Epigenetics in cancer stem cells: mechanism, characteristics, and therapies

By Chuyue Zhang

Since their discovery as a key tumour-initiating subset of cancer cells, cancer stem cells (CSCs) have attracted considerable interest. They are a population that contributes to intratumor heterogeneity with self-renewal ability, intrinsic therapy resistance, and the capability to give rise to differentiated progeny (O’Flaherty et al., 2012). Along with genetic determinants and microenvironments, epigenetic mechanisms including DNA methylation, chromatin remodelling, histone modifications, and non-coding RNAs also play a role in regulating stem cells. Understanding the common types of epigenetic mechanisms and how they affect CSC characteristics can help develop improved therapeutics and reduce disease progression and recurrence (Kreso & Dick, 2014).

Proteins involved in DNA methylation have been identified as drivers of CSC formation. DNA methyltransferases DNMT3a and DNMT3b are important in regulating stem cell characteristics as they set the pattern of gene methylation by targeting unmethylated CpG sites. Mutated DNMT3a is present in 25% of acute myeloid leukemia (AML) patients and was observed to inhibit enzyme activities, leading to expansion of pre-leukemic stem cells (LSCs) (Russler-Germain et al., 2014). Similar consequences can also result from the loss-of-function mutation of TET proteins that antagonise the function of DNMTs and IDH protein mutations that indirectly affect DNA methylation patterns (The Cancer Genome Atlas Research Network, 2013). Mutations of DNMTs and IDHs were also observed in solid tumours. DNMT3b, on the other hand, functions to maintain CSCs in undifferentiated states when present at higher levels at the CpG-islands of hypermethylated genes (Jin et al., 2012).

Inactivating mutations of chromatin remodelling complexes that control the expression of genes are found frequently in various types of cancers, and are linked to aberrant activation of stem-cell related pathways (Wilson & Roberts, 2011). Especially the SWI/SNF family, which mutates in 20% of tumours of many types of cancer because they have a decisive role in chromatin remodelling (Lee & Roberts, 2013). Mutated structural proteins like cohesins that regulate chromatin higher-order structure can also initiate stem cell transcriptional programs and affect LSC emergence (Mazumdar et al., 2015).

In addition to CSC formation, the chromatin remodelling complex SWI/SNF is required for maintaining self-renewing LSCs in mixed lineage leukemia (MLL) by sustaining high levels of c-MYC and regulating enhancer function (Shi et al., 2013). Furthermore, BRG1, which is involved in coding for SWI/SNF complex’s ATPase subunits, has mutations in non-small lung cancer at a frequency of 20-40%, which suggests its role as a tumour suppressor in lung tumorigenesis (Wu, 2011).

Chromatin status contributes to another key characteristic of CSCs called intrinsic plasticity. This allows CSCs to quickly respond to signals and switch cellular states by changing phenotypes so that some differentiated cancer cells can revert to CSC states. For example, in basal carcinomas of the breast, TGF-β stimulation can convert non-tumorigenic cells into CSCs, which is dependent on the chromatin status of the ZEB1 promoter (Chaffer et al., 2013).

As for histone modifications, they contribute to chromatin status along with chromatin remodelling proteins. In studies of glioblastoma, one-third of pediatric cases showed gain-of-function mutations in histone H3 genes. Linker histone H1.0 restricts cancer cell long-term proliferative potential as low levels are expressed in CSCs but high levels in differentiated cells. CSCs repressing H1.0 can thus preserve a chromatin configuration with self-renewal capacity. High levels of histone demethylase KDM5B were found to extinguish LSC potential