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Protein kinase Cι (PKCι) is an atypical PKC isoform and participates in multiple aspects of the transformed phenotype in human cancer cells. We previously reported that frequent amplification and overexpression of PKCι were correlated with lymph node metastasis in primary esophageal squamous cell carcinomas (ESCC). In the present study, short interfering RNA–mediated silencing of PKCι revealed that this enzyme was required for cell migration, invasion, and resistance to anoikis. In vivo experiments showed that PKCι suppression decreased tumor growth in esophageal cancer xenografts and lung metastases in nude mice. At the molecular level, knockdown of PKCι in suspended ESCC cells caused a decrease in S-phase kinase-associated protein 2 (SKP2) that had been reported to promote resistance to anoikis via the PI3K/AKT pathway. AKT phosphorylation was abolished after PKCι suppression, but AKT activation could be refreshed by PKCι upregulation, suggesting that PKCι enhanced cell resistance to anoikis via the PKCι-SKP2-PI3K/AKT pathway. Addition of the proteasome inhibitor MG132 prevented the decrease of SKP2 in PKCι silenced cells, and polyubiquitin-SKP2 was elevated after PKCι depletion, showing that PKCι might regulate the expression of SKP2 through the ubiquitin-proteasome pathway in suspended cells. Furthermore, overexpression of SKP2 in PKCι-downregulated cells restored cell resistance to anoikis. Most importantly, PKCι expression significantly correlated with SKP2 in 133 ESCC tissues (P = 0.031). Taken together, our data show that PKCι promotes tumorigenicity and metastasis of human esophageal cancer and that SKP2 is a candidate downstream effector of PKCι signaling in ESCC. Mol Cancer Res; 9(4); 390–402. ©2011 AACR.

Supplementary Figures S1-S5.

Supplementary Table 1. NRP1 expression positively correlates with CRT in ESCC

Purpose: Our aim was to identify frequent genomic aberrations in both esophageal squamous cell carcinoma (ESCC) and esophageal dysplasia and to discover important copy number-driving genes and microRNAs (miRNA) in ESCC.Experimental Design: We conducted array-based comparative genomic hybridization (array CGH) on 59 ESCC resection samples and 16 dysplasia biopsy samples. Expression of genes at 11q13.3 was analyzed by real-time PCR (RT-PCR) and immunohistochemistry (IHC). Integrated analysis was conducted to identify genes or miRNAs with copy number-expression correlations.Results: Array CGH identified 11 amplifications and eight homozygous deletions in ESCC. Integrated analysis of array CGH data with matched gene expression microarray data showed that 90 overexpressed genes and 24 underexpressed genes were consistent with DNA copy number changes, including 12 copy number-driving miRNAs. In esophageal dysplasia, six gains, four losses, 12 amplifications, and four homozygous deletions were detected. Amplifications of 7p11.2 and 11q13.2–11q13.3 (CCND1) and homozygous deletion at 9p21.3 (CDKN2A) were consistent genomic changes in both dysplasia and carcinoma. ANO1 at 11q13.3 was overexpressed at the mRNA and protein levels in tumors, and higher mRNA expression was correlated with the copy number increase. In particular, ANO1 expression was elevated in moderate dysplasia compared with normal esophageal epithelium. IHC revealed that ANO1 overexpression was positively correlated with lymph node metastasis and advanced clinical stage. Knockdown of ANO1 significantly inhibited the proliferation of KYSE30 and KYSE510 cells.Conclusion: Copy number aberrations in both esophageal dysplasia and ESCC may be useful as potential biomarkers for early detection. In addition, ANO1 may be a candidate target gene in esophageal tumorigenesis. Clin Cancer Res; 19(21); 5867–78. ©2013 AACR.

Supplementary Fig. 1. Mutational spectrum of pediatric ALL.

