Characterizing isoform switching events in esophageal adenocarcinoma

Yun Zhang; Katherine M. Weh; Connor L. Howard; Jean Jack Riethoven; Jennifer L. Clarke; Kiran H. Lagisetty; Jules Lin; Rishindra M. Reddy; Andrew C. Chang; David G. Beer; Laura A. Kresty

doi:10.1016/j.omtn.2022.08.018

Characterizing isoform switching events in esophageal adenocarcinoma

Yun Zhang, Katherine M. Weh, Connor L. Howard, Jean Jack Riethoven, Jennifer L. Clarke, Kiran H. Lagisetty, Jules Lin, Rishindra M. Reddy, Andrew C. Chang, David G. Beer, Laura A. Kresty

Research output: Contribution to journal › Article › peer-review

2 Scopus citations

Abstract

Isoform switching events with predicted functional consequences are common in many cancers, but characterization of switching events in esophageal adenocarcinoma (EAC) is lacking. Next-generation sequencing was used to detect levels of RNA transcripts and identify specific isoforms in treatment-naïve esophageal tissues ranging from premalignant Barrett's esophagus (BE), BE with low- or high-grade dysplasia (BE.LGD, BE.HGD), and EAC. Samples were stratified by histopathology and TP53 mutation status, identifying significant isoform switching events with predicted functional consequences. Comparing BE.LGD with BE.HGD, a histopathology linked to cancer progression, isoform switching events were identified in 75 genes including KRAS, RNF128, and WRAP53. Stratification based on TP53 status increased the number of significant isoform switches to 135, suggesting switching events affect cellular functions based on TP53 mutation and tissue histopathology. Analysis of isoforms agnostic, exclusive, and shared with mutant TP53 revealed unique signatures including demethylation, lipid and retinoic acid metabolism, and glucuronidation, respectively. Nearly half of isoform switching events were identified without significant gene-level expression changes. Importantly, two TP53-interacting isoforms, RNF128 and WRAP53, were significantly linked to patient survival. Thus, analysis of isoform switching events may provide new insight for the identification of prognostic markers and inform new potential therapeutic targets for EAC.

Original language	English (US)
Pages (from-to)	749-768
Number of pages	20
Journal	Molecular Therapy - Nucleic Acids
Volume	29
DOIs	https://doi.org/10.1016/j.omtn.2022.08.018
State	Published - Sep 13 2022

Keywords

Barrett's esophagus
MT: Bioinformatics
Mortality-linked isoforms
TP53
esophageal adenocarcinoma
isoform switching
transcriptomics

ASJC Scopus subject areas

Molecular Medicine
Drug Discovery

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10.1016/j.omtn.2022.08.018

Cite this

@article{be49333363c24be9a9a8dc71dd4d2246,

title = "Characterizing isoform switching events in esophageal adenocarcinoma",

abstract = "Isoform switching events with predicted functional consequences are common in many cancers, but characterization of switching events in esophageal adenocarcinoma (EAC) is lacking. Next-generation sequencing was used to detect levels of RNA transcripts and identify specific isoforms in treatment-na{\"i}ve esophageal tissues ranging from premalignant Barrett's esophagus (BE), BE with low- or high-grade dysplasia (BE.LGD, BE.HGD), and EAC. Samples were stratified by histopathology and TP53 mutation status, identifying significant isoform switching events with predicted functional consequences. Comparing BE.LGD with BE.HGD, a histopathology linked to cancer progression, isoform switching events were identified in 75 genes including KRAS, RNF128, and WRAP53. Stratification based on TP53 status increased the number of significant isoform switches to 135, suggesting switching events affect cellular functions based on TP53 mutation and tissue histopathology. Analysis of isoforms agnostic, exclusive, and shared with mutant TP53 revealed unique signatures including demethylation, lipid and retinoic acid metabolism, and glucuronidation, respectively. Nearly half of isoform switching events were identified without significant gene-level expression changes. Importantly, two TP53-interacting isoforms, RNF128 and WRAP53, were significantly linked to patient survival. Thus, analysis of isoform switching events may provide new insight for the identification of prognostic markers and inform new potential therapeutic targets for EAC.",

keywords = "Barrett's esophagus, MT: Bioinformatics, Mortality-linked isoforms, TP53, esophageal adenocarcinoma, isoform switching, transcriptomics",

author = "Yun Zhang and Weh, {Katherine M.} and Howard, {Connor L.} and Riethoven, {Jean Jack} and Clarke, {Jennifer L.} and Lagisetty, {Kiran H.} and Jules Lin and Reddy, {Rishindra M.} and Chang, {Andrew C.} and Beer, {David G.} and Kresty, {Laura A.}",

note = "Funding Information: With the exception of RNF128, isoform switching events have been largely unexplored in BE and EAC. Herein, we identify large numbers of events linked to advanced pathology, as well as TP53 mutation (as detailed in Tables S6, S7, and S8). Two reports characterizing isoform switching events in ESCC identified numerous unique isoforms with only MINDY1 as a gene in common with our isoform switch analysis, supporting divergent molecular changes based on esophageal cancer subtype.36,49 MINDY-1, also known as FAM63A, is a newly identified deubiquitinating protein that preferentially removes K48-linked ubiquitin molecules.50 In BE and EAC, MINDY1 was identified in a set of 90 genes significantly predicting disease progression by distinguishing EAC progressors from patients with non-dysplastic BE.51 Moreover, MINDY-1 was found to promote bladder cancer progression by stabilizing YAP,52 a key tumorigenesis pathway member that can also be induced by conjugated bile acids in EAC.53 In our analysis, increased usage of the MINDY1 transcript that lacks deubiquitinase (DUB) domains and a decreased usage of the MINDY1 transcript that contains five DUB domains are observed, potentially suggesting impaired MINDY-1 function during EAC progression. Despite the fact that the MINDY1 transcripts involved in isoform switching are different in our analysis than the data published in ESCC,36 a similar switching trend is observed in that the isoform transcript that does not contain DUB domains is increased while the usage is decreased for a DUB domain-containing transcript. These data suggest that MINDY1 isoforms lacking deubiquitinase domains may be important for both EAC and ESCC progression.Interestingly, isoform switch analysis in this study also identified significant isoform-switched genes without significant differences in gene-level expression (Tables 1 and 2). Thirty-two isoform switching events (32 of 75, or 42.7%) were identified in comparing BE.LGD with BE.HGD, whereas BE.LGD TP53 WT compared with BE.HGD TP53 MUT resulted in identification of 62 isoform switching events (62 of 135, or 45.9%) without overall gene expression changes. GO enrichment analysis revealed similar enriched biological processes for those genes compared with enrichment analysis of all isoform-switched genes (data not shown). Isoform-switched genes that occur in the absence of altered gene-level expression include KDM6B, UGT2B7, WRAP53, and TRIM29. KDM6B, also known as lysine-specific demethylase 6B, is a histone demethylase that can act as either a tumor suppressor or oncogene in cancer.62 KDM6B is reportedly overexpressed in pancreatic premalignancy with its expression decreasing with progression to PDAC, supporting a role for KDM6B in pancreatic carcinogenesis.63 Although overall expression differences were not significant between patients with BE.LGD compared with BE.HGD, expression of the KDM6B coding transcript decreased while the expression of the non-coding transcript increased, suggesting KDM6B might also be important during early EAC progression. Conversely, KDM6B has been shown to promote ESCC progression and KDM6B levels significantly increased in patients with lymph node metastasis.64The Venn diagram and subsequent GO biological process analysis in Figure 3 show differences in pathway enrichment based on pathology and TP53 mutation status, suggesting that patients stratified into different subgroups may potentially benefit from different treatment regimens. As a proof-of-principle, we used two FDA-approved drugs that target the 9-cis-retinoic acid pathway and TP53 mutation to test this hypothesis. Two EAC cell lines, OE33 and JHAD1, were treated with the RARβ and RARγ agonist, adapalene, and results indicated that adapalene was a potent inducer of cell death in both cell lines. Considering RARβ and RARγ are two of the receptors of 9-cis-retinoic acid, these data suggest activation of 9-cis-retinoic acid pathway may hold potential for targeting EAC.86 Moreover, to test the potential benefit of blocking mutant TP53 expression in EAC, OE33 and JHAD1 cells (both TP53 mutant) were treated with APR-246, a small molecule that restores TP53 WT functionality.44 Results showed that post-treatment with APR-246, both EAC cell lines experienced significant loss of viability, which is in alignment with previously published results in EAC and ESCC, although a discrepancy in effective treatment concentration of OE33 cell was noticed.87,88 Therefore, treatment with APR-246 suggests that inhibition of mutant TP53 expression and isoform expression linked to mutant TP53 may be a viable preventive or treatment approach for inhibiting EAC. Moreover, enrichment of the lipid metabolism process is also observed in our analysis for BE.HGD patients that carry the TP53 mutation. Metformin, a lipid metabolism modulating drug, was also shown to inhibit proliferation of EAC cell lines,89 further supporting the plausibility of targeting specific pathways identified in the analysis and the importance of patient stratification based on TP53 mutational status. Finally, epidemiological and limited preclinical data point to statins as esophageal cancer inhibitors, in alignment with agents impacting lipid metabolism holding promise in a subset of EACs. 90–94We thank the National Institutes of Health and National Cancer Institute (U54CA163059) and the University of Michigan (U057239) for supporting this study. This study was additionally supported by the John and Carla Klein Family research fund awarded to L.A.K. We also thank Dr. James R. Eshleman (Johns Hopkins University, Baltimore, MD) for sharing the JH-EsoAd1 cell line used in this study. The graphical abstract was created using BioRender (BioRender.com). Conceptualization, Y.Z. K.M.W. C.L.H. and L.A.K.; methodology, Y.Z. C.L.H. J.R. J.L.C. D.G.B. and L.A.K.; software, Y.Z. C.L.H. and J.R.; validation, Y.Z. C.L.H. K.M.W. and L.A.K.; formal analysis, Y.Z. and C.L.H.; investigation, Y.Z. K.M.W. C.L.H. and L.A.K.; resources, K.H.L. J.L. R.M.R. A.C.C. D.G.B. and L.A.K.; data curation, Y.Z. K.M.W. C.L.H. J.R. D.G.B. and L.A.K.; writing—original draft preparation, Y.Z. K.M.W. C.L.H. and L.A.K.; writing—review and editing, Y.Z. K.M.W. C.L.H. J.R. J.L.C. K.H.L. J.L. R.M.R. A.C.C. D.G.B. and L.A.K.; visualization, Y.Z.; supervision, L.A.K.; project administration, L.A.K.; funding acquisition, L.A.K. All authors have read and agreed to the published version of the manuscript. The authors declare no competing interests. Funding Information: We thank the National Institutes of Health and National Cancer Institute ( U54CA163059 ) and the University of Michigan ( U057239 ) for supporting this study. This study was additionally supported by the John and Carla Klein Family research fund awarded to L.A.K.. We also thank Dr. James R. Eshleman (Johns Hopkins University, Baltimore, MD) for sharing the JH-EsoAd1 cell line used in this study. The graphical abstract was created using BioRender ( BioRender.com ). Publisher Copyright: {\textcopyright} 2022 The Author(s)",

year = "2022",

month = sep,

day = "13",

doi = "10.1016/j.omtn.2022.08.018",

language = "English (US)",

volume = "29",

pages = "749--768",

journal = "Molecular Therapy - Nucleic Acids",

issn = "2162-2531",

publisher = "Nature Publishing Group",

}

TY - JOUR

T1 - Characterizing isoform switching events in esophageal adenocarcinoma

AU - Zhang, Yun

AU - Weh, Katherine M.

AU - Howard, Connor L.

AU - Riethoven, Jean Jack

AU - Clarke, Jennifer L.

AU - Lagisetty, Kiran H.

AU - Lin, Jules

AU - Reddy, Rishindra M.

AU - Chang, Andrew C.

AU - Beer, David G.

AU - Kresty, Laura A.

N1 - Funding Information: With the exception of RNF128, isoform switching events have been largely unexplored in BE and EAC. Herein, we identify large numbers of events linked to advanced pathology, as well as TP53 mutation (as detailed in Tables S6, S7, and S8). Two reports characterizing isoform switching events in ESCC identified numerous unique isoforms with only MINDY1 as a gene in common with our isoform switch analysis, supporting divergent molecular changes based on esophageal cancer subtype.36,49 MINDY-1, also known as FAM63A, is a newly identified deubiquitinating protein that preferentially removes K48-linked ubiquitin molecules.50 In BE and EAC, MINDY1 was identified in a set of 90 genes significantly predicting disease progression by distinguishing EAC progressors from patients with non-dysplastic BE.51 Moreover, MINDY-1 was found to promote bladder cancer progression by stabilizing YAP,52 a key tumorigenesis pathway member that can also be induced by conjugated bile acids in EAC.53 In our analysis, increased usage of the MINDY1 transcript that lacks deubiquitinase (DUB) domains and a decreased usage of the MINDY1 transcript that contains five DUB domains are observed, potentially suggesting impaired MINDY-1 function during EAC progression. Despite the fact that the MINDY1 transcripts involved in isoform switching are different in our analysis than the data published in ESCC,36 a similar switching trend is observed in that the isoform transcript that does not contain DUB domains is increased while the usage is decreased for a DUB domain-containing transcript. These data suggest that MINDY1 isoforms lacking deubiquitinase domains may be important for both EAC and ESCC progression.Interestingly, isoform switch analysis in this study also identified significant isoform-switched genes without significant differences in gene-level expression (Tables 1 and 2). Thirty-two isoform switching events (32 of 75, or 42.7%) were identified in comparing BE.LGD with BE.HGD, whereas BE.LGD TP53 WT compared with BE.HGD TP53 MUT resulted in identification of 62 isoform switching events (62 of 135, or 45.9%) without overall gene expression changes. GO enrichment analysis revealed similar enriched biological processes for those genes compared with enrichment analysis of all isoform-switched genes (data not shown). Isoform-switched genes that occur in the absence of altered gene-level expression include KDM6B, UGT2B7, WRAP53, and TRIM29. KDM6B, also known as lysine-specific demethylase 6B, is a histone demethylase that can act as either a tumor suppressor or oncogene in cancer.62 KDM6B is reportedly overexpressed in pancreatic premalignancy with its expression decreasing with progression to PDAC, supporting a role for KDM6B in pancreatic carcinogenesis.63 Although overall expression differences were not significant between patients with BE.LGD compared with BE.HGD, expression of the KDM6B coding transcript decreased while the expression of the non-coding transcript increased, suggesting KDM6B might also be important during early EAC progression. Conversely, KDM6B has been shown to promote ESCC progression and KDM6B levels significantly increased in patients with lymph node metastasis.64The Venn diagram and subsequent GO biological process analysis in Figure 3 show differences in pathway enrichment based on pathology and TP53 mutation status, suggesting that patients stratified into different subgroups may potentially benefit from different treatment regimens. As a proof-of-principle, we used two FDA-approved drugs that target the 9-cis-retinoic acid pathway and TP53 mutation to test this hypothesis. Two EAC cell lines, OE33 and JHAD1, were treated with the RARβ and RARγ agonist, adapalene, and results indicated that adapalene was a potent inducer of cell death in both cell lines. Considering RARβ and RARγ are two of the receptors of 9-cis-retinoic acid, these data suggest activation of 9-cis-retinoic acid pathway may hold potential for targeting EAC.86 Moreover, to test the potential benefit of blocking mutant TP53 expression in EAC, OE33 and JHAD1 cells (both TP53 mutant) were treated with APR-246, a small molecule that restores TP53 WT functionality.44 Results showed that post-treatment with APR-246, both EAC cell lines experienced significant loss of viability, which is in alignment with previously published results in EAC and ESCC, although a discrepancy in effective treatment concentration of OE33 cell was noticed.87,88 Therefore, treatment with APR-246 suggests that inhibition of mutant TP53 expression and isoform expression linked to mutant TP53 may be a viable preventive or treatment approach for inhibiting EAC. Moreover, enrichment of the lipid metabolism process is also observed in our analysis for BE.HGD patients that carry the TP53 mutation. Metformin, a lipid metabolism modulating drug, was also shown to inhibit proliferation of EAC cell lines,89 further supporting the plausibility of targeting specific pathways identified in the analysis and the importance of patient stratification based on TP53 mutational status. Finally, epidemiological and limited preclinical data point to statins as esophageal cancer inhibitors, in alignment with agents impacting lipid metabolism holding promise in a subset of EACs. 90–94We thank the National Institutes of Health and National Cancer Institute (U54CA163059) and the University of Michigan (U057239) for supporting this study. This study was additionally supported by the John and Carla Klein Family research fund awarded to L.A.K. We also thank Dr. James R. Eshleman (Johns Hopkins University, Baltimore, MD) for sharing the JH-EsoAd1 cell line used in this study. The graphical abstract was created using BioRender (BioRender.com). Conceptualization, Y.Z. K.M.W. C.L.H. and L.A.K.; methodology, Y.Z. C.L.H. J.R. J.L.C. D.G.B. and L.A.K.; software, Y.Z. C.L.H. and J.R.; validation, Y.Z. C.L.H. K.M.W. and L.A.K.; formal analysis, Y.Z. and C.L.H.; investigation, Y.Z. K.M.W. C.L.H. and L.A.K.; resources, K.H.L. J.L. R.M.R. A.C.C. D.G.B. and L.A.K.; data curation, Y.Z. K.M.W. C.L.H. J.R. D.G.B. and L.A.K.; writing—original draft preparation, Y.Z. K.M.W. C.L.H. and L.A.K.; writing—review and editing, Y.Z. K.M.W. C.L.H. J.R. J.L.C. K.H.L. J.L. R.M.R. A.C.C. D.G.B. and L.A.K.; visualization, Y.Z.; supervision, L.A.K.; project administration, L.A.K.; funding acquisition, L.A.K. All authors have read and agreed to the published version of the manuscript. The authors declare no competing interests. Funding Information: We thank the National Institutes of Health and National Cancer Institute ( U54CA163059 ) and the University of Michigan ( U057239 ) for supporting this study. This study was additionally supported by the John and Carla Klein Family research fund awarded to L.A.K.. We also thank Dr. James R. Eshleman (Johns Hopkins University, Baltimore, MD) for sharing the JH-EsoAd1 cell line used in this study. The graphical abstract was created using BioRender ( BioRender.com ). Publisher Copyright: © 2022 The Author(s)

PY - 2022/9/13

Y1 - 2022/9/13

N2 - Isoform switching events with predicted functional consequences are common in many cancers, but characterization of switching events in esophageal adenocarcinoma (EAC) is lacking. Next-generation sequencing was used to detect levels of RNA transcripts and identify specific isoforms in treatment-naïve esophageal tissues ranging from premalignant Barrett's esophagus (BE), BE with low- or high-grade dysplasia (BE.LGD, BE.HGD), and EAC. Samples were stratified by histopathology and TP53 mutation status, identifying significant isoform switching events with predicted functional consequences. Comparing BE.LGD with BE.HGD, a histopathology linked to cancer progression, isoform switching events were identified in 75 genes including KRAS, RNF128, and WRAP53. Stratification based on TP53 status increased the number of significant isoform switches to 135, suggesting switching events affect cellular functions based on TP53 mutation and tissue histopathology. Analysis of isoforms agnostic, exclusive, and shared with mutant TP53 revealed unique signatures including demethylation, lipid and retinoic acid metabolism, and glucuronidation, respectively. Nearly half of isoform switching events were identified without significant gene-level expression changes. Importantly, two TP53-interacting isoforms, RNF128 and WRAP53, were significantly linked to patient survival. Thus, analysis of isoform switching events may provide new insight for the identification of prognostic markers and inform new potential therapeutic targets for EAC.

AB - Isoform switching events with predicted functional consequences are common in many cancers, but characterization of switching events in esophageal adenocarcinoma (EAC) is lacking. Next-generation sequencing was used to detect levels of RNA transcripts and identify specific isoforms in treatment-naïve esophageal tissues ranging from premalignant Barrett's esophagus (BE), BE with low- or high-grade dysplasia (BE.LGD, BE.HGD), and EAC. Samples were stratified by histopathology and TP53 mutation status, identifying significant isoform switching events with predicted functional consequences. Comparing BE.LGD with BE.HGD, a histopathology linked to cancer progression, isoform switching events were identified in 75 genes including KRAS, RNF128, and WRAP53. Stratification based on TP53 status increased the number of significant isoform switches to 135, suggesting switching events affect cellular functions based on TP53 mutation and tissue histopathology. Analysis of isoforms agnostic, exclusive, and shared with mutant TP53 revealed unique signatures including demethylation, lipid and retinoic acid metabolism, and glucuronidation, respectively. Nearly half of isoform switching events were identified without significant gene-level expression changes. Importantly, two TP53-interacting isoforms, RNF128 and WRAP53, were significantly linked to patient survival. Thus, analysis of isoform switching events may provide new insight for the identification of prognostic markers and inform new potential therapeutic targets for EAC.

KW - Barrett's esophagus

KW - MT: Bioinformatics

KW - Mortality-linked isoforms

KW - TP53

KW - esophageal adenocarcinoma

KW - isoform switching

KW - transcriptomics

UR - http://www.scopus.com/inward/record.url?scp=85137098681&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85137098681&partnerID=8YFLogxK

U2 - 10.1016/j.omtn.2022.08.018

DO - 10.1016/j.omtn.2022.08.018

M3 - Article

C2 - 36090744

AN - SCOPUS:85137098681

SN - 2162-2531

VL - 29

SP - 749

EP - 768

JO - Molecular Therapy - Nucleic Acids

JF - Molecular Therapy - Nucleic Acids

ER -

Characterizing isoform switching events in esophageal adenocarcinoma

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