Unraveling epigenetic regulation of epithelial mesenchymal transition

Epithelial mesenchymal transition (EMT) is a naturally occurred transdifferentiation of epithelial cells that encompasses their plasticity and involves conversion from epithelial to mesenchymal phenotypes (1-3). EMT is a common program in embryonic development, organ fibrogenesis and cancer metastasis. The cells undergone EMT have been evidenced with cancer cell stemness and resistance to chemotherapy (4). Several recent studies on EMT, particularly in cancer entity, have moved forward to better understand molecular mechanisms underlying EMT and its regulation. Since EMT can be modulated by several factors at multiple steps, integrative information of EMT regulation at transcription, post-transcription, post-translation and epigenetic levels is thus required for the new approach on EMT-related cancer therapy (5). Beyond transcriptional control, DNA methylation, histone modification and microRNA (miRNA) have been recognized as epigenetic modifications that can regulate EMT-related genes (5). One of the driving forces for epigenetic modifications in cancer-related EMT is the adaptive mechanism of the cancer cells to alter their metabolism to survive under energy deprivation stage during tumorigenesis (6). Additionally, dysregulated metabolism can be affected by genetic mutations of metabolic enzymes, e.g., succinate dehydrogenase (SDH), fumarate hydratase (FH) and isocitrate dehydrogenase (IDH), that subsequently cause alterations in cellular metabolites and finally initiate cancer development and progression (7). The small metabolites with the potential to trigger cancers are termed “oncometabolites” (8,9). One of the well-known oncometabolites related to cancer progression is fumarate, which has been evidenced to be associated with the development of hereditary leiomyomatosis and renal cell cancer (HLRCC) (8).

HLRCC is an autosomal dominant hereditary cancer syndrome caused by germline mutation of the gene encoding FH, a tricarboxylic acid cycle (TCA) enzyme, which catalyzes hydration of fumarate to malate (10). The consequences of FH inactivation in kidney cancer cells include TCA cycle imbalance, fumarate accumulation, impaired oxidative phosphorylation, and metabolic reprogramming to aerobic glycolysis (also known as Warburg effect), which in turn promote tumor growth (10). Furthermore, stabilization of hypoxia-inducible factor 1 (HIF1), inactivation of AMP-activated protein kinase (AMPK), and dysregulation of Keap1-Nrf2 antioxidant system have been reported in FH-deficient kidney cancers (11-13). Although adaptive responses to fumarate accumulation are partially known, there is still a huge gap in understanding epigenetic regulation of tumorigenesis in HLRCC.

The most recent study by Sciacovelli et al. (14), has established a connection between an oncometabolite (fumarate) and epigenetic regulation of miR-200 family that plays important roles in EMT program of HLRCC. The authors primarily characterized the EMT phenotypes by proteome and mRNA profilings of Fh1^−/− (murine) and UOK262 (human) FH-deficient cells (note that the latter was derived from HLRCC patient). They demonstrated that a mesenchymal marker, vimentin, was the most increased protein, while a counterpart epithelial marker, E-cadherin, was significantly decreased in the FH-deficient cells, suggesting the acquired mesenchymal signature in these cell lines concomitant with the increased cell migratory activity. Interestingly, epithelial features were re-gained and cell migratory activity was decreased when the FH-deficient cells were reintroduced with full-length FH. In addition, several EMT transcription factors, including SNAI1, SNAI2, TWIST1, ZEB1 and ZEB2, were elevated in the FH-deficient cells (14). It is known that the loss of FH in renal cancer can result in accumulation of fumarate and inhibition of HIF prolyl hydroxylase, thereby stabilizing HIF1 (11). Moreover, silencing of HIF1α in UOK262 significantly reduces invasiveness of the cancer cells (12). Unexpectedly, Sciacovelli et al. (14) found that silencing of HIF1β could not suppress EMT phenotypes, indicating that the involvement of HIF1 in cancer development/progression is (iso)form-specific.

Numerous miRNAs are supposed to be involved in EMT by epigenetic modulation of their mRNA targets especially through DNA methylation at the CpG promoter island or histone acetylation. Among these, ZEB-miR-200 axis has gained wide attention on its role for regulation of transcription factors in cancer EMT (15). miR-200 family is a potent antimetastatic miRNA cluster that suppresses expression of EMT-transcription factors as well as tumor initiation and metastatic cascade (16). Sciacovelli et al. (14) hypothesized that epigenetic modification driven by accumulation of fumarate could affect miR200-mediated regulation of EMT-related genes in HLRCC. In concordance with elevated ZEB1 and ZEB2 expression, they also showed that miR-200 family was the most down-regulated miRNA in FH-deficient cells and FH re-expression successfully rescued the expression of miR-200ab and E-cadherin, while suppressed vimentin expression (14). As expected, quantitative PCR (qPCR) revealed hypermethylation of CpG43 in FH-deficient cells that could be returned to unmethylated state by FH reconstitution (14).