Purpose: We previously revealed that the calreticulin (CRT) gene is a candidate oncogene promoting cell migration and invasion and that neuropilin-1 (NRP1) is a possible effector downstream of CRT in esophageal squamous carcinoma cells. This study aims to explore the mechanisms underlying the migration and invasion of esophageal cancer cells regulated by CRT through NRP1.Experimental Design: Quantitative reverse-transcription polymerase chain reaction, Western blot analysis, chromatin immunoprecipitation, and reporter gene assays were used to investigate the relationship between CRT and NRP1. In vitro and in vivo assays were carried out to evaluate the effects of NRP1 on malignant phenotypes of ESCC cells and tumor metastasis in NOD/SCID mice. Immunohistochemistry was performed to analyze the expression of CRT and NRP1 in esophageal squamous cell carcinomas (ESCC).Results: Knockdown of CRT decreased the expression of NRP1. Inhibition of NRP1 reduced ESCC cell motility in vitro and experimental metastasis in vivo. Ectopic expression of NRP1 rescued the defects of cell migration and invasion in CRT-shRNA cells. CRT depletion inhibited STAT5A phosphorylation at the Y694 site via a CaMKII-independent pathway. Moreover, STAT5A directly regulated NRP1 transcription. Knockdown of CRT or NRP1 led to a downregulation of MMP2, MMP9, and FAK. Notably, positive correlation was found between CRT and NRP1 expression in ESCC tissues (P = 5.87 × 10−5). CRT and NRP1 coexpression was significantly associated with lymph node metastasis (P = 0.025).Conclusions: Our findings suggest that NRP1 is a critical downstream effector of CRT in promoting cell migration and invasion, which might contribute to the metastasis of ESCC. Clin Cancer Res; 20(23); 6153–62. ©2014 AACR.

Effect of ANO1 on cell proliferation. Knock-down the expression of ANO1 inhibited the cell proliferation of KYSE30 (A) and KYSE510 cells (B). The absorbance at 450 nm was measured.

Nasopharyngeal carcinoma (NPC) is an invasive cancer with particularly high incidence in Southeast Asia and Southern China. The pathogenic mechanisms of NPC, particularly those involving epigenetic dysregulation, remain largely elusive, hampering clinical management of this malignancy. To identify novel druggable targets, we carried out an unbiased high-throughput chemical screening and observed that NPC cells were highly sensitive to inhibitors of cyclin-dependent kinases (CDK), especially THZ1, a covalent inhibitor of CDK7. THZ1 demonstrated pronounced antineoplastic activities both in vitro and in vivo. An integrative analysis using both whole-transcriptome sequencing and chromatin immunoprecipitation sequencing pinpointed oncogenic transcriptional amplification mediated by super-enhancers (SE) as a key mechanism underlying the vulnerability of NPC cells to THZ1 treatment. Further characterization of SE-mediated networks identified many novel SE-associated oncogenic transcripts, such as BCAR1, F3, LDLR, TBC1D2, and the long noncoding RNA TP53TG1. These transcripts were highly and specifically expressed in NPC and functionally promoted NPC malignant phenotypes. Moreover, DNA-binding motif analysis within the SE segments suggest that several transcription factors (including ETS2, MAFK, and TEAD1) may help establish and maintain SE activity across the genome. Taken together, our data establish the landscape of SE-associated oncogenic transcriptional network in NPC, which can be exploited for the development of more effective therapeutic regimens for this disease. Cancer Res; 77(23); 6614–26. ©2017 AACR.

Supplementary Fig. 5. Kaplan-Meier analysis of the survival of patients with different mutations.

Supplementary Table 2. Relationship between NRP1/CRT co-expression and lymph node metastasis in 216 ESCC patients

Legends for Supplementary Figures

Supplementary Figure 2. Quantitative real-time PCR analysis of STAT5A and STAT5B expression in ESCC cells transiently transfected with non-silencing siRNA, STAT5A siRNA or STAT5B siRNA. Columns, mean; Bars, SD (n = 3); **, P < 0.01.

Supplementary Data. Supplementary Materials and Methods, Supplementary Figure Legends

The IC50 (nM) values, RNA-seq, ChIP seq and motif analysis in NPC cell lines

Supplementary Figure 1. Distribution of putative targets of the top-ranked small molecules Supplementary Figure 2. CDK7 silencing significantly inhibited cells growth Supplementary Figure 3. Cell viability assay of C666-1 cells treated with THZ1 Supplementary Figure 4. Cell-cycle arrested at G2/M stage in C666-1 and HK1 cells treated with THZ1 Supplementary Figure 5. Cell viability (A) and apoptosis assay (B) showing the effects of THZ1 treatment in PNHNC Supplementary Figure 6. THZ1 potently suppressed xenografts growth and lung metastasis of NPC cells in NOD/SCID mice Supplementary Figure 7. THZ1 exhibited prominent anti-NPC activity in NOD/SCID mice Supplementary Figure 8. THZ1 inhibited RNAPII-mediated transcription in NPC Supplementary Figure 9. Representative SE-associated genes in NPC and PNHNC Supplementary Figure 10. Gene Ontology enrichment analysis of SE-associated genes in PNHNC cell

