties, and improves resistance to mechanical forces in circulatory system. CONCLUSION: CDC25A significantly increased the cholesterol metabolism through endosome pathway in quiescent cancer cells, leading to the significant changes in cytoskeleton and membrane properties so as to enhance the resistance of mechanical force in circulatory system, facilitating lung metastasis. In high-density cultivation, quiescent cancer cells, up-regulate cholesterol metabolism by CDC25A through endosome pathway, enhancing the resistance to mechanical force in circulatory system, facilitating lung metastasis.

APC/C-regulated CPT1C promotes tumor progression by upregulating the energy supply and accelerating the G1/S transition.

BACKGROUND: In addition to functioning as a precise monitoring mechanism in cell cycle, the anaphase-promoting complex/cyclosome (APC/C) is reported to be involved in regulating multiple metabolic processes by facilitating the ubiquitin-mediated degradation of key enzymes. Fatty acid oxidation is a metabolic pathway utilized by tumor cells that is crucial for malignant progression; however, its association with APC/C remains to be explored. METHODS: Cell cycle synchronization, immunoblotting, and propidium iodide staining were performed to investigate the carnitine palmitoyltransferase 1 C (CPT1C) expression manner. Proximity ligation assay and co-immunoprecipitation were performed to detect interactions between CPT1C and APC/C. Flow cytometry, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium, inner salt (MTS) assays, cell-scratch assays, and transwell assays and xenograft transplantation assays were performed to investigate the role of CPT1C in tumor progression in vitro and in vivo. Immunohistochemistry was performed on tumor tissue microarray to evaluate the expression levels of CPT1C and explore its potential clinical value. RESULTS: We identified CPT1C as a novel APC/C substrate. CPT1C protein levels exhibited cell cycle-dependent fluctuations, peaking at the G1/S boundary. Elevated CPT1C accelerated the G1/S transition, facilitating tumor cell proliferation in vitro and in vivo. Furthermore, CPT1C enhanced fatty acid utilization, upregulated ATP levels, and decreased reactive oxygen species levels, thereby favoring cell survival in a harsh metabolic environment. Clinically, high CPT1C expression correlated with poor survival in patients with esophageal squamous cell carcinoma. CONCLUSIONS: Overall, our results revealed a novel interplay between fatty acid utilization and cell cycle machinery in tumor cells. Additionally, CPT1C promoted tumor cell proliferation and survival by augmenting cellular ATP levels and preserving redox homeostasis, particularly under metabolic stress. Therefore, CPT1C could be an independent prognostic indicator in esophageal squamous cell carcinoma.

Combined Wee1 and EGFR inhibition reveals synergistic antitumor effect in esophageal squamous cell carcinoma.

Epidermal growth factor receptor (EGFR) is one of the most common amplified and overexpressed oncogenes in esophageal squamous cell carcinoma (ESCC), while the clinical efficacy of EGFR-targeted therapy in ESCC is dismal. Here, we evaluated the efficacy of dual blockage using monoclonal antibody against EGFR (Nimotuzumab) and an Wee1 inhibitor (AZD1775) in ESCC. We found that the mRNA and protein expression of EGFR and Wee1 were positively correlated in ESCC. Nimotuzumab-AZD1775 co-treatment inhibited tumor growth in PDX models with different drug susceptibility. Transcriptome sequencing and mass spectrometry analysis indicated that higher sensitive models showed enrichment of the PI3K/Akt or MAPK signaling pathway in Nimotuzumab-AZD1775 group compared with control group. In vitro experiments showed that the combination further inhibit PI3K/Akt and MAPK pathways compared to their monotherapy as indicated by downregulation of pAKT, pS6, pMEK, pErk and p-p38 MAPK. Furthermore, AZD1775 potentiated Nimotuzumab's antitumor effect through inducing apoptosis. Meanwhile, the bioinformatics analysis suggests the POLR2A might be candidate molecule of EGFR/Wee1 downstream. In conclusion, our work uncovers that EGFR-mAb Nimotuzumab combined with Wee1 inhibitor AZD1775 elicited potentiated anticancer activity against ESCC cell line and PDXs partially through PI3K/Akt and MAPK pathways blockade. These preclinical data raise the promising that ESCC patients may benefit from dual target EGFR and Wee1.

Correction: Molecular predictors of brain metastasis-related microRNAs in lung adenocarcinoma.

[This corrects the article DOI: 10.1371/journal.pgen.1007888.].

