Esophageal Adenocarcinoma-Derived Extracellular Vesicle MicroRNAs Induce a Neoplastic Phenotype in Gastric Organoids.

There have been no reports describing the effects of cancer cell-derived extracellular vesicles (EVs) on three-dimensional organoids. In this study, we delineated the proneoplastic effects of esophageal adenocarcinoma (EAC)-derived EVs on gastric organoids (gastroids) and elucidated molecular mechanisms underlying these effects. EVs were identified using PKH-67 staining. Morphologic changes, Ki-67 immunochemistry, cell viability, growth rates, and expression levels of miR-25 and miR-210, as well as of their target mRNAs, were determined in gastroids co-cultured with EAC-derived extracellular vesicles (c-EVs). C-EVs were efficiently taken up by gastroids. Notably, c-EV-treated gastroids were more crowded, compact, and multilayered and contained smaller lumens than did those cultured in organoid medium alone or in EAC-conditioned medium that had been depleted of EVs. Moreover, c-EV-treated gastroids manifested increased proliferation and cellular viability relative to medium-only or EV-depleted controls. Expression levels of miR-25 and miR-210 were significantly higher, and those of PTEN and AIFM3 significantly lower, in c-EV-treated versus medium-only or EV-depleted control groups. Inhibitors of miR-25 and miR-210 reversed the increased cell proliferation induced by c-exosomes in co-cultured gastroids by lowering miR-25 and miR-210 levels. In conclusion, we have constructed a novel model system featuring the co-culture of c-EVs with three-dimensional gastroids. Using this model, we discovered that cancer-derived EVs induce a neoplastic phenotype in gastroids. These changes are due, at least in part, to EV transfer of miR-25 and miR-210.

RNA sequencing of esophageal adenocarcinomas identifies novel fusion transcripts, including NPC1-MELK, arising from a complex chromosomal rearrangement.

BACKGROUND: Studies of chromosomal rearrangements and fusion transcripts have elucidated mechanisms of tumorigenesis and led to targeted cancer therapies. This study was aimed at identifying novel fusion transcripts in esophageal adenocarcinoma (EAC). METHODS: To identify new fusion transcripts associated with EAC, targeted RNA sequencing and polymerase chain reaction (PCR) verification were performed in 40 EACs and matched nonmalignant specimens from the same patients. Genomic PCR and Sanger sequencing were performed to find the breakpoint of fusion genes. RESULTS: Five novel in-frame fusion transcripts were identified and verified in 40 EACs and in a validation cohort of 15 additional EACs (55 patients in all): fibroblast growth factor receptor 2 (FGFR2)-GRB2-associated binding protein 2 (GAB2) in 2 of 55 or 3.6%, Niemann-Pick C1 (NPC1)-maternal embryonic leucine zipper kinase (MELK) in 2 of 55 or 3.6%, ubiquitin-specific peptidase 54 (USP54)-calcium/calmodulin dependent protein kinase II γ (CAMK2G) in 2 of 55 or 3.6%, megakaryoblastic leukemia (translocation) 1 (MKL1)-fibulin 1 (FBLN1) in 1 of 55 or 1.8%, and CCR4-NOT transcription complex subunit 2 (CNOT2)-chromosome 12 open reading frame 49 (C12orf49) in 1 of 55 or 1.8%. A genomic analysis indicated that NPC1-MELK arose from a complex interchromosomal translocation event involving chromosomes 18, 3, and 9 with 3 rearrangement points, and this was consistent with chromoplexy. CONCLUSIONS: These data indicate that fusion transcripts occur at a stable frequency in EAC. Furthermore, our results indicate that chromoplexy is an underlying mechanism that generates fusion transcripts in EAC. These and other fusion transcripts merit further study as diagnostic markers and potential therapeutic targets in EAC. Cancer 2017;123:3916-24. © 2017 American Cancer Society.

The novel fusion transcript NR5A2-KLHL29FT is generated by an insertion at the KLHL29 locus.