Current standard of care for patients with pediatric acute lymphoblastic leukemia (ALL) is mainly effective, with high remission rates after treatment. However, the genetic perturbations that give rise to this disease remain largely undefined, limiting the ability to address resistant tumors or develop less toxic targeted therapies. Here, we report the use of next-generation sequencing to interrogate the genetic and pathogenic mechanisms of 240 pediatric ALL cases with their matched remission samples. Commonly mutated genes fell into several categories, including RAS/receptor tyrosine kinases, epigenetic regulators, transcription factors involved in lineage commitment, and the p53/cell-cycle pathway. Unique recurrent mutational hotspots were observed in epigenetic regulators CREBBP (R1446C/H), WHSC1 (E1099K), and the tyrosine kinase FLT3 (K663R, N676K). The mutant WHSC1 was established as a gain-of-function oncogene, while the epigenetic regulator ARID1A and transcription factor CTCF were functionally identified as potential tumor suppressors. Analysis of 28 diagnosis/relapse trio patients plus 10 relapse cases revealed four evolutionary paths and uncovered the ordering of acquisition of mutations in these patients. This study provides a detailed mutational portrait of pediatric ALL and gives insights into the molecular pathogenesis of this disease. Cancer Res; 77(2); 390–400. ©2016 AACR.

Supplementary Fig. 4. p53 pathway is dysregulated in ALL.

Supplementary Fig. 2. Mutations found in the ASXL family of genes.

Supplementary Figure 3. Western blot analysis in ESCC cells treated with or without 100 μmol/L KN-93 for 24 hours.

Supplementary Figure 1. Quantitative real-time PCR analysis of CRT expression in ESCC cells transiently transfected with non-silencing siRNA or CRT siRNA. Columns, mean; Bars, SD (n = 3); **, P < 0.01.

The Highest Expression Genes in NPC Compare With RNA-seq Results From 675 Human Cancer Cell Lines

Supplement file: Materials and Methods

Alternative lengthening of telomeres (ALT) supports telomere maintenance in 10-15% of cancers, thus representing a compelling target for therapy. By performing anti-cancer compound library screen on isogenic cell lines and using extrachromosomal telomeric C-circles, as a bona fide marker of ALT activity, we identify a receptor tyrosine kinase inhibitor ponatinib that deregulates ALT mechanisms, induces telomeric dysfunction, reduced ALT-associated telomere synthesis, and targets, in vivo, ALT-positive cells. Using RNA-sequencing and quantitative phosphoproteomic analyses, combined with C-circle level assessment, we find an ABL1-JNK-JUN signalling circuit to be inhibited by ponatinib and to have a role in suppressing telomeric C-circles. Furthermore, transcriptome and interactome analyses suggest a role of JUN in DNA damage repair. These results are corroborated by synergistic drug interactions between ponatinib and either DNA synthesis or repair inhibitors, such as triciribine. Taken together, we describe here a signalling pathway impacting ALT which can be targeted by a clinically approved drug. Ministry of Health (MOH) Nanyang Technological University National Medical Research Council (NMRC) National Research Foundation (NRF) Published version This work was funded or supported by: Nanyang Technological University Presidential Postdoctoral Fellowship (M.J.); Singapore Ministry of Health’s National Medical Research Council (NMRC) (Open Fund—Young Individual Research Grant to M.J. (MOH-00534) and Singapore Translational Research (STaR) Investigator Award to H.P.K. (NMRC/STaR/0021/2014)); National Institute of Health (NIH) R01 CA200992-04 grant (H.P.K.); NMRC Centre Grant Programme awarded to National University Cancer Institute of Singapore (NMRC/CG/012/2013 and CGAug16M005); and the National Research Foundation Singapore and the Singapore Ministry of Education under its Research Centres of Excellence initiatives; LMSarcoma Direct Research Foundation (H.P.K.); and National Research Foundation (NRF) Singapore, NRF Competitive Research Programme (CRP), Grant Award Number NRF-CRP21-2018-0002 (P.Dr.).

Super-enhancers (SEs) are congregated enhancer clusters with high level of loading of transcription factors (TFs), cofactors and epigenetic modifications. Through direct co-occupancy at their own SEs as well as each other's, a small set of so called "master" TFs form interconnected core regulatory circuitry (CRCs) to orchestrate transcriptional programs in both normal and malignant cells. These master TFs can be predicted mathematically using epigenomic methods. In this Review, we summarize the identification of SEs and CRCs in cancer cells, the mechanisms by which master TFs and SEs cooperatively regulate cancer-type-specific expression programs, and the cancer-type- and subtype-specificity of CRC and the significance in cancer biology.

Proteasome inhibitors, such as bortezomib and carfilzomib, have shown efficacy in anti-cancer therapy in hematological diseases but not in solid cancers. Here, we found that liposarcomas (LPS) are susceptible to proteasome inhibition, and identified drugs that synergize with carfilzomib, such as selinexor, an inhibitor of XPO1-mediated nuclear export. Through quantitative nuclear protein profiling and phospho-kinase arrays, we identified potential mode of actions of this combination, including interference with ribosome biogenesis and inhibition of pro-survival kinase PRAS40. Furthermore, by assessing global protein levels changes, FADS2, a key enzyme regulating fatty acids synthesis, was found down-regulated after proteasome inhibition. Interestingly, SC26196, an inhibitor of FADS2, synergized with carfilzomib. Finally, to identify further combinational options, we performed high-throughput drug screening and uncovered novel drug interactions with carfilzomib. For instance, cyclosporin A, a known immunosuppressive agent, enhanced carfilzomib’s efficacy in vitro and in vivo. Altogether, these results demonstrate that carfilzomib and its combinations could be repurposed for LPS clinical management. Electronic supplementary material The online version of this article (10.1007/s00018-020-03620-w) contains supplementary material, which is available to authorized users.

Nuclear import, mediated in part by karyopherin-α (KPNA)/importin-α subtypes, regulates transcription factor access to the genome and determines cell fate. However, the cancer-specific changes of KPNA subtypes and the relevancy in cancer biology remain largely unknown. Here, we report that KPNA4, encoding karyopherin-α4 (KPNA4), is exclusively amplified and overexpressed in head and neck of squamous cell carcinoma (HNSCC). Depletion of KPNA4 attenuated nuclear localization signal-dependent transport activity and suppressed malignant phenotypes and induced epidermal differentiation. Mechanistically, KPNA4-mediated nuclear transport of Ras-responsive element-binding protein (RREB1), which sustains Ras/ERK pathway signaling through repressing miR-143/145 expression. Notably, MAPK signaling enhanced trafficking activity of KPNA4 via phosphorylation of KPNA4 at Ser60. These data reveal that KPNA4 establishes a feed-forward cascade that potentiates Ras/ERK signaling in HNSCC.

Squamous cell carcinomas (SCCs) comprise one of the most common histologic types of human cancer. Transcriptional dysregulation of SCC cells is orchestrated by tumor protein p63 (TP63), a master transcription factor (TF) and a well-researched SCC-specific oncogene. In the present study, both Gene Set Enrichment Analysis (GSEA) of SCC patient samples and in vitro loss-of-function assays establish fatty-acid metabolism as a key pathway downstream of TP63. Further studies identify sterol regulatory element binding transcription factor 1 (SREBF1) as a central mediator linking TP63 with fatty-acid metabolism, which regulates the biosynthesis of fatty-acids, sphingolipids (SL), and glycerophospholipids (GPL), as revealed by liquid chromatography tandem mass spectrometry (LC-MS/MS)-based lipidomics. Moreover, a feedback co-regulatory loop consisting of SREBF1/TP63/Kruppel like factor 5 (KLF5) is identified, which promotes overexpression of all three TFs in SCCs. Downstream of SREBF1, a non-canonical, SCC-specific function is elucidated: SREBF1 cooperates with TP63/KLF5 to regulate hundreds of cis-regulatory elements across the SCC epigenome, which converge on activating cancer-promoting pathways. Indeed, SREBF1 is essential for SCC viability and migration, and its overexpression is associated with poor survival in SCC patients. Taken together, these data shed light on mechanisms of transcriptional dysregulation in cancer, identify specific epigenetic regulators of lipid metabolism, and uncover SREBF1 as a potential therapeutic target and prognostic marker in SCC., The relevance and underlying molecular mechanisms of epigenetic regulation in squamous cell carcinomas (SCC) await further characterization. Here, the authors show a transcriptional regulatory loop involving SREBF1, TP63 and KLF5 driving tumourigenesis in SCC through fatty acid, ERBB and mTOR pathway regulation.