Aqueous-soluble components of sporoderm-removed Ganoderma lucidum spore powder promote ferroptosis in oral squamous cell carcinoma.

Objective: Ferroptosis is a novel cell death process which displays a promising role in cancer treatment. However, clinically available drugs targeting ferroptosis are rarely used, and yet there are no studies reporting on inducing ferroptosis via Chinese herbal extracts. Here we explored the tumor inhibition effects of Ganoderma lucidum (G. lucidum) on oral squamous cell carcinoma (OSCC). Specifically, we aimed to clarify the biological mechanism of components in the dietary, aqueous-soluble sporoderm-removed G. lucidum spore powder (A-GSP). Methods: Preliminary transcriptome analysis revealed the significant enrichment of the ferroptosis pathway. Cellular Fe2+, glutathione (GSH), malondialdehyde (MDA), reactive oxygen species (ROS) and lipid peroxide levels were measured to identify ferroptosis occurrence. Western blotting was used to measure ferroptosis-related proteins. Changes in mitochondria morphology and function were observed with transmission electron microscopy (TEM) and ATP detection assays. Ferroptosis inhibitor ferrostatin-1 was then used to verify the anti-tumor effects of A-GSP. Finally, nude mice xenograft models of oral cancer confirmed that A-GSP inhibited tumor growth. Results: A-GSP promoted ferroptosis in oral cancer cells by inducing Fe2+ influx, GSH depletion, as well as lipid peroxide and ROS accumulation. Ferroptosis-related proteins exhibited corresponding changes, particularly Acyl-coA synthetase long chain family member 4 (ACSL4) increase and glutathione peroxidase 4 (GPX4) decrease. A-GSP considerably lowered mitochondrial volume and ridge number, while significantly decreasing ATP production. Ferrostatin-1 reversed all of these A-GSP-induced changes. In vivo, A-GSP exerted a ferroptosis-mediated tumor-suppressing effect without observable adverse reactions. Conclusions: Our findings demonstrate the therapeutic potential of A-GSP for treating patients with OSCC by targeting ferroptosis.

A liquid biopsy signature predicts lymph node metastases in T1 oesophageal squamous cell carcinoma: implications for precision treatment strategy.

BACKGROUND: The treatment strategies for T1 oesophageal squamous cell carcinoma (ESCC) patients with or without lymph node metastasis (LNM) are different. Given the advantages of the minimally invasive, sensitive and real-time detection, liquid biopsy has become an important cancer diagnostic and prognostic tool. METHODS: MiRNA array and small-RNA sequencing were performed. Then, 222 formalin-fixed and paraffin-embedded tumour samples and 229 pretreatment serum samples from T1 ESCC patients were used to verify and evaluate the results. RESULTS: We demonstrated that serum miR-20b-5p could predict LNM in T1 ESCC patients. The AUC for serum miR-20b-5p was higher (0.827) than those for lymphovascular invasion (LVI) (0.751, P = 0.2128), invasion depth (0.662, P = 0.0027) and tumour differentiation grade (0.634, P = 0.0019). A nomogram for predicting LNM with three independent significant predictors (miR-20b-5p, LVI and invasion depth) was constructed with a concordance index of 0.931. Serum miR-20b-5p was also significantly correlated with disease-free survival (P < 0.001). An algorithm of improved T1 ESCC treatment strategy after biopsy and/or after endoscopic resection based on serum miR-20b-5p level was constructed. CONCLUSIONS: This study suggests that serum miR-20b-5p is a potential biomarker for predicting LNM and can be helpful for precise clinical decision-making strategies and improve treatment outcomes for T1 ESCC patients.

PTBP1 promotes IRES-mediated translation of cyclin B1 in cancer.

Cyclin B1 is an essential cyclin-dependent protein that involves in the G2/M transition. Multiple studies report that cyclin B1 is upregulated in cancers and promotes cancer progression. However, the mechanism of cyclin B1 upregulation remains unclear. Here we report that the 5'UTR of cyclin B1 mRNA contains an internal ribosome entry site (IRES) by using a bicistronic fluorescent reporter. We show that IRES can initiate the translation of cyclin B1, and the IRES-mediated translation is further activated under cell stress. Interacting trans-acting factors (ITAFs) are required by most IRES to initiate the translation. We find that PTBP1 promotes the IRES-mediated translation of cyclin B1 by binding to the 5'UTR of cyclin B1. On top of that, PTBP1 promotes the malignancy of ESCC cells. Our data suggest that the IRES-mediated translation of cyclin B1 plays an essential role in the cyclin B1 upregulation in cancers.

