Scholarly article on topic 'SOX15 Governs Transcription in Human Stratified Epithelia and a Subset of Esophageal Adenocarcinomas'

SOX15 Governs Transcription in Human Stratified Epithelia and a Subset of Esophageal Adenocarcinomas Academic research paper on "Biological sciences"

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{"Barrett’s Esophagus" / "Esophageal Gene Regulation" / "Esophageal Transcriptome" / "SOX15 Cistrome"}

Abstract of research paper on Biological sciences, author of scientific article — Rita Sulahian, Justina Chen, Zoltan Arany, Unmesh Jadhav, Shouyong Peng, et al.

Background & Aims Intestinal metaplasia (Barrett’s esophagus, BE) is the principal risk factor for esophageal adenocarcinoma (EAC). Study of the basis for BE has centered on intestinal factors, but loss of esophageal identity likely also reflects the absence of key squamous-cell factors. As few determinants of stratified epithelial cell-specific gene expression have been characterized, identifying the necessary transcription factors is important. Methods We tested regional expression of mRNAs for all putative DNA-binding proteins in the mouse digestive tract and verified the esophagus-specific factors in human tissues and cell lines. Integration of diverse data defined a human squamous esophagus-specific transcriptome. We used chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) to locate transcription factor binding sites, computational approaches to profile the transcripts in cancer data sets, and immunohistochemistry to reveal protein expression. Results The transcription factor Sex-determining region Y-box 15 (SOX15) is restricted to esophageal and other murine and human stratified epithelia. SOX15 mRNA levels are attenuated in BE, and its depletion in human esophageal cells reduces esophageal transcripts significantly and specifically. SOX15 binding is highly enriched near esophagus-expressed genes, indicating direct transcriptional control. SOX15 and hundreds of genes coexpressed in squamous cells are reactivated in up to 30% of EAC specimens. Genes normally confined to the esophagus or intestine appear in different cells within the same malignant glands. Conclusions These data identify a novel transcriptional regulator of stratified epithelial cells and a subtype of EAC with bi-lineage gene expression. Broad activation of squamous-cell genes may shed light on whether EACs arise in the native stratified epithelium or in ectopic columnar cells.

Academic research paper on topic "SOX15 Governs Transcription in Human Stratified Epithelia and a Subset of Esophageal Adenocarcinomas"



S0X15 Governs Transcription in Human Stratified Epithelia d*


and a Subset of Esophageal Adenocarcinomas

Rita Sulahian,1'2 Justina Chen,1 Zoltan Arany,3 Unmesh Jadhav,1'2 Shouyong Peng,1

4 12 5 5

Anil K. Rustgi,4 Adam J. Bass,12 Amitabh Srivastava,5 Jason L. Hornick, and Ramesh A. Shivdasani1,2

department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts; 2Department of Medicine, Harvard Medical School, Harvard University, Boston, Massachusetts; 3Cardiovascular Institute and 4Division of Gastroenterology, Departments of Medicine and Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; 5Department of Pathology, Brigham & Women's Hospital, and Department of Pathology, Harvard Medical School, Harvard University, Boston, Massachusetts


This study identifies SOX15 as a direct transcriptional regulator of a substantial fraction of cell type-specific genes in stratified epithelial cells. SOX15 expression is attenuated in intestinal metaplasia (Barrett's esophagus) but is active in many esophageal adenocarcinomas.

BACKGROUND & AIMS: Intestinal metaplasia (Barrett's esophagus, BE) is the principal risk factor for esophageal adenocarcinoma (EAC). Study of the basis for BE has centered on intestinal factors, but loss of esophageal identity likely also reflects the absence of key squamous-cell factors. As few determinants of stratified epithelial cell-specific gene expression have been characterized, identifying the necessary transcription factors is important.

METHODS: We tested regional expression of mRNAs for all putative DNA-binding proteins in the mouse digestive tract and verified the esophagus-specific factors in human tissues and cell lines. Integration of diverse data defined a human squa-mous esophagus-specific transcriptome. We used chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) to locate transcription factor binding sites, computational approaches to profile the transcripts in cancer data sets, and immunohistochemistry to reveal protein expression.

RESULTS: The transcription factor Sex-determining region Y-box 15 (SOX15) is restricted to esophageal and other murine and human stratified epithelia. SOX15 mRNA levels are attenuated in BE, and its depletion in human esophageal cells reduces esophageal transcripts significantly and specifically. SOX15 binding is highly enriched near esophagus-expressed genes, indicating direct transcriptional control. SOX15 and hundreds of genes coexpressed in squamous cells are reactivated in up to 30% of EAC specimens. Genes normally confined to the esophagus or intestine appear in different cells within the same malignant glands.

CONCLUSIONS: These data identify a novel transcriptional regulator of stratified epithelial cells and a subtype of EAC with bi-lineage gene expression. Broad activation of squamous-cell genes may shed light on whether EACs arise in the native stratified epithelium or in ectopic columnar cells. (Cell Mol

Gastroenterol Hepatol 2015;1:598-609; 10.1016/j.jcmgh.2015.07.009)

Keywords: Barrett's Esophagus; Esophageal Gene Regulation; Esophageal Transcriptome; SOX15 Cistrome.