CpG Island promoter genes make up more than half of human genes, and a subset regulated by Polycomb-Repressive Complex 2 (PRC2+-CGI) become DNA hypermethylated and silenced in cancer. Here, we perform a systematic analysis of CGI genes across TCGA cancer types, finding that PRC2+-CGI genes are frequently prone to transcriptional upregulation as well. These upregulated PRC2+-CGI genes control important pathways such as Epithelial-Mesenchymal Transition (EMT) and TNFα-associated inflammatory response, and have greater cancer-type specificity than other CGI genes. Using publicly available chromatin datasets and genetic perturbations, we show that transcription factor binding sites (TFBSs) within distal enhancers underlie transcriptional activation of PRC2+-CGI genes, coinciding with loss of the PRC2-associated mark H3K27me3 at the linked promoter. In contrast, PRC2-free CGI genes are predominantly regulated by promoter TFBSs which are common to most cancer types. Surprisingly, a large subset of PRC2+-CGI genes that are upregulated in one cancer type are also hypermethylated/silenced in at least one other cancer type, underscoring the high degree of regulatory plasticity of these genes, likely derived from their complex regulatory control during normal development., A subset of CpG Island promoter genes are regulated by Polycomb-Repressive Complex 2 (PRC2+-CGI), which become DNA hypermethylated and silenced in cancer. Here, the authors investigate the transcriptomic and epigenomic characteristics of PRC2-occupied CGI and free CGI across pan-cancer types.

As the second most common malignant bone tumor in children and adolescents, Ewing sarcoma is initiated and exacerbated by a chimeric oncoprotein, most commonly, EWS-FLI1. In this study, we apply epigenomic analysis to characterize the transcription dysregulation in this cancer, focusing on the investigation of super-enhancer and its associated transcriptional regulatory mechanisms. We demonstrate that super-enhancer-associated transcripts are significantly enriched in EWS-FLI1 target genes, contribute to the aberrant transcriptional network of the disease, and mediate the exceptional sensitivity of Ewing sarcoma to transcriptional inhibition. Through integrative analysis, we identify MEIS1 as a super-enhancer-driven oncogene, which co-operates with EWS-FLI1 in transcriptional regulation, and plays a key pro-survival role in Ewing sarcoma. Moreover, APCDD1, another super-enhancer-associated gene, acting as a downstream target of both MEIS1 and EWS-FLI1, is also characterized as a novel tumor-promoting factor in this malignancy. These data delineate super-enhancer-mediated transcriptional deregulation in Ewing sarcoma, and uncover numerous candidate oncogenes which can be exploited for further understanding of the molecular pathogenesis for this disease.

Characterising the activated oncogenic signalling that leads to advanced breast cancer is of clinical importance. Here, we showed that SET domain, bifurcated 1 (SETDB1), a histone H3 lysine 9 methyltransferase, is aberrantly expressed and behaves as an oncogenic driver in breast cancer. SETDB1 enhances c-MYC and cyclin D1 expression by promoting the internal ribosome entry site (IRES)-mediated translation of MYC/CCND1 mRNA, resulting in prominent signalling of c-MYC to promote cell cycle progression, and provides a growth/self-renewal advantage to breast cancer cells. The activated c-MYC-BMI1 axis is essential for SETDB1-mediated breast tumourigenesis, because silencing of either c-MYC or BMI1 profoundly impairs the enhanced growth/colony formation conferred by SETDB1. Furthermore, c-MYC directly binds to the SETDB1 promoter region and enhances its transcription, suggesting a positive regulatory interplay between SETDB1 and c-MYC. In this study, we identified SETDB1 as a prominent oncogene and characterised the underlying mechanism whereby SETDB1 drives breast cancer, providing a therapeutic rationale for targeting SETDB1-BMI1 signalling in breast cancer. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Esophageal squamous cell carcinoma (ESCC) is one of the malignancies in digestive system, with a low 5-year survival rate. We previously revealed that Sequestosome 1 (SQSTM1/p62) protein levels were upregulated in ESCC tissues. However, it is unclear about the function of p62 and the underlying mechanism. Here, we used immunofluorescence and immunohistochemistry to investigate the expression of p62 in ESCC. Western blotting, quantitative RT-PCR, colony formation assay, flow cytometry, immunoprecipitation and xenograft tumor assay were used to analyze the role of p62 in vitro and vivo. Here, we showed that p62 serves as a regulator of cell apoptosis under serum starvation condition in ESCC cells. Through activating the protein kinase C iota (PKCiota)-S-phase kinase-associated protein 2 (SKP2) signaling pathway, p62 enhances cell apoptosis resistance and colony formation in vitro and tumor growth in mouse models. Through interaction with the domains PB1, p62 upregulated the expression of PKCiota and then depressed the ubiquitin-mediated proteasomal degradation of SKP2. p62-silencing combined with a PKCiota inhibitor ATM significantly enhanced cell apoptosis and inhibited cell survival. Immunohistochemical analysis revealed a positive association between the expression of p62 and SKP2 in primary ESCC tissues. And importantly, p62 presented a markedly cytoplasmic translocation in cancerous cells, including in 16 (30.76%) tumors at stage T1, as compared with its nuclear location in normal esophageal epithelial cells. In summary, p62 plays an anti-apoptotic role in ESCC cells via stabilizing SKP2 under serum starvation condition. These data suggest that p62 might be an early biomarker and a candidate therapeutic target of ESCC.

p63, more specifically its ΔNp63α isoform, plays essential roles in squamous cell carcinomas (SCCs), yet the mechanisms controlling its nuclear transport remain unknown. Nucleoporins (NUPs) are a family of proteins building nuclear pore complexes (NPC) and mediating nuclear transport across the nuclear envelope. Recent evidence suggests a cell type-specific function for certain NUPs; however, the significance of NUPs in SCC biology remains unknown. In this study, we show that nucleoporin 62 (NUP62) is highly expressed in stratified squamous epithelia and is further elevated in SCCs. Depletion of NUP62 inhibits proliferation and augments differentiation of SCC cells. The impaired ability to maintain the undifferentiated status is associated with defects in ΔNp63α nuclear transport. We further find that differentiation-inducible Rho kinase reduces the interaction between NUP62 and ΔNp63α by phosphorylation of phenylalanine-glycine regions of NUP62, attenuating ΔNp63α nuclear import. Our results characterize NUP62 as a gatekeeper for ΔNp63α and uncover its role in the control of cell fate through regulation of ΔNp63α nuclear transport in SCC.

Nasopharyngeal carcinoma (NPC) is an invasive cancer with particularly high incidence in Southeast Asia and Southern China. The pathogenic mechanisms of NPC, particularly those involving epigenetic dysregulation, remain largely elusive, hampering clinical management of this malignancy. To identify novel druggable targets, we carried out an unbiased high-throughput chemical screening and observed that NPC cells were highly sensitive to inhibitors of cyclin-dependent kinases (CDK), especially THZ1, a covalent inhibitor of CDK7. THZ1 demonstrated pronounced antineoplastic activities both in vitro and in vivo. An integrative analysis using both whole-transcriptome sequencing and chromatin immunoprecipitation sequencing pinpointed oncogenic transcriptional amplification mediated by super-enhancers (SE) as a key mechanism underlying the vulnerability of NPC cells to THZ1 treatment. Further characterization of SE-mediated networks identified many novel SE-associated oncogenic transcripts, such as BCAR1, F3, LDLR, TBC1D2, and the long noncoding RNA TP53TG1. These transcripts were highly and specifically expressed in NPC and functionally promoted NPC malignant phenotypes. Moreover, DNA-binding motif analysis within the SE segments suggest that several transcription factors (including ETS2, MAFK, and TEAD1) may help establish and maintain SE activity across the genome. Taken together, our data establish the landscape of SE-associated oncogenic transcriptional network in NPC, which can be exploited for the development of more effective therapeutic regimens for this disease. Cancer Res; 77(23); 6614–26. ©2017 AACR.

Minichromosome Maintenance (MCM) proteins play essential roles in various cancers. We previously reported that MCM7 could be a prognostic biomarker in non-small cell lung cancer (NSCLC). The purpose of current study is to explore roles of other MCM proteins in NSCLC and their correlation with clinico-pathologic parameters of NSCLC patients. We evaluated the expression of MCM2, MCM5 and MCM6 immunohistochemically in 571 primary NSCLC samples. High expression of MCM2, MCM5 and MCM6 was detected in 42.2%, 38.3% and 52.9% of tumor tissues, respectively. The expression of MCM2, MCM5 and MCM6 was significantly associated with gender (P = 0.00004, 0.00004, 0.008), tumor type (P < 0.00001, < 0.00001, 0.00001) and smoking history (P = 0.009, 0.00043, 0.002). MCM2 and MCM5 were detected more in central-type lung cancer (P< 0.006, 0.016). Higher labeling index (LI) of MCM2 was observed more frequently in aged patients (P = 0.023) and in those at later stage (P = 0.001). Higher MCM5 LIs was detected more in patients with distant metastasis (P = 0.008). Kaplan-Meier curves indicated that early-stage (stage I/II) patients with higher MCM2 LIs had a poorer OS compared to those with lower LIs (P = 0.021). And lung squamous cell carcinoma (SCC) patients presenting high MCM5 expression had shorter OS (P = 0.015). Multivariate Cox regression analysis showed that MCM5 was an independent prognostic indicator (P = 0.035, HR = 1.586, 95%CI: 1.032-2.437). We reported for the first time that higher MCM5 LIs could be an independent adverse prognostic biomarker for SCC patients.

ObjectivesOesophageal squamous cell carcinoma (OSCC) and adenocarcinoma (OAC) are distinct cancers in terms of a number of clinical and epidemiological characteristics, complicating the design of clinical trials and biomarker developments. We analysed 1048 oesophageal tumour-germline pairs from both subtypes, to characterise their genomic features, and biological and clinical significance.DesignPreviously exome-sequenced samples were re-analysed to identify significantly mutated genes (SMGs) and mutational signatures. The biological functions of novel SMGs were investigated using cell line and xenograft models. We further performed whole-genome bisulfite sequencing and chromatin immunoprecipitation (ChIP)-seq to characterise epigenetic alterations.ResultsOSCC and OAC displayed nearly mutually exclusive sets of driver genes, indicating that they follow independent developmental paths. The combined sample size allowed the statistical identification of a number of novel subtype-specific SMGs, mutational signatures and prognostic biomarkers. Particularly, we identified a novel mutational signature similar to Catalogue Of Somatic Mutations In Cancer (COSMIC)signature 16, which has prognostic value in OSCC. Two newly discovered SMGs, CUL3 and ZFP36L2, were validated as important tumour-suppressors specific to the OSCC subtype. We further identified their additional loss-of-function mechanisms. CUL3 was homozygously deleted specifically in OSCC and other squamous cell cancers (SCCs). Notably, ZFP36L2 is associated with super-enhancer in healthy oesophageal mucosa; DNA hypermethylation in its super-enhancer reduced active histone markers in squamous cancer cells, suggesting an epigenetic inactivation of a super-enhancer-associated SCC suppressor.ConclusionsThese data comprehensively contrast differences between OSCC and OAC at both genomic and epigenomic levels, and reveal novel molecular features for further delineating the pathophysiological mechanisms and treatment strategies for these cancers.

Gastrointestinal adenocarcinomas (GIAC) of the tubular gastrointestinal (GI) tract including esophagus, stomach, colon, and rectum comprise most GI cancers and share a spectrum of genomic features. However, the unified epigenomic changes specific to GIAC are poorly characterized. Using 907 GIAC samples from The Cancer Genome Atlas, we applied mathematical algorithms to large-scale DNA methylome and transcriptome profiles to reconstruct transcription factor (TF) networks and identify a list of functionally hyperactive master regulator (MR) TF shared across different GIAC. The top candidate HNF4A exhibited prominent genomic and epigenomic activation in a GIAC-specific manner. A complex interplay between the HNF4A promoter and three distal enhancer elements was coordinated by GIAC-specific MRTF including ELF3, GATA4, GATA6, and KLF5. HNF4A also self-regulated its own promoter and enhancers. Functionally, HNF4A promoted cancer proliferation and survival by transcriptional activation of many downstream targets, including HNF1A and factors of interleukin signaling, in a lineage-specific manner. Overall, our study provides new insights into the GIAC-specific gene regulatory networks and identifies potential therapeutic strategies against these common cancers. Significance: These findings show that GIAC-specific master regulatory transcription factors control HNF4A via three distal enhancers to promote GIAC cell proliferation and survival.

ObjectiveWhile oesophageal squamous cell carcinoma remains infrequent in Western populations, the incidence of oesophageal adenocarcinoma (EAC) has increased sixfold to eightfold over the past four decades. We aimed to characterise oesophageal cancer-specific and subtypes-specific gene regulation patterns and their upstream transcription factors (TFs). DesignTo identify regulatory elements, we profiled fresh-frozen oesophageal normal samples, tumours and cell lines with chromatin immunoprecipitation sequencing (ChIP-Seq). Mathematical modelling was performed to establish (super)-enhancers landscapes and interconnected transcriptional circuitry formed by master TFs. Coregulation and cooperation between master TFs were investigated by ChIP-Seq, circularised chromosome conformation capture sequencing and luciferase assay. Biological functions of candidate factors were evaluated both in vitro and in vivo.ResultsWe found widespread and pervasive alterations of the (super)-enhancer reservoir in both subtypes of oesophageal cancer, leading to transcriptional activation of a myriad of novel oncogenes and signalling pathways, some of which may be exploited pharmacologically (eg, leukemia inhibitory factor (LIF) pathway). Focusing on EAC, we bioinformatically reconstructed and functionally validated an interconnected circuitry formed by four master TFs—ELF3, KLF5, GATA6 and EHF—which promoted each other’s expression by interacting with each super-enhancer. Downstream, these master TFs occupied almost all EAC super-enhancers and cooperatively orchestrated EAC transcriptome. Each TF within the transcriptional circuitry was highly and specifically expressed in EAC and functionally promoted EAC cell proliferation and survival.ConclusionsBy establishing cancer-specific and subtype-specific features of the EAC epigenome, our findings promise to transform understanding of the transcriptional dysregulation and addiction of EAC, while providing molecular clues to develop novel therapeutic modalities against this malignancy.

SUMMARYTranscriptional network is controlled by master transcription factors (TFs) andcis-regulatory elements through interacting with target sequences and recruiting epigenetic regulators. By integration of enhancer profiling and chromatin accessibility, we establish super-enhancer (SE) mediated core regulatory circuitry (CRC) for esophageal squamous cell carcinoma (ESCC) and identify tumor cells-dependent CRC TFs-TP63, SOX2 and KLF5. They preferentially co-occupy SE loci and form a positive interconnected auto-regulatory loop through SEs to orchestrate chromatin and transcriptional programming. SE-associated oncogene-ALDH3A1is identified as a novel CRC target contributing to ESCC viability. Using circular chromosome conformation capture sequencing (4C-seq) and CRISPR/Cas9 genome editing, the direct interaction betweenTP63promoter and functional enhancers which is mediated by CRC TFs is identified. Deletion of each enhancer decreases expression of CRC TFs and impairs cell viability, phenocopying the knockdown of each CRC TF. Targeting epigenetic regulation by inhibition of either the BET bromodomain or HDAC disrupts the CRC program and its dependent global epigenetic modification, consequently suppressing ESCC tumor growth. Importantly, combination of both compounds result in synergistic anti-tumor effect.Graphical AbstractHIGHLIGHTSSuper-enhancers mediated transcriptional regulatory circuitry is established for ESCCTP63, SOX2 and KLF5 as CRC TFs co-localize super-enhancer loci to orchestrate chromatin accessibility and transcriptional dysregulationComplex interaction between functional enhancers andTP63promoter is mediated by CRC TFsALDH3A1is a key downstream target of ESCC CRC and is essential for ESCC cell survivalBET degrader-ARV-771 and HDAC inhibitor-Romidepsin synergistically inhibit ESCC tumor growth