Molecular predictors of brain metastasis-related microRNAs in lung adenocarcinoma.

Brain metastasis (BM) is a major complication of lung adenocarcinoma (LAD). An investigation of the pathogenic mechanisms of BM, as well as the identification of appropriate molecular markers, is necessary. The aim of this study was to determine the expression patterns of microRNAs (miRNAs) in LAD with BM, and to investigate the biological role of these miRNAs during tumorigenesis. miRNA array profiles were used to identify BM-associated miRNAs. These miRNAs were independently validated in 155 LAD patients. Several in vivo and in vitro assays were performed to verify the effects of miRNAs on BM. We identified six miRNAs differentially expressed in patients with BM as compared to patients with BM. Of these, miR-4270 and miR-423-3p were further investigated. miR-4270 and miR-423-3p directly targeted MMP19 and P21, respectively, to influence cell viability, migration, and colony formation in vitro. miR-4270 downregulation and miR-423-3p upregulation was associated with an increased risk of BM in LAD patients. Thus, our results suggested that miR-4270 and miR-423-3p might play an important role in BM pathogenesis in LAD patients, and that these miRNAs might be useful prognostic and clinical treatment targets.

Deregulated expression and subcellular localization of CPSF6, a circRNA-binding protein, promote malignant development of esophageal squamous cell carcinoma.

Objective: Cleavage and polyadenylation specific factor 6 (CPSF6) has been documented as an oncoprotein in different types of cancer. However, functions of CPSF6 have not been investigated yet in esophageal squamous cell carcinoma (ESCC). Here, we aimed to investigate the potential clinical values and biological functions of CPSF6 in ESCC. Methods: For determining the expression level of CPSF6 in ESCC patients, we analyzed published data, performed quantitative real-time polymerase chain reaction (RT-qPCR) and immunohistochemistry assays. Kaplan-Meier curves and log-rank tests were used for survival analyses. GO and KEGG analyses were done for CPSF6-related genes. Cell proliferation, colony formation and xenograft assays were conducted to verify the effects of CPSF6 on ESCC. In addition, cell cycle and apoptosis assays were also performed to manifest the functions of CPSF6 and circCPSF6. RNA pulldown and radioimmunoprecipitation (RIP) assays were used for confirming the interaction between circCPSF6 (hsa_circ_0000417) and CPSF6 protein. The regulatory relationship between CPSF6 protein and circCPSF6 was determined by RT-qPCR. Results: We found that CPSF6 was upregulated in ESCC tissues and overexpression of cytoplasmic CPSF6 was associated with poor prognosis. GO and KEGG analyses suggested that CPSF6 could mainly affect cell division in ESCC. Further experiments manifested that CPSF6 promoted cell proliferation and colony formationin vitro. Xenograft assay showed that knockdown of CPSF6 significantly decreased tumor growth rate in vivo. Subsequently, we verified that depletion of CPSF6 led to cell cycle arrest and apoptosis. Finally, we validated that CPSF6, as a circRNA-binding protein, interacted with and regulated its circular isoform circCPSF6 (hsa_circ_0000417), of which depletion also resulted in cell cycle arrest and cell apoptosis in ESCC. Conclusions: These findings gave us insight that overexpression of cytoplasmic CPSF6 protein is associated with poor prognosis in ESCC and CPSF6 may function as an oncoprotein, at least in part, through regulating circCPSF6 expression.

Focal adhesion kinase (FAK) inhibitor-defactinib suppresses the malignant progression of human esophageal squamous cell carcinoma (ESCC) cells via effective blockade of PI3K/AKT axis and downstream molecular network.