The same group of investigators also demonstrated that suppression of miR-200 cluster was a consequence of inhibition of ten-eleven translocation (Tet)-mediated demethylation (14). Tet family of dioxygenase comprises three proteins, including Tet1, Tet2 and Tet3, which are responsible for catalyzing the conversion of 5-methylcytosine (5mc) to 5-hydroxymethylcytosine (5hmc). The product 5hmc can be used to implicate the reversible process of DNA methylation/demethylation (17). This group of investigators revealed that combined silencing of Tet2 and Tet3 (Fh1^fl/fl + shTet2-3) resulted in decreased miR-200abc and E-cadherin expression, suggesting the role of Tet-mediated demethylation in EMT regulation (14). The same results were observed when dioxygenase was reactivated in Fh1^fl/fl + shTet2-3 cells by using dimethyl alpha-ketoglutarate (DM-a-KG) (14). To decipher whether this molecular scenario occurred in FH-deficient cells, the authors performed chromatin immunoprecipitation (ChIP) assay in combination with chromosome conformation capture (3C) analysis to pin-point the region of histone modification, indicating the opened-closed state of chromatin structure. It was clearly shown that the region adjacent to CpG43 and transcriptional start site of miR-200ba429 was hypermethylated in FH-deficient cells, which could be restored to basal level by reintroduction of FH (14). Accordingly, the marked decrease in Tet-catalyzed 5hmC was also observed in FH-deficient cells (14). Although they did not determine the basal levels of Tet proteins in FH-deficient cells, these findings collectively suggest the role of Tet-mediated demethylation in the production of miR-200 family in FH-deficient cells. Alternatively, they confirmed the effects of oncometabolite on EMT regulation by using monomethyl fumarate (MMF), a fumarate derivative, in wild-type FH (Fh1^fl/fl) and HK2 cells. As expected, treatment of MMF up-regulated EMT transcription factors and down-regulated miR-200abc and E-cadherin expression in both cell lines, whereas vimentin was increased only in Fh1^fl/fl cells (14). To avoid the effect of fumarate by-products (i.e., succinic GSH and 2-succinic cysteine), they employed SDHb-deficient cell line and demonstrated that only succinate accumulation could raise EMT phenotypes similar to those of FH-deficient cells. However, it was questionable that whether triggering EMT in SDHb-deficient cells engaged the same molecular mechanism as revealed in FH-deficient cells, since hypermethylation of CpG43 was not predominant. Finally, the effects of oncometabolite fumarate and epigenetic regulation of EMT were validated in tumor samples collected from HLRCC patients (only two cases included in their own study) (14) and other cancers related to FH-mutation, including papillary renal cell carcinoma and clear cell renal cell carcinoma from the published data. By this strategy, it was convincing that the decrease of FH was correlated with down-regulation of miR-200 cluster due to hypermethylation, leading to EMT features. Last but not least, the decreased FH expression was related to the worsen prognosis of the patients (14).

In summary, the study by Sciacovelli et al. (14) provided a missing piece of the puzzle and demonstrated that oncometabolite fumarate could induce epigenetic regulation of EMT in FH-deficient kidney cancers. All these findings raise the possibility that aberrant epigenetics by inhibition of Tet-mediated demethylation may be generalized in other cancers related to miR-200 suppression or loss of FH. Considering epigenetic modifications for anticancer drug design, it is still challenging to figure out the unique set of epigenetic markers specific to EMT (e.g., histone modifications, histone marks, etc.) and chromatin modifying enzymes for the precise targeting of EMT cells. In addition to the discovered linkage, further functional analyses on transcriptome, proteome, and miRNA profiling data provided in this report and by other groups are still necessary for unlocking and conveying the comprehensive message for specific therapeutics of cancers related to FH-mutation. However, it should be noted that epigenetic regulation is not limited only to the cancer entity of EMT but also organ fibrogenesis. Several studies have shown a proof-of-concept that aberrant DNA promoter methylation causing gene silencing contributes to many fibrotic diseases, including pulmonary, liver, cardiac, and kidney fibrosis (18). For example, hypermethylation of anti-fibrotic RASAL1 promoter mediated via Tet activity has been recently demonstrated in cardiac and kidney fibrosis (19,20). Nonetheless, this molecular mechanism has been revealed only in differentiated/activated fibroblasts and coronary endothelial cells. Therefore, there is still a plenty of space for investigating the role of epigenetic regulations of EMT in fibrogenesis, including a link between Tet-mediated demethylation and expression of miR-200 cluster or other genes. The study provided by Sciacovelli et al. (14) thus sheds light onto the emerging role of epigenetic modifications of EMT process not only in cancers but also for in organ fibrogenesis and may hold a promise for the novel therapeutic strategy in cancers and also in fibrotic diseases in the future.

Acknowledgments

Funding: This work was supported by Mahidol University research grant, Office of the Higher Education Commission and Mahidol University under the National Research Universities Initiative, and the Thailand Research Fund (RTA5680004, TRG5780294 and IRG5980006). R Kanlaya and V Thongboonkerd are also supported by the “Chalermphrakiat” Grant, Faculty of Medicine Siriraj Hospital.

