A distinct mechanism of epigenetic reprogramming silences PAX2 and initiates endometrial carcinogenesis
Subhransu S. Sahoo, Susmita G. Ramanand, Ileana C. Cuevas, Yunpeng Gao, Sora Lee, Ahmed Abbas, Xunzhi Zhang, Ashwani Kumar, Prasad Koduru, Sambit Roy, Russell R. Broaddus, Victoria L. Bae-Jump, Andrew B. Gladden, Jayanthi Lea, Elena Lucas, Chao Xing, Akio Kobayashi, Ram S. Mani, Diego H. Castrillon
Subhransu S. Sahoo, Susmita G. Ramanand, Ileana C. Cuevas, Yunpeng Gao, Sora Lee, Ahmed Abbas, Xunzhi Zhang, Ashwani Kumar, Prasad Koduru, Sambit Roy, Russell R. Broaddus, Victoria L. Bae-Jump, Andrew B. Gladden, Jayanthi Lea, Elena Lucas, Chao Xing, Akio Kobayashi, Ram S. Mani, Diego H. Castrillon
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Research Article Oncology Reproductive biology

A distinct mechanism of epigenetic reprogramming silences PAX2 and initiates endometrial carcinogenesis

Abstract

Functional inactivation of tumor suppressor genes drives cancer initiation, progression, and treatment responses. Most tumor suppressor genes are inactivated through 1 of 2 well-characterized mechanisms: DNA-level mutations, such as point mutations or deletions, and promoter DNA hypermethylation. Here, we report a distinct third mechanism of tumor suppressor inactivation based on alterations to the histone rather than DNA code. We demonstrated that PAX2 is an endometrial tumor suppressor recurrently inactivated by a distinct epigenetic reprogramming event in more than 80% of human endometrial cancers. Integrative transcriptomic, epigenomic, 3D genomic, and machine learning analyses showed that PAX2 transcriptional downregulation is associated with replacement of open/active chromatin features (H3K27ac/H3K4me3) with inaccessible/repressive chromatin features (H3K27me3) in a framework dictated by 3D genome organization. The spread of the repressive H3K27me3 signal resembled a pearl necklace, with its length modulated by cohesin loops, thereby preventing transcriptional dysregulation of neighboring genes. This mechanism, involving the loss of a promoter-proximal superenhancer, was shown to underlie transcriptional silencing of PAX2 in human endometrial cancers. Mouse and human preclinical models established PAX2 as a potent endometrial tumor suppressor. Functionally, PAX2 loss promoted endometrial carcinogenesis by rewiring the transcriptional landscape via global enhancer reprogramming. The discovery that most endometrial cancers originate from a recurring epigenetic alteration carries profound implications for their diagnosis and treatment.

Authors

Subhransu S. Sahoo, Susmita G. Ramanand, Ileana C. Cuevas, Yunpeng Gao, Sora Lee, Ahmed Abbas, Xunzhi Zhang, Ashwani Kumar, Prasad Koduru, Sambit Roy, Russell R. Broaddus, Victoria L. Bae-Jump, Andrew B. Gladden, Jayanthi Lea, Elena Lucas, Chao Xing, Akio Kobayashi, Ram S. Mani, Diego H. Castrillon

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Figure 1

Emergence of PAX2-deficient clones in endometrial epithelium is age dependent and associated with carcinogenesis.

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Emergence of PAX2-deficient clones in endometrial epithelium is age depe...
(A) PAX2 immunolocalization of endometrial tissue section from younger (18–25 y/o) patient group. No PAX2-deficient clones were detected across entire specimen; representative region shown. Scale bar: 200 μm. (B) EC from 63 y/o patient showing complete loss of PAX2, which occurs in 80% of EC. Residual normal (non-neoplastic) gland in lower left corner underscores striking and complete loss of PAX2 expression in EC. Scale bar: 200 μm. (C) Endometrial tissue section from older (44–45 y/o) patient group. Dashed red circle highlights single gland in entire specimen with PAX2 loss; only portion of section shown. Right panel, magnification of boxed area showing complete (clonal) loss in all cells of the gland. Scale bars: 100 μm. (D) Parts of whole plots show cases with PAX2 protein loss among younger (n = 27) and older (n = 32) patients. P value per 2-sided Fisher’s exact test. (E) PAX2 expression in normal proliferative endometrium and loss in most (>80%) ECs of grades 1–3. G1, grade 1; G2, grade 2; G3, grade 3. Scale bars: 100 μm. (F) Box-and-whisker plots of PAX2 protein expression levels per H-scores in normal endometrium (n = 8) and ECs (grade 1, n = 45; grade 2, n = 37; grade 3, n = 33). ****P < 0.0001 per Dunnett’s multiple-comparison test. (G) Western blot analysis of human EC cell line panel (n = 13) with same PAX2 monoclonal antibody used for immunolocalization. Only 2/13 lines (AN3CA and EI) expressed normal levels of PAX2, consistent with the observed loss in approximately 80% of primary EC. (H) PAX2 mRNA expression levels across human EC lines per qPCR (n = 3, mean ± SEM).