Intestinal metaplasia of the esophagus (Barrett's esophagus, BE) is a common, chronic condition in which an epithelium containing intestinal goblet and other columnar cells replaces the native stratified squamous mucosa.1 BE results from chronic acid and bile reflux. Over time, the metaplastic tissue may become dysplastic, and it progresses to invasive cancer in three to five cases per 1000 person-years.2 Esophageal adenocarcinoma (EAC) arises principally in the setting of BE, and the incidence of this cancer in the West increased about eightfold between 1970 and 2010, with about 18,000 new U.S. cases and 15,000 deaths expected in 2015 (

Investigation into the mechanisms of BE has centered largely on determinants of intestinal identity,3 particularly the intestine-restricted transcription factors (TFs) Caudal type homeobox 1 (CDX1) and CDX2, which specify the embryonic intestine.4 Forced expression of CDX2 or CDX1 in the mouse stomach induces ectopic intestinal differentia-tion,5,6 and both factors are implicated in activating intestinal genes in BE,7,8 though forced CDX2 expression in the

Abbreviations used in this paper: BE, Barrett's esophagus; CDX1/2, caudal type homeobox 1/2; ChIP, chromatin immunoprecipitation; ChIP-seq, chromatin immunoprecipitation with high-throughput sequencing; EAC, esophageal adenocarcinoma; G-E, gastroesopha-geal; KRT5, keratin 5, type II; KRT6A, keratin 6A, type II; PAX9, paired box 9; PBS, phosphate-buffered saline; qRT-PCR, quantitative reverse-transcription polymerase chain reaction; shRNA, small hairpin RNA; SIM2, single-minded family bHLH transcription factor 2; SOX2, 15, sex-determining region Y-box 2, -box 15; TCGA, The Cancer Genome Atlas; TF, transcription factor; TP63, tumor protein P63; TRIM29, tripartite motif containing 29.

(j) Most current article

© 2015 The Authors. Published by Elsevier Inc. on behalf of the AGA Institute. This is an open access article under the CC BY-NC-ND license ( '/ ). 2352-345X

mouse esophagus does not induce BE per se.9 Loss of esophagus-specific transcripts and of stratified squamous morphology probably reflects parallel loss of transcriptional determinants of the native epithelium, which are largely unknown. Tumor protein P63 (TP63) regulates differentiation of all stratified epithelia, such as those in the esophagus and skin,10,11 acting in part through another transcription factor, basonuclin 1 (BNC1).12 Sex-determining region Y-box 2 (SOX2) controls esophageal differentiation in embryos13 and growth of adult progenitor cells,14,15 an activity in which Kruppel-like factor 4 (KLF4) and KLF5 also may participate.16 Forkhead box A2 (FOXA2) is expressed in embryonic but not in adult esophageal cells.17 We sought to identify other tissue-restricted TFs that might control the characteristic stratified epithelium.

Among all putative DNA-binding proteins, we searched first for those with esophagus-restricted expression among digestive epithelia and then for factors with attenuated expression in BE. We identified sex-determining region Y-box 15 (SOX15) as such a TF and we show that it directly controls transcription of a large fraction of human

esophagus-expressed genes. SOX15 is absent from most EACs, but up to 30% of cases retain expression of SOX15 and its target genes, coexpressing representative intestinal and squamous-specific genes within the same tissue. Together, these data identify a novel regulator of stratified epithelial genes and a subtype of EAC with bi-lineage gene expression.

Materials and Methods

Tissue Preparation and Transcription Factor Expression Screen

We isolated epithelial sheets from the esophagus, gastric corpus-antrum, and duodenum of 1-month old CD1 and C57BL/6 mice. Before peeling the mucosa using fine forceps, the esophagus was treated with 0.1% collagenase-dispase (cat. no. 11097113001; Roche Applied Science, Indianapolis, IN) in phosphate-buffered saline (PBS) for 15 minutes at 37° C, whereas stomach and duodenum were incubated in 1 mM ethylenediaminetetraacetic acid (EDTA) in PBS at 37°C. To determine the relative transcript levels (Figure 1A-C), we used quantitative reverse-transcription

Fold Increase

Esophagus vs Intestine

Intestine I/E I/S Stomach S/E S/I



Fold difference > 1000

> 100 > 32

Fold dll




Figure 1. Differential transcription factor (TF) expression in the normal mouse gut and other tissues. (A) Distribution of all TFs in wild-type mouse digestive epithelia, as revealed in a quantitative reverse-transcription polymerase chain reaction (qRT-PCR) screen. Expression of 1880 Tf mRNAs was assessed in epithelial cell isolates from adult CD1 mouse esophagus (red), stomach and intestine (blue). (B) TFs restricted to intestinal (I), stomach (S) or esophageal (E) epithelium, with the fold-excess over other tissues represented in shades of blue. (C) Relative expression of Sim2, Pax9, Sox15, Trim29, Elf5, and Tcfap2a mRNAs in mouse tissues. The fold-excess values are represented in shades of color as indicated in the key. (D) Products of qRT-PCRforthe six most highly esophagus-specific TF mRNAs in 12 adult mouse organs, showing selective expression in the esophagus and of some factors in the skin.

Figure 2. Differential transcription factor (TF) expression in normal and metaplastic human esophagus. (A) Expression profiles of PAX9, SIM2, SOX15, and TRIM29 in 65 human organs. Data from necropsies,21 analyzed using Oncomine tools,31 show selective expression in the esophagus and other stratified epithelia such as the oropharyngeal mucosa and skin derivatives. (B) Relative expression of esophagus-active keratin genes in the human Barrett's esophagus (BE) cell line series (CP-A, CP-B, and CP-C) with increasing dysplasia. Results of quantitative reverse-transcription polymerase chain reaction analysis are represented with respect to transcript levels in the immortalized human esophageal cell line EPC2-hTert.20 (C) Relative expression of esophagus-specific TF mRNAs in human BE cell lines CP-A, CP-B and CP-C, expressed in relation to levels in EPC2-hTert cells. (D) Fold-enriched expression of esophagus-specific TF mRNAs in fresh human esophageal epithelial biopsy samples8 relative to areas of BE in the same patients. (E) Expression of esophagus-specific TFs and intestine-specific genes in normal human esophagus and BE resection specimens. Data used22 were analyzed using Oncomine tools.

polymerase chain reaction (qRT-PCR) and a library containing oligonucleotide primers specific to 1880 known and putative TFs.18 Tissue-specific TFs were identified using the comparative CT method.19 To further determine the tissue specificity (Figure 1D), other whole organs were harvested from adult C57BL/6 mice.

Cell Lines

We cultured CP-A (KR-42421), CP-B (CP-52731), and CP-C (CP-94251) cells (American Type Culture Collection,

Manassas, VA) in MCDB-153 medium (cat. no. M7403; Sigma-Aldrich, St. Louis, MO) supplemented with 0.4 mg/mL hydrocortisone, 20 ng/mL recombinant human epidermal growth factor (cat. no. E9644; Sigma-Aldrich), 8.4 mg/L cholera toxin (cat. no. H0135; Sigma-Aldrich), 20 mg/L adenine (cat. no. A2786; Sigma-Aldrich), 140 mg/mL bovine pituitary extract (cat. no. P1476; Sigma-Aldrich), ITS supplement (cat. no. I1884; Sigma-Aldrich) (final concentrations: 5 mg/mL insulin, 5 mg/mL transferrin, 5 ng/mL sodium selenite), 4 mM glutamine, and 5% fetal bovine serum. EPC2-hTert cells20 were cultured in Keratinocyte-

SFM medium (GIBCO/Life Technologies, Grand Island, NY) supplemented with bovine pituitary extract and recombinant human epidermal growth factor (GIBCO). Soybean trypsin inhibitor (Sigma-Aldrich) was used to quench trypsin activity during cell passage.

Gene Analyses

Figures 2A, D, and E and selected other figures show analyses of relative mRNA expression levels from published studies of 65 adult human tissues,21 of human esophageal biopsy specimens,8 and of human normal esophagus, BE,

and EAC samples.22 The data were reanalyzed with respect to SOX15 using Oncomine tools (Compendia Bioscience, Ann Arbor, MI;, considering all samples in each data set. Genes significantly associated with SOX15 were ranked on the basis of correlation values. Enriched Gene Ontology terms were determined using DAVID tools (

We examined processed RNA-seq data from a Cancer Genome Atlas (TCGA) study on stomach cancer,23 first isolating 30 CIN+ tumors arising at the gastroesophageal (G-E) junction or gastric cardia for unsupervised clustering (Supplementary Figure 1). To this group we applied hierarchical clustering (using hclust from the R package; http:// on the 1000 most variable transcripts normalized according to expression z-scores, followed by a second hierarchic clustering on the set of 317 genes coex-pressed with SOX15. To assess the specificity of SOX15 overexpression in tumors of the gastric cardia, we compared with RNA-seq data from TCGA studies on colon24 and distal gastric adenocarcinomas.23

Experimental RNA Analyses

Total RNA was isolated using TRIzol (Invitrogen/Life Technologies, Carlsbad, CA), treated with the RNeasy Mini Kit (Qiagen, Valencia, CA), and DNA was digested using Turbo DNA-Free (Ambion/Life Technologies, Austin, TX). For qRT-PCR analysis (Figure 1A-C, Figure 2B and C, etc. 1 mg of total RNA was reverse-transcribed with Superscript III First Strand Synthesis System (Invitrogen), and cDNA was amplified using SYBRGreen PCR Master Mix (Applied Biosystems, Foster City, CA). RNA-seq libraries (the full data set is deposited in the Gene Expression Omnibus with accession number GSE62909) were prepared from 300 ng of total RNA using TruSeq RNA Sample Preparation kits (Illumina, San Diego, CA), and 75-base pair (bp) single-end sequences were obtained on a NextSeq 500 instrument (Illumina). The reads were aligned to human genome build Hg19 using TopHat v2.0.6 ( Expression levels of transcripts in duplicate samples were calculated as fragments per kb per 106 mapped reads (FPKM) using Cufflinks v2.0.2 ( cufflinks/), and differential expression was determined using CuffDiff (

cuffdiff/).25 Chi-square tests with 1 degree of freedom and two-tailed P values were used to assess significance. Log2 (FPKM + 1) values for control and SOX15-depleted samples were plotted to display differential expression.

Depletion of SOX15 and Expression of Biotin-Tagged SOX15

The cells were infected with lentiviruses generated from the pLKO.1 vector (Open Biosystems/GE Dharmacon, Huntsville, AL) carrying either a SOX15-targeting small hairpin RNA (shRNA) (TGCCTGGCAGCTATGGCTCTT) or a control, nonspecific shRNA that does not complement any human gene and is not toxic to cultured human cells (CCTAAGGTTAAGTCGCCCTCG). Human SOX15 cDNA was cloned into the pUltra vector (cat. no. 24129; Addgene, Cambridge, MA) together with cassettes for the T2A sequence, biotin, and BirA-V5 (gift of Ben Ebert, Brigham & Women's Hospital, Boston, MA).

Chromatin Immunoprecipitation and Chromatin Immunoprecipitation With High-Throughput Sequencing

Cells were cross-linked with 2 mM disuccinimidyl gluta-rate (catalog no. 20593; Pierce Biotechnology, Rockford, IL) in PBS for 45 minutes, followed by 10 minutes with 1% formaldehyde (Pierce Biotechnology) in PBS at room temperature. Chromatin immunoprecipitation (ChIP) and chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) were performed as described previously elsewhere26 using a 30-mL slurry of streptavidin-conjugated magnetic beads (cat. no. 65601; Invitrogen). We used Cistrome tools ( to identify and annotate TF binding sites, generate wiggle files and conservation plots, identify enriched sequence motifs and linked genes, and compare data across ChIP-seq libraries. Wiggle traces were projected on the Integrative Genome Viewer ( Functions of genes within 50 kb of SOX15 occupancy were determined using GREAT ( ChIP-seq data are deposited in the Gene Expression Omnibus (GEO) database with accession number GSE62909 (www.ncbi.nlm.nih. gov/geo/).

Figure 3. (See previous page). Impact of SOX15 depletion on esophageal gene expression. (A) Delineation of the human esophageal transcriptome. The mRNAs expressed in human esophageal necropsy specimens (left) were compared against transcripts from 7 other postmortem organs,21 and mRNAs present in fresh esophageal biopsy specimens (right) were compared against transcripts from fresh Barrett's esophagus (BE) and intestinal biopsies.8 Numbers in each box represent squamous esophagus-specific genes relative to that tissue. We identified 362 and 300 esophagus-specific genes, respectively, with a significant 114-gene overlap (P < .0001, chi-square test). The top Gene Ontology (GO) terms in each case are highly related to stratified epithelia. (B) SOX15 mRNA depletion in 3 representative experiments in which CPA cells were infected with lentiviruses carrying SOX15-specific or a nonspecific (NS) 21-bp shRNAs. Knockdown efficiency, assessed by quantitative reverse-transcription polymerase chain reaction 72 hours after infection, was >8- to 10-fold in every experiment.

(C) Results of duplicate RNA-seq analysis of genes differentially expressed in CP-A cells treated with SOX15-specific (y-axis) or control, nonspecific (x-axis) shRNAs. Grey dots mark differential expression (log2 >1.5-fold, q < .05); genes present in the union (548 genes) or intersection (114) sets of esophagus-specific genes are represented by red and blue dots, respectively.

(D) Fraction of esophagus-specific transcripts reduced upon SOX15 depletion (red, 548 union-set genes; blue, 114 intersection-set genes) compared with five random sets of equal numbers of genes expressed in CP-A cells (grey bars). The table lists GO terms enriched among SOX15-dependent genes.

Figure 4. Genome-wide SOX15 occupancy and gene dependence in human esophageal cells. (A) Summary of chromatin immunoprecipitation (ChIP) analysis for biotinylated SOX15 in CP-A cells, showing high sequence conservation and significant enrichment of a canonical SOX recognition motif ACAA(A/T)G among 4864 identified binding sites. SOX15 mainly binds DNA far from promoters. (B) Gene Ontology (GO) terms enriched among the two nearest genes within 50 kb of SOX15-binding sites, determined using GREAT.28 (C) Percentages of esophagus-specific genes (as determined in Figure 2A, red: union; blue: intersection set) that bind SOX15 within 50 kb of the transcription start site (TSS), compared with five random gene sets of equivalent size (grey bars, P < .0001). The table lists GO terms enriched among genes from the esophagus transcriptome that lie within 50 kb of SOX15 binding sites. (D) SOX15 binding (orange dots) within 50 kb of genes expressed in SOX15-depleted and control CP-A cells (grey dots, q < .05, as in Figure 2B). Dashed lines demarcate genes unaffected by SOX15 loss. (E) Genes reduced or increased in SOX15-depleted cells are significantly enriched for nearby SOX15 binding. Together with the proportions of orange dots in D, the data imply direct SOX15 activation of many, and direct repression of fewer genes. (F) Integrated Genome Viewer representation of esophageal gene KRT6A, showing SOX15 binding at the locus (top rows, blue, ChIP-seq tags) and reduced expression in SOX15-depleted CP-A cells (bottom rows, grey, RNA-seq tags). Numbers represent the height of the y-axis.


We baked 4-mm-thick tissue paraffin sections overnight at 37° C; they then were deparaffinized in xylenes, rehydrated, and peroxidase activity was blocked with 1.5% H2O2 in methanol for 10 minutes. Slides were treated with 0.01 M citrate buffer, pH 6.0, in a pressure cooker at 120°C for 30

minutes for antigen retrieval, then transferred to Tris-buffered saline. Sections were first incubated with mouse CDX2 Ab (clone CDX2-88, Biogenex mu392A-uc, 1:200) for 40 minutes, followed by Dako Envision+ Mouse (Dako K4007; Dako, Carpinteria, CA) secondary Ab for 30 minutes, and developed with 3,3'-diaminobenzidine (Dako). Sections were

Figure 5. SOX15 expression in esophageal adenocarcinomas (EACs). (A) Gene coexpression profiles for SOX15 (left) and CDX2 (right) in a large collection of normal, Barrett's esophagus (BE), and EAC epithelium.22 We found that 317 transcripts correlated strongly (r >0.81) with SOX15 mRNA levels in normal esophagus and in approximately one-third of 75 EACs in this series. The 100 most highly correlated genes are shown. (B) Gene Ontology (GO) term enrichment among these 317 SOX15-coexpressed genes. (C) Top: Fraction of SOX15 coexpressed genes showing SOX15 occupancy (observed) within 50 kb compared with the fraction expected for appropriate random gene sets of equal size. Bottom: Fraction of SOX15 coexpressed genes affected by SOX15 depletion (observed) compared with the fraction expected among random gene sets of equal size. (D) Ranges of SOX15 mRNA expression extracted from RNA-seq data on the Cancer Genome Atlas collection of cancers of the gastric cardia, fundus/body, and antrum,23 or colon and rectum.24 Statistical significance of the differences was determined by t-test.

then incubated with mouse KRT5 Ab (clone XM26, Leica NCL-L-CK, 1:500) for 40 minutes, followed by PowerVision AP mouse (catalog no. PV6110; Leica Biosystems, Buffalo Grove, IL) secondary Ab for 30 minutes, developed with Permanent Red, and counterstained with Mayer's hematoxylin. To stain resection specimens that carried areas of BE, slides were treated with the same mouse KRT5 Ab, followed by Dako Envision+ Mouse (Dako K4007) secondary Ab for 30 minutes, and developed with 3,3'-diaminobenzidine (Dako).


Identification of Transcription Factors That Are Specific to the Esophageal Epithelium and Attenuated in Barrett's Esophagus

To identify candidate regulators of esophageal squamous identity, we first examined epithelia isolated from different regions of the mouse alimentary tract—esophagus, glandular stomach, and intestine (duodenum)—with a goal to identify TF mRNAs expressed selectively in the stratified esophageal epithelium (Figure 1A). Among 1880 known and

putative DNA-binding proteins, those showing >32-fold higher expression in the intestinal mucosa included the known intestinal factors Atoh1, Cdx1, Creb3l3, Hnf4g, and Isx,29 underscoring the fidelity of the experimental approach (Figure 1B). Forty factors and 59 TF genes showed considerably higher expression in esophageal cells, compared with the gastric corpus and the intestine, respectively (Figure 1A), and 21 TFs were common to the two esophagus-specific groups (Figure 1B).

To exclude variability among mouse strains and to assess specificity relative to nondigestive organs, we measured expression of these 21 mRNAs in nine diverse tissues from C57BL/6 mice, including the skin. Six TFs gave consistent evidence of high tissue specificity (Figure 1C and D). Sim2 (single-minded family bHLH transcription factor 2) and Pax9 (paired box 9) showed the greatest specificity, followed by Sox15 and Trim29 (tripartite motif containing 29), which showed some expression in murine skin. Additional data from 65 adult human tissues21 revealed robust expression of each of these four TF mRNAs in the esophagus, with varying levels in other stratified squamous

Figure 6. Bi-lineage gene expression in a subset of esophageal adenocarcinomas (EACs). (A-B) High correlation (r = 0.97) of SOX15 and KRT5 mRNAs in normal esophagus, Barrett's esophagus (BE), and EAC, validating KRT5 as a proxy for SOX15 and other stratified epithelium-specific genes. (B) Table of IHC results for KRT5 and CDX2 in 99 cases of EAC. (C-D) Representative immunohisto-chemistry for KRT5 (red, a surrogate marker for SOX15 and other squamous-specific gene products) and CDX2 (brown, a representative intestine-specific marker) in two separate cases (C and D) of human EAC. High KRT5 expression is evident, with almost mutually exclusive distribution of KRT5 (++ to +++) and CDX2 (+++ to ++++) in the same malignant glands. Original magnifications: Top, 200x; Bottom, 400x. (E) Absence of KRT5 immunostaining in areas of BE. Adjoining areas of normal stratified epithelium provide a positive control and contrast. IHC, immunohistochemistry.

tissues, such as the tongue, mouth, pharynx, and skin derivatives (Figure 2A).

To determine whether these TFs may function in the identity of stratified epithelia, we examined expression data from immortalized EPC2-hTert esophageal keratinocytes20 and found high expression of each factor except ELF5 (data not shown). Next we tested a series of three cell lines: CP-A, which represents nondysplastic BE, and CP-B and CP-C, which represent BE with high-grade dysplasia. This cell line series replicates disease progression30 with reduced levels of multiple keratin mRNAs (Figure 2B).

We observed a concomitant decline in SOX15 and TRIM29 levels, matching or exceeding that of TP63 mRNA, with little variance in the other factors (Figure 2C). Although these findings do not in isolation give robust information about a relation to mucosal dysplasia per se, they reveal the squamous cell specificity of SOX15 and TRIM29. Furthermore, gene expression data from a collection of human esophageal biopsy specimens8 showed significantly fewer SOX15 and TRIM29 mRNAs in primary BE compared with adjacent normal esophageal mucosa (Figure 2D).

Finally, we used Oncomine tools31 to analyze mRNA data from an independent series of 28 frozen human normal esophagus and 15 frozen BE biopsy specimens.22 Levels of PAX9, SOX15, and TRIM29 were uniformly high in normal esophagus and attenuated in BE specimens (Figure 2E).

Together, these data identify SOX15, PAX9, and TRIM29 as conserved candidate determinants of squamous cell identity. PAX9 levels were similar in CP-A, CP-B and CP-C cells (Figure 2C), and although TRIM29 has a putative DNA-binding domain, its role in transcriptional regulation is poorly defined and uncertain.32 By contrast, SOX proteins control differentiation of diverse tissues, often in conjunction with other family members,33 and related factors such as SOX2 and SOX7 are known to regulate aspects of esophageal organogenesis and squamous cell cancer.13,14 We therefore concentrated on human SOX15, which shares 85% homology (100% in the DNA-binding domain) with the mouse protein. SOX15 was previously noted as one among hundreds of genes in various expression profiling stud-ies,34-36 and we proceeded to investigate its functions.

SOX15 Depletion Affects Genes Specific to Stratified Epithelium

To test whether SOX15 might regulate genes specific to the stratified human esophageal epithelium, we needed to delineate the corresponding transcriptome. To this end, first we considered public data from adult human postmortem tissues21 (see Figure 2A) and identified 362 genes that express at greater than threefold higher levels (P < .05) in the esophagus than in any of seven diverse tissues, including glandular stomach, from the same collection of postmortem samples (Figure 3A). Second, we identified 300 genes with greater than threefold higher mRNA levels (P < .05) in normal fresh human esophagus biopsy specimens than in adjacent areas of BE or in fresh intestinal biopsies from the same study.8 Consistent with specific roles in stratified epithelia, both gene sets were highly enriched for functions

related to ectodermal, epidermal, and keratinocyte differentiation, and they shared 114 genes (Figure 3A). Accordingly, we regard the union set of 548 genes as a good representation of human esophagus-specific transcripts and the intersection set of 114 genes as an especially robust subset.

To determine whether SOX15 regulates any part of this transcriptome, we used lentiviral delivered shRNA to deplete the TF in CP-A cells. These cells express keratin and TF genes specific to stratified epithelia, including SOX15, at levels similar to immortalized EPC2-hTert esophageal epithelial cells (Figure 2B and C), and they tolerate lentiviral infection and drug selection. Because SOX15 depletion retarded CP-A cell growth and survival, we harvested cells 72 hours after infection, when they appeared healthy but the SOX15 mRNA levels were appreciably reduced (Figure 3B).

RNA-seq analysis showed reduced and increased levels of 2950 and 717 transcripts, respectively, compared with cells treated with a nonspecific shRNA (Figure 3C). In agreement with the deficit in cell growth, these genes were enriched for Gene Ontology terms related to the cell cycle (Supplementary Table 1). More importantly, genes reduced in SOX15-depleted cells included 26.4% of the human esophagus-specific "union" transcriptome, compared with 15.34% overlap with multiple sets of 2950 random genes expressed in CP-A cells (Figure 3D; P < .0001). Correspondence was even higher for the esophagus-specific "intersection" transcriptome, where 31.5% of genes were reduced in SOX15-depleted cells compared with 14.68% of random genes (P < .0001). SOX15-dependent genes were highly enriched for functions related to stratified epithelia (Figure 3D). None of the 114 genes in the esophagus-specific "intersection set," and only 23 genes in the "union set" were increased in SOX15-depleted CP-A cells, and we observed no increase in intestinal genes. Rather, the 717 increased transcripts were enriched for functions such as apoptosis and vesicular transport (Supplementary Table 1). Thus, beyond cell survival or proliferation, a substantial portion of the esophageal transcriptome depends on SOX15.

SOX15 Directly Regulates Esophagus-Specific Genes

Depletion of SOX15 could affect transcript levels as a consequence of its cis-regulatory activity or indirectly. To determine whether SOX15 might regulate dependent genes directly, we used ChIP-seq to map its cistrome. Because available antibodies performed poorly in ChIP assays, we expressed biotin-tagged SOX15 stably in CP-A cells and precipitated chromatin using streptavidin beads. The nearly 5000 high-confidence binding sites we identified by this approach showed high sequence conservation and greatest enrichment for the SOX consensus motif, which was present in >97% of sites, implying direct TF occupancy (Figure 4A). Similar to other tissue-specific TFs, SOX15 occupied few promoters (6.2% of all binding sites) and bound DNA predominantly in intergenic regions and introns (Figure 4A).

GREAT analysis28 of the nearest flanking genes within 50 kb of SOX15 occupancy revealed enrichment of pathways known to be vital in stratified epithelia, such as epidermal

growth factor and Rho/Rac signaling, and in cell survival (Figure 4B). Moreover, 20.9% of genes in the human esophagus-specific transcriptome and 31.5% of genes common to the two esophagus transcript sources showed at least one SOX15-binding site within 50 kb of the transcription start site, compared with about 5% of comparable numbers of random genes (P < .0001, Figure 4C).

Gene Ontology terms related to stratified epithelia were further enriched among SOX15-bound genes (Figure 4C). Most importantly, genes affected by SOX15 depletion in CP-A cells were highly enriched for nearby SOX15 binding, compared with random gene sets of equal size (P < .0001), and SOX15-bound genes reduced in SOX15-depleted cells far outnumbered genes that were increased (Figure 4D and E).

Taken together, these data indicate direct SOX15 regulation of genes specific to the stratified squamous epithelium, with a strong bias toward gene activation. Canonical esophageal genes such as KRT6A (keratin 6A, type II) illustrate SOX15 occupancy at putative cis-regulatory sites and reduced expression in SOX15-depleted cells (Figure 4F).

SOX15 in Human Esophageal Adenocarcinoma

SOX15 is expressed highly in normal human esophagus, but not in the BE cell lines CP-B and CP-C (Figure 2C) or in areas of intestinal metaplasia in vivo (Figure 2D and E). To our surprise, RNA expression data from a large collection of frozen primary esophagus, BE, and EAC biopsy specimens22 revealed high SOX15 mRNA levels in up to one-third of EACs (Figure 3A, left; note that all samples in this study, including EAC, were frozen biopsy specimens). Moreover, at least 317 transcripts that are strongly coexpressed with SOX15 in the normal esophagus (r > 0.81; Supplementary Table 2) were also present in the same EAC specimens (Figure 5A, which shows the 100 genes with highest correlation), suggesting broad activation of the squamous cell transcriptional program.

Accordingly, functions related to stratified epithelia were significantly enriched among the genes coexpressed with SOX15 (Figure 5B). The canonical intestinal marker CDX2 and its coexpressed genes (r > 0.81) were expressed in many SOX15+ and also in SOX15~ specimens (Figure 5A, right), revealing coexpression of esophageal and intestinal genes in some cases. Moreover, 21.6% of genes coexpressed with SOX15 in this analysis showed SOX15 binding within 50 kb in CP-A cells, compared with ~6% of random genes (P < .0001; Figure 5C, top), which implies that many of these genes are direct transcriptional targets. Indeed, the effects of SOX15 depletion were statistically significantly greater on these genes than on random sets of genes expressed in CP-A cells (P < .017; Figure 5C, bottom). These features collectively suggest direct SOX15 regulation of many esophagus-restricted genes that are silent in BE and reactivated in up to one-third of human EACs.

To exclude the possibility that EACs expressing SOX15 were simply contaminated with normal SOX15+ esophageal cells, we studied cases from an independent collection, the Cancer Genome Atlas (TCGA), where nonmalignant cells were meticulously minimized.23 Cancers of the G-E junction

typically arise in a background of BE and, when associated with chromosomal instability (CIN), usually represent distal EACs. Among 30 cases of CIN+ tumors from the G-E junction or gastric cardia in the TCGA collection of gastric cancers, some samples showed robust levels of SOX15 and of genes coexpressed with SOX15 in normal esophageal epithelium (Supplementary Figure 1A and B). Transcripts specific to the squamous esophageal epithelium were thus again evident in a fraction of EACs. To determine whether this extent of SOX15 expression is specific to EACs, we evaluated other gastrointestinal cancers in the TCGA collection: gastric fundus, body, and antrum, and colorectal tumors. Extreme outliers for SOX15 mRNA expression were present only among tumors of the gastric cardia (Figure 5D).

Finally, we examined 99 separate EACs by immunohis-tochemistry on resection specimens. Because several antibodies failed to detect SOX15, we used KRT5 (keratin 5, type II) as a proxy for expression of SOX15 and other stratified cell-specific products, noting nearly total concordance of SOX15 and KRT5 mRNA expression in the large aforementioned tissue collection22 (Figure 6A; r = 0.97). We also stained the same samples for the intestinal marker CDX2. Nineteen cases (19%) showed cytoplasmic KRT5 expression within malignant glands, and most of these cases coex-pressed nuclear CDX2 (Figure 6B). Levels of KRT5 were variable (Figure 6B) but did not correlate with tumor grade or other pathologic features such as mucin production. Importantly, KRT5 was not expressed in rare pockets of squamous differentiation but rather in bona fide glandular structures. In fact, and of particular note, cytoplasmic KRT5 and nuclear CDX2 were almost always present in different cells within the same glands (see Figures 6C and D for examples from two different cases). Coexpression of esophagus- and intestine-specific genes within individual glands reveals the malignant cells' potential to express genes from distinct cell lineages.

To corroborate the observation that mRNA levels of stratified cell genes are low in BE but elevated in many EACs (Figures 5A and 6A), we used immunohistochemistry to assess areas of BE that were present in 24 of the 99 resection specimens. KRT5 was uniformly absent from these areas, though the signal was clear in adjoining stratified epithelium (Figure 6E). These findings extend previous reports of absent expression of stratified epithelium-specific keratins in BE37-39 and low-level expression of squamous cell products in EACs.40 Our delineation of a squamous cell-restricted transcriptome (Figure 3A), coupled with reanalysis of published RNA expression data (Figure 5A and B) and investigation of additional cases by immunohistochemical analysis (Figure 6B-D), reveals for the first time the extent and breadth of an aberrant stratified-cell program in EACs.


Implications for Esophageal Squamous Differentiation and Esophageal Adenocarcinoma

Insights into transcriptional control of the esophageal squamous epithelium are largely limited to the broad functions of TP63 and SOX2.10,13 Our identification of SOX15

as a novel, conserved, and likely direct regulator of many human stratified epithelial genes extends our understanding of esophageal differentiation and pathology. The lack of overt esophageal defects in Sox15 mutant mice41,42 is compatible with the considerable known redundancies among SOX-family TFs.33

There is much debate whether BE and particularly EAC originate in the native esophageal epithelium through bona fide metaplasia or in ectopic cells that may colonize the esophagus from the gastric cardia, as in mice.11,43 Clearly, the best way to answer the question is through lineage-tracing studies, which are possible in animals but not in humans. When lineage tracing is not feasible, cell-specific transcript patterns offer clues, and the expression of SOX15 and other squamous epithelial genes may be informative. Consider, for example, the observation that the BE cell lines CP-A/B/C show reduced levels of SOX15, esopha-geal keratins, TP63, and other esophagus-specific TF genes, implying loss of an esophageal program, but some esophagus-restricted TF genes such as SIM2 and TFAP2A (transcription factor activating enhancer binding protein 2a) are highly expressed in these cells (Figure 2C) and in BE biopsy specimens (Figure 2D and E). Moreover, >300 esophageal genes, including SOX15, are active in up to 30% of human EACs (Figure 3A), with intestinal genes such as CDX2 often coexpressed in the same glands as esophageal genes (Figure 4C and D). These findings in BE and EAC could indicate residual squamous cell-specific transcription or fortuitous ectopic gene activity. If diseased cells are better equipped to express genes from their native transcriptional program than are genes from a heterologous cell lineage, because native genes and their cis-regulatory elements are inherently primed and accessible, then the first possibility may be more likely. Our findings do not of course rule out the alternative model, which will require additional, independent lines of evidence.

EAC is a particularly recalcitrant disease, with poor 5-year survival rates. Surgery and empiric cytotoxic chemotherapy anchor current treatment approaches,44,45 and although disease heterogeneity is apparent in the clinic, the underlying determinants are unclear. We show here that one-fifth to one-third of EACs simultaneously express products specific to the esophageal squamous epithelium and columnar intestinal cells. It will be important in the future to identify the clinical and genetic correlates of these EACs showing bi-lineage gene expression and to determine whether they reflect a distinctive pathophysiology or harbor unique therapeutic vulnerabilities.


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Received December 5, 2014. Accepted July 27, 2015. Correspondence

Address correspondence to: Ramesh A. Shivdasani, MD, PhD, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, Massachusetts 02215. e-mail:; fax: (617) 582-7198.


The authors thank Rishi Puram and Ben Ebert for BirA-V5 vectors and Austin Dulak and Hiroshi Nakagawa for assistance with esophageal cell culture.

Conflicts of interest

The authors disclose no conflicts.


This study was funded by the DFCI-Novartis Drug Discovery Program, generous gifts from the Lind family, and National Institutes of Health grants R01DK081113 (R.A.S), P01CA098101 (A.J.B. and A.K.R.), and P50CA127003 (Dana-Farber/Harvard).

Supplementary Figure 1. Genes coex-pressed with SOX15 in the Cancer Genome Atlas (TCGA) data set of gastric cancers. (A) Correlations of all mRNAs with SOX15 levels. The 317 genes coexpressed with SOX15 in esophageal epithelium are marked with red lines. (B) Expression of SOX15 coexpressed genes in 30 cases of CIN+ (chromosomal instability) ade-nocarcinomas from the gastroesophageal junction or gastric cardia.


Supplementary Table 1.Top Gene Ontology (GO) terms for transcripts altered after SOX15 depletion in CPA cells

Term RNAseq Down P Value Term RNAseq UP P Value

G0:0000278 w mitotic cell cycle 3.26E- 20 G0:0000079 w regulation of cyclin-dependent 4.40E- -06

protein kinase activity

G0:0051301 w cell division 6.39E- 20 G0:0016192 w vesicle-mediated transport 6.89E- -06

G0:0000280 w nuclear division 1.39E- 19 G0:0051726 w regulation of cell cycle 8.53E- -06

G0:0007067 w mitosis 1.39E- 19 G0:0009991 w response to extracellular stimulus 3.09E- 05

G0:0022403 w cell cycle phase 1.48E- 19 G0:0006091 w generation of precursor metabolites 5.73E 05

and energy

G0:0000087 w M phase of mitotic cell cycle 1.52E- 19 G0:0045767 w regulation of antiapoptosis 7.05E- 05

G0:0022402 w cell cycle process 4.58E- 19 G0:0031667 w response to nutrient levels 1.71E- 04

G0:0007049 w cell cycle 2.49E- 18 G0:0007584 w response to nutrient 2.63E- 04

G0:0000279 w M phase 2.96E- 18 G0:0008219 w cell death 5.87E- 04

G0:0048285 w organelle fission 3.08E- 18 G0:0010033 w response to organic substance 6.29E- 04

G0:0007059 w chromosome segregation 1.93E- 10 G0:0042981 w regulation of apoptosis 6.32E- 04

G0:0008104 w protein localization 4.68E- 10 G0:0055114 w oxidation reduction 6.36E- 04

G0:0045184 w establishment of protein localization 5.39E- 10 G0:0042127 w regulation of cell proliferation 6.77E- 04

G0:0008654 w phospholipid biosynthetic process 6.48E- 10 G0:0016265 w death 6.77E- 04

G0:0015031 w protein transport 2.46E- 09 G0:0043067 w regulation of programmed cell death 7.56E- 04

Supplementary Table 2. Genes Coexpressed With SOX15 in a Large Collection of Primary Esophagus, Barrett's Esophagus, and Esophageal Adenocarcinoma Specimens (r > 0.81)

Gene Correlation With SOX15 Expression

SOX15 1.000

GRHL3 0.981

FAM46B 0.981

ZNF750 0.981

LYPD3 0.981

CAPNS2 0.979

IL20RB 0.978

BNIPL 0.978

ANXA8L2 0.975

GPR87 0.971

TMPRSS11D 0.971

LY6D 0.971

LASS3 0.971

CLCA2 0.971

PKP1 0.971

KRT5 0.971

GSDMC 0.970

TMEM40 0.970

FAM83C 0.970

TP63 0.970

DSC3 0.970

TGM5 0.964

TMPRSS11A 0.964

GJB6 0.964

KLK13 0.964

LYNX1 0.964

SPINK5 0.961

ENDOU 0.961

RNF222 0.961

PRSS27 0.961

KRT78 0.961

CRCT1 0.961

SCEL 0.961

A2ML1 0.961

SLURP1 0.961

c9orf169 0.961

CSTA 0.961

MAL 0.961

KRT6C 0.961

ARHGAP6 0.961

DSG3 0.961

TGM1 0.961

SBSN 0.961

SPRR1B 0.961

CLCA4 0.961

CALML3 0.961

RHCG 0.961

Supplementary Table 2. Continued

Gene Correlation With SOX15 Expression

KRT4 0.961

SERPINB13 0.961

GBP6 0.961

NCCRP1 0.961

TMRSS11B 0.961

CNFN 0.961

TGM3 0.961

CRNN 0.961

HSPB8 0.961

SERPINB2 0.961

S100A2 0.961

LGALS7B 0.952

LGALS7 0.952

DUOX1 0.949

DUOXA1 0.949

CSNK1E 0.944

CLIC3 0.942

HOPX 0.940

SERPINB4 0.939

SERPINB3 0.939

ECM1 0.937

Trim29 0.937

SLC39A2 0.937

RAET1G 0.937

AQP3 0.937

TMEM154 0.937

GNA15 0.937

SULT2B1 0.937

ALDH3B2 0.937

EVPL 0.937

GRHL1 0.937

KAZ 0.937

PITX1 0.937

TMEM79 0.937

DENND2C 0.937

VSIG10L 0.937

ZNF185 0.937

PPL 0.937

GJB5 0.937

KRT15 0.937

c10orf99 0.931

EPHX3 0.928

AIF1L 0.928

CRABP2 0.928

PPP1R3C 0.917

ZNF365 0.916

CPA4 0.916

SPINK7 0.916

RNASE7 0.916

LOC643479 0.916

Supplementary Table 2. Continued

Gene Correlation With SOX15 Expression

TIAM1 0.912

ARL4D 0.912

LASS4 0.912

IVL 0.912

P2RY1 0.912

DLK2 0.912

ANXA8 0.912

BBOX1 0.911

CYP4F22 0.911

SCNN18 0.911

MUC15 0.911

CWH43 0.911

CALML5 0.909

CST6 0.906

FAM83A 0.904

CDA 0.904

KRT80 0.904

LYPD2 0.901

FGF11 0.899

PPP2R2C 0.899

TLE3 0.881

DOCK9 0.881

PLD2 0.881

PYGL 0.881

BNIP3 0.881

TUBB6 0.881

NDUFA4L2 0.881

BDKRB1 0.881

NDRG4 0.881

CBR3 0.881

SLC22A17 0.881

SRPX2 0.881

FRMD6 0.881

MID2 0.881

EFS 0.881

PARD6G 0.881

c3orf54 0.881

RGMA 0.881

RRAGD 0.881

ANKRD35 0.881

TNFAIP8L3 0.881

ELOVL4 0.881

CRYAB 0.881

GPC1 0.881

ZNF385A 0.881

WDFY2 0.881

NOD2 0.881

PTPN13 0.881

TFAP2C 0.881

CDK5R1 0.881

Supplementary Table 2. Continued




















Correlation With SOX15 Expression

Supplementary Table 2. Continued

Gene Correlation With SOX15 Expression

MTSS1 0.824

WDR47 0.824

ELL2 0.824

RORA 0.824

SLC9A9 0.824

SEMA4A 0.824

MAF 0.824

BEX4 0.824

SERPINB8 0.824

ZDHHC21 0.824

ZNF425 0.824

SNX24 0.824

ALDH4A1 0.824

NOTCH2NL 0.824

DUSP22 0.824

MBD2 0.824

C4ORF3 0.824

CNOT1 0.824


CUL4B 0.824

ADK 0.824

MOSPD1 0.824

PDZD2 0.824

CAB39P 0.824

ZNF431 0.824

RASAL2 0.824

TMOD3 0.824

ARHGAP10 0.824

DAPP1 0.824

SLC2A6 0.824

ZNF426 0.824

RIT1 0.824

UBE2H 0.824

SPTLC1 0.824

KAT2B 0.824


KIAA1370 0.824

RNF11 0.824

WDR26 0.824

RANBP9 0.824

ABHD5 0.824

YOD1 0.824

SEPT10 0.824

UBE2G1 0.824

SASH1 0.824

GAB1 0.824

PARD3 0.824

RSC1A1 0.824

TPD52L1 0.824

IL34 0.824

Supplementary Table 2. Continued














Correlation With SOX15 Expression







Supplementary Table 2. Continued

Gene Correlation With SOX15 Expression

CRISP3 0.824

EHD3 0.824

ACYP2 0.824

C2ORF54 0.824

KLF8 0.824

KREMN1 0.824

TPRG1 0.824

PAX9 0.824

SUSD4 0.824

DAPL1 0.824

ARSF 0.816

AL0X15B 0.816

C180RF26 0.816

FMO2 0.816

FAM63A 0.806

SH3GL3 0.806

LY6G6C 0.806

DSG1 0.806

CLDN17 0.806

KPRP 0.806

IGFL1 0.806