BACKGROUND: Novel fusion transcripts (FTs) caused by chromosomal rearrangement are common factors in the development of cancers. In the current study, the authors used massively parallel RNA sequencing to identify new FTs in colon cancers. METHODS: RNA sequencing (RNA-Seq) and TopHat-Fusion were used to identify new FTs in colon cancers. The authors then investigated whether the novel FT nuclear receptor subfamily 5, group A, member 2 (NR5A2)-Kelch-like family member 29 FT (KLHL29FT) was transcribed from a genomic chromosomal rearrangement. Next, the expression of NR5A2-KLHL29FT was measured by quantitative real-time polymerase chain reaction in colon cancers and matched corresponding normal epithelia. RESULTS: The authors identified the FT NR5A2-KLHL29FT in normal and cancerous epithelia. While investigating this transcript, it was unexpectedly found that it was due to an uncharacterized polymorphic germline insertion of the NR5A2 sequence from chromosome 1 into the KLHL29 locus at chromosome 2, rather than a chromosomal rearrangement. This germline insertion, which occurred at a population frequency of 0.40, appeared to bear no relationship to cancer development. Moreover, expression of NR5A2-KLHL29FT was validated in RNA specimens from samples with insertions of NR5A2 at the KLHL29 gene locus, but not from samples without this insertion. It is interesting to note that NR5A2-KLH29FT expression levels were significantly lower in colon cancers than in matched normal colonic epithelia (P =.029), suggesting the potential participation of NR5A2-KLHL29FT in the origin or progression of this tumor type. CONCLUSIONS: NR5A2-KLHL29FT was generated from a polymorphism insertion of the NR5A2 sequence into the KLHL29 locus. NR5A2-KLHL29FT may influence the origin or progression of colon cancer. Moreover, researchers should be aware that similar FTs may occur due to transchromosomal insertions that are not correctly annotated in genome databases, especially with current assembly algorithms. Cancer 2017;123:1507-1515. © 2017 American Cancer Society.

Long non-coding RNA HNF1A-AS1 regulates proliferation and migration in oesophageal adenocarcinoma cells.

OBJECTIVES: Long non-coding RNAs (lncRNA) have been shown to play important roles in the development and progression of cancer. However, functional lncRNAs and their downstream mechanisms are largely unknown in the molecular pathogenesis of oesophageal adenocarcinoma (EAC) and its progression. DESIGN: lncRNAs that are abnormally upregulated in EACs were identified by RNA-sequencing analysis, followed by quantitative RT-PCR (qRTPCR) validation using tissues from 25 EAC patients. Cell biological assays in combination with small interfering RNA-mediated knockdown were performed in order to probe the functional relevance of these lncRNAs. RESULTS: We discovered that a lncRNA, HNF1A-AS1, is markedly upregulated in human primary EACs relative to their corresponding normal oesophageal tissues (mean fold change 10.6, p<0.01). We further discovered that HNF1A-AS1 knockdown significantly inhibited cell proliferation and anchorage-independent growth, suppressed S-phase entry, and inhibited cell migration and invasion in multiple in vitro EAC models (p<0.05). A gene ontological analysis revealed that HNF1A-AS1 knockdown preferentially affected genes that are linked to assembly of chromatin and the nucleosome, a mechanism essential to cell cycle progression. The well known cancer-related lncRNA, H19, was the gene most markedly inhibited by HNF1A-AS1 knockdown. Consistent to this finding, there was a significant positive correlation between HNF1A-AS1 and H19 expression in primary EACs (p<0.01). CONCLUSIONS: We have discovered abnormal upregulation of a lncRNA, HNF1A-AS1, in human EAC. Our findings suggest that dysregulation of HNF1A-AS1 participates in oesophageal tumorigenesis, and that this participation may be mediated, at least in part, by modulation of chromatin and nucleosome assembly as well as by H19 induction.

Hypomethylation of noncoding DNA regions and overexpression of the long noncoding RNA, AFAP1-AS1, in Barrett's esophagus and esophageal adenocarcinoma.

BACKGROUND & AIMS: Alterations in methylation of protein-coding genes are associated with Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC). Dysregulation of noncoding RNAs occurs during carcinogenesis but has never been studied in BE or EAC. We applied high-resolution methylome analysis to identify changes at genomic regions that encode noncoding RNAs in BE and EAC. METHODS: We analyzed methylation of 1.8 million CpG sites using massively parallel sequencing-based HELP tagging in matched EAC, BE, and normal esophageal tissues. We also analyzed human EAC (OE33, SKGT4, and FLO-1) and normal (HEEpic) esophageal cells. RESULTS: BE and EAC exhibited genome-wide hypomethylation, significantly affecting intragenic and repetitive genomic elements as well as noncoding regions. These methylation changes targeted small and long noncoding regions, discriminating normal from matched BE or EAC tissues. One long noncoding RNA, AFAP1-AS1, was extremely hypomethylated and overexpressed in BE and EAC tissues and EAC cells. Its silencing by small interfering RNA inhibited proliferation and colony-forming ability, induced apoptosis, and reduced EAC cell migration and invasion without altering the expression of its protein-coding counterpart, AFAP1. CONCLUSIONS: BE and EAC exhibit reduced methylation that includes noncoding regions. Methylation of the long noncoding RNA AFAP1-AS1 is reduced in BE and EAC, and its expression inhibits cancer-related biologic functions of EAC cells.