The clinical therapeutic efficacy toward esophageal squamous cell carcinoma (ESCC) is undesirable, due to the lack of targeted agents. Focal adhesion kinase (FAK), a nonreceptor tyrosine kinase involved in multiple fields of tumorigenesis, recently has been indicated as a promising therapeutic target in ESCC treatment. Here, we revealed that defactinib, a specific FAK inhibitor, effectively suppressed the malignancy of ESCC cells. Mechanistically, defactinib dose and time-dependently induced the dissociation of phosphoinositide-3-kinase (PI3K) from FAK, resultantly led to blockade of protein kinase B (AKT) signaling, and the expression of several oncogenes, such as SOX2, MYC, EGFR, MET, MDM2, or TGFBR2, identified by microarray and real-time polymerase chain reaction assay. Specifically, this FAK inhibition-mediated suppression of PI3K/AKT signaling and downstream ESCC specific biomarkers was maintained to 24 h in in vitro experiments to guarantee the treatment durability and efficacy. Importantly, defactinib suppressed tumor growth, metastatic ability, and increased overall survival of xenografted animals without producing significantly systematic toxicity. Our data suggest that FAK inhibition provides an excellent targeted therapy toward ESCC by effectively inhibiting PI3K/AKT pathway and downstream molecular network.

Feed-forward activation of STAT3 signaling limits the efficacy of c-Met inhibitors in esophageal squamous cell carcinoma (ESCC) treatment.

c-Hepatocyte growth factor receptor (Met) inhibitors have demonstrated clinical benefits in some types of solid tumors. However, the efficacy of c-Met inhibitors in esophageal squamous cell carcinoma (ESCC) remains unclear. In this study, we discovered that c-Met inhibitors induced "Signal Transducer and Activator of Transcription (STAT3)-addiction" in ESCC cells, and the feedback activation of STAT3 in ESCC cells limits the tumor response to c-Met inhibition. Mechanistically, c-Met inhibition increased the autocrine of several cytokines, including CCL2, interleukin 8, or leukemia inhibitory factor, and facilitated the interactions between the receptors of these cytokines and Janus Kinase1/2 (JAK1/2) to resultantly activate JAKs/STAT3 signaling. Pharmacological inhibition of c-Met together with cytokines/JAKs/STAT3 axis enhanced cancer cells regression in vitro. Importantly, combined c-Met and STAT3 inhibitors synergistically suppressed tumor growth and promoted the apoptosis of tumor cells without producing systematic toxicity. These findings suggest that inhibition of the STAT3 feedback loop may augment the response to c-Met inhibitors via the STAT3-mediated oncogene addiction in ESCC cells.

Exome sequencing identifies new somatic alterations and mutation patterns of tongue squamous cell carcinoma in a Chinese population.

Tongue squamous cell carcinoma (TSCC) is an aggressive group of tumors characterized by high rates of regional lymph node metastasis and local recurrence. Emerging evidence has revealed genetic variations of TSCC across different geographical regions due to the impact of multiple risk factors such as chewing betel-quid. However, we know little of the mutational processes of TSCC in the Chinese population without the history of chewing betel-quid/tobacco. To explore the mutational spectrum of this disease, we performed whole-exome sequencing of sample pairs, comprising tumors and normal tissue, from 82 TSCC patients. In addition to identifying seven previously known TSCC-associated genes (TP53, CDKN2A, PIK3CA, NOTCH1, ASXL1, USH2A, and CSMD3), the analysis revealed six new genes (GNAQ, PRG4, RP1, ZNF16, NBEA, and PTPRC) that had not been reported previously in TSCC. Our in vitro experiments identified ZNF16 for the first time as a solid tumor associated gene to promote malignancy of TSCC cells. We also identified a microRNA (miR-585-5p) encoded by the 5q35.1 region and characterized it as a tumor suppressor by targeting SOX9. At least one non-silent mutation of genes involved in the 10 canonical oncogenic pathways (Notch, RTK-RAS, PI3K, Wnt, Cell cycle, p53, Myc, Hippo, TGFß, and Nrf2) was found in 82.9% of cases. Collectively, our data extend the spectrum of TSCC mutations and define novel diagnosis markers and potential clinical targets for TSCC. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

OncoBase: a platform for decoding regulatory somatic mutations in human cancers.

Whole-exome and whole-genome sequencing have revealed millions of somatic mutations associated with different human cancers, and the vast majority of them are located outside of coding sequences, making it challenging to directly interpret their functional effects. With the rapid advances in high-throughput sequencing technologies, genome-scale long-range chromatin interactions were detected, and distal target genes of regulatory elements were determined using three-dimensional (3D) chromatin looping. Herein, we present OncoBase (http://www.oncobase.biols.ac.cn/), an integrated database for annotating 81 385 242 somatic mutations in 68 cancer types from more than 120 cancer projects by exploring their roles in distal interactions between target genes and regulatory elements. OncoBase integrates local chromatin signatures, 3D chromatin interactions in different cell types and reconstruction of enhancer-target networks using state-of-the-art algorithms. It employs informative visualization tools to display the integrated local and 3D chromatin signatures and effects of somatic mutations on regulatory elements. Enhancer-promoter interactions estimated from chromatin interactions are integrated into a network diffusion system that quantitatively prioritizes somatic mutations and target genes from a large pool. Thus, OncoBase is a useful resource for the functional annotation of regulatory noncoding regions and systematically benchmarking the regulatory effects of embedded noncoding somatic mutations in human carcinogenesis.

ZNF292 suppresses proliferation of ESCC cells through ZNF292/SKP2/P27 signaling axis.

OBJECTIVE: Increasing evidence has demonstrated that ZNF292 plays a suppressive role in cancer, however, little is known about its function and exact mechanism in esophageal squamous cell carcinoma (ESCC). METHODS: Bioinformatic analysis and immunohistochemistry (IHC) were performed to analyze the role of ZNF292 in affecting the prognosis of ESCC. Cell proliferation and colony formation ability assays were performed to analyze cell growth after inferring the expression of ZNF292. Flow cytometry was used to analyze changes in the cell cycle upon the depletion of ZNF292. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis were used to determine the alteration of cell cycle related RNAs and proteins after knocking down ZNF292. MG-132, cycloheximide (CHX) treatment experiments were performed to analyze the change and half-life time of P27 after knockdown of ZNF292. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays were used to analyze the transcriptional regulation of SKP2 by ZNF292. RESULTS: We report that low expression of ZNF292 is associated with poor prognosis, and ZNF292 emerges to be highly expressed in adjacent and normal tissues rather than tumor tissues in ESCC. Knockdown of ZNF292 significantly boosts cell growth and S phase entry in ESCC cells. ZNF292 depletion will decrease the expression and half-life time of P27, while knockdown of SKP2 will result in elevated expression of P27. ZNF292 can bind to the promoter region of SKP2, and knockdown of ZNF292 will boost the expression of SKP2. CONCLUSIONS: Knockdown of ZNF292 mediates G1/S cell cycle procession by activating SKP2/P27 signaling in ESCC cells. ZNF292 knockdown promotes SKP2 expression at the transcriptional level, thereby boosting P27 ubiquitin-degradation, and eventually facilitating the S phase entrance.

MTA1 promotes tumorigenesis and development of esophageal squamous cell carcinoma via activating the MEK/ERK/p90RSK signaling pathway.

Metastasis-associated protein 1 (MTA1) is upregulated in multiple malignancies and promotes cancer proliferation and metastasis, but whether and how MTA1 promotes esophageal squamous cell carcinoma (ESCC) tumorigenesis remain unanswered. Here, we established an ESCC model in MTA1 transgenic mice induced by the chemical carcinogen 4-nitroquinoline 1-oxide (4-NQO) and found that MTA1 promotes ESCC tumorigenesis in mice. MTA1 overexpression was observed in ESCC cells and clinical ESCC samples. Overexpressed MTA1 increased colony formation and the invasiveness and migration of ESCC cells, whereas knock down of MTA1 in ESCC cells significantly decreased colony formation, invasion and migration in vitro and inhibited the growth of xenograft tumors in vivo. RNA sequencing (RNA-seq) analysis combined with western blot assays revealed that MTA1 promotes carcinogenesis by enhancing MEK/ERK/p90RSK signaling. The phosphorylation of MEK, ERK and their downstream target p90RSK was significantly decreased after MTA1 knockdown in ESCC cells and was increased in MTA1-overexpressing cells. Moreover, colony formation, invasion and migration potential were dramatically suppressed when cells overexpressing MTA1 were treated with MEK (PD0325901) or ERK (SCH772948) inhibitors. In conclusion, MTA1 plays a pivotal oncogenic role in ESCC tumorigenesis and development through activating the MEK/ERK/p90RSK pathway.

Telomeric epigenetic response mediated by Gadd45a regulates stem cell aging and lifespan.

Progressive attrition of telomeres triggers DNA damage response (DDR) and limits the regenerative capacity of adult stem cells during mammalian aging. Intriguingly, telomere integrity is not only determined by telomere length but also by the epigenetic status of telomeric/sub-telomeric regions. However, the functional interplay between DDR induced by telomere shortening and epigenetic modifications in aging remains unclear. Here, we show that deletion of Gadd45a improves the maintenance and function of intestinal stem cells (ISCs) and prolongs lifespan of telomerase-deficient mice (G3Terc-/-). Mechanistically, Gadd45a facilitates the generation of a permissive chromatin state for DDR signaling by inducing base excision repair-dependent demethylation of CpG islands specifically at sub-telomeric regions of short telomeres. Deletion of Gadd45a promotes chromatin compaction in sub-telomeric regions and attenuates DDR initiation at short telomeres of G3Terc-/- ISCs. Treatment with a small molecule inhibitor of base excision repair reduces DDR and improves the maintenance and function of G3Terc-/- ISCs. Taken together, our study proposes a therapeutic approach to enhance stem cell function and prolong lifespan by targeting epigenetic modifiers.

Identification of genomic alterations in oesophageal squamous cell cancer.

Oesophageal cancer is one of the most aggressive cancers and is the sixth leading cause of cancer death worldwide. Approximately 70% of global oesophageal cancer cases occur in China, with oesophageal squamous cell carcinoma (ESCC) being the histopathological form in the vast majority of cases (>90%). Currently, there are limited clinical approaches for the early diagnosis and treatment of ESCC, resulting in a 10% five-year survival rate for patients. However, the full repertoire of genomic events leading to the pathogenesis of ESCC remains unclear. Here we describe a comprehensive genomic analysis of 158 ESCC cases, as part of the International Cancer Genome Consortium research project. We conducted whole-genome sequencing in 17 ESCC cases and whole-exome sequencing in 71 cases, of which 53 cases, plus an additional 70 ESCC cases not used in the whole-genome and whole-exome sequencing, were subjected to array comparative genomic hybridization analysis. We identified eight significantly mutated genes, of which six are well known tumour-associated genes (TP53, RB1, CDKN2A, PIK3CA, NOTCH1, NFE2L2), and two have not previously been described in ESCC (ADAM29 and FAM135B). Notably, FAM135B is identified as a novel cancer-implicated gene as assayed for its ability to promote malignancy of ESCC cells. Additionally, MIR548K, a microRNA encoded in the amplified 11q13.3-13.4 region, is characterized as a novel oncogene, and functional assays demonstrate that MIR548K enhances malignant phenotypes of ESCC cells. Moreover, we have found that several important histone regulator genes (MLL2 (also called KMT2D), ASH1L, MLL3 (KMT2C), SETD1B, CREBBP and EP300) are frequently altered in ESCC. Pathway assessment reveals that somatic aberrations are mainly involved in the Wnt, cell cycle and Notch pathways. Genomic analyses suggest that ESCC and head and neck squamous cell carcinoma share some common pathogenic mechanisms, and ESCC development is associated with alcohol drinking. This study has explored novel biological markers and tumorigenic pathways that would greatly improve therapeutic strategies for ESCC.

GADD45G Interacts with E-cadherin to Suppress the Migration and Invasion of Esophageal Squamous Cell Carcinoma.

BACKGROUND/AIMS: Esophageal squamous cell carcinoma (ESCC) is one of the most prevalent cancers with poor prognosis. Metastasis is the leading cause of cancer-related deaths. The growth arrest and DNA damage-inducible 45 gamma (GADD45G) has been reported to correlate with survival, invasion, and metastasis of ESCC. This study was aimed to investigate the role and mechanism of GADD45G in ESCC cell migration and invasion. METHODS: Both the effects of GADD45G and its need for E-cadherin to function on ESCC cell migration and invasion were determined through loss- and gain-of-function approaches via Transwell assays. The interaction between GADD45G and E-cadherin was detected by GST-pull down and IP assays. The expression of E-cadherin upon GADD45G overexpression was evaluated by RT-qPCR and western blot. The level of E-cadherin in cytoplasmic, nuclear, and membrane fractions was examined by western blot following subcellular fractionation. RESULTS: Knockdown of GADD45G increased the migration and invasion abilities of KYSE150 cells, while overexpression of GADD45G showed the opposite effects on YES2 and KYSE30 cells. GADD45G could interact with E-cadherin and enhanced its membrane level. Knockdown of E-cadherin abolished the inhibitory effects of GADD45G on ESCC cell migration and invasion. Intriguingly, dimer-dissociating mutant of GADD45G could not interact with E-cadherin and almost lost its ability to suppress the ESCC cell migration and invasion. CONCLUSIONS: This study reveals a novel role for GADD45G in inhibiting the ESCC cell migration and invasion, which will provide a new insight in understanding the ESCC metastatic mechanism.