Footnote

Provenance and Peer Review: This article was commissioned and reviewed by the Section Editor Peng Zhang (Department of Urology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China).

Conflicts of Interest: Both authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/tcr.2016.11.59). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Ye X, Weinberg RA. Epithelial-Mesenchymal Plasticity: A Central Regulator of Cancer Progression. Trends Cell Biol 2015;25:675-86. [Crossref] [PubMed]
2. Kanlaya R, Khamchun S, Kapincharanon C, et al. Protective effect of epigallocatechin-3-gallate (EGCG) via Nrf2 pathway against oxalate-induced epithelial mesenchymal transition (EMT) of renal tubular cells. Sci Rep 2016;6:30233. [Crossref] [PubMed]
3. Kanlaya R, Sintiprungrat K, Thongboonkerd V. Secreted products of macrophages exposed to calcium oxalate crystals induce epithelial mesenchymal transition of renal tubular cells via RhoA-dependent TGF-β1 pathway. Cell Biochem Biophys 2013;67:1207-15. [Crossref] [PubMed]
4. Smith BN, Bhowmick NA. Role of EMT in Metastasis and Therapy Resistance. J Clin Med 2016;5:E17 [Crossref] [PubMed]
5. Serrano-Gomez SJ, Maziveyi M, Alahari SK. Regulation of epithelial-mesenchymal transition through epigenetic and post-translational modifications. Mol Cancer 2016;15:18. [Crossref] [PubMed]
6. Sullivan LB, Gui DY, Vander Heiden MG. Altered metabolite levels in cancer: implications for tumour biology and cancer therapy. Nat Rev Cancer 2016;16:680-93. [Crossref] [PubMed]
7. Laurenti G, Tennant DA. Isocitrate dehydrogenase (IDH), succinate dehydrogenase (SDH), fumarate hydratase (FH): three players for one phenotype in cancer? Biochem Soc Trans 2016;44:1111-6. [Crossref] [PubMed]
8. Yang M, Soga T, Pollard PJ, et al. The emerging role of fumarate as an oncometabolite. Front Oncol 2012;2:85. [Crossref] [PubMed]
9. Nowicki S, Gottlieb E. Oncometabolites: tailoring our genes. FEBS J 2015;282:2796-805. [Crossref] [PubMed]
10. Linehan WM, Rouault TA. Molecular pathways: Fumarate hydratase-deficient kidney cancer--targeting the Warburg effect in cancer. Clin Cancer Res 2013;19:3345-52. [Crossref] [PubMed]
11. Isaacs JS, Jung YJ, Mole DR, et al. HIF overexpression correlates with biallelic loss of fumarate hydratase in renal cancer: novel role of fumarate in regulation of HIF stability. Cancer Cell 2005;8:143-53. [Crossref] [PubMed]
12. Tong WH, Sourbier C, Kovtunovych G, et al. The glycolytic shift in fumarate-hydratase-deficient kidney cancer lowers AMPK levels, increases anabolic propensities and lowers cellular iron levels. Cancer Cell 2011;20:315-27. [Crossref] [PubMed]
13. Ooi A, Wong JC, Petillo D, et al. An antioxidant response phenotype shared between hereditary and sporadic type 2 papillary renal cell carcinoma. Cancer Cell 2011;20:511-23. [Crossref] [PubMed]
14. Sciacovelli M, Gonçalves E, Johnson TI, et al. Fumarate is an epigenetic modifier that elicits epithelial-to-mesenchymal transition. Nature 2016;537:544-7. [Crossref] [PubMed]
15. Tang J, Li Y, Wang J, et al. Molecular mechanisms of microRNAs in regulating epithelial-mesenchymal transitions in human cancers. Cancer Lett 2016;371:301-13. [Crossref] [PubMed]
16. Humphries B, Yang C. The microRNA-200 family: small molecules with novel roles in cancer development, progression and therapy. Oncotarget 2015;6:6472-98. [Crossref] [PubMed]
17. Pastor WA, Aravind L, Rao A. TETonic shift: biological roles of TET proteins in DNA demethylation and transcription. Nat Rev Mol Cell Biol 2013;14:341-56. [Crossref] [PubMed]
18. Neary R, Watson CJ, Baugh JA. Epigenetics and the overhealing wound: the role of DNA methylation in fibrosis. Fibrogenesis Tissue Repair 2015;8:18. [Crossref] [PubMed]
19. Xu X, Tan X, Tampe B, et al. Epigenetic balance of aberrant Rasal1 promoter methylation and hydroxymethylation regulates cardiac fibrosis. Cardiovasc Res 2015;105:279-91. [Crossref] [PubMed]
20. Tampe B, Tampe D, Müller CA, et al. Tet3-mediated hydroxymethylation of epigenetically silenced genes contributes to bone morphogenic protein 7-induced reversal of kidney fibrosis. J Am Soc Nephrol 2014;25:905-12. [Crossref] [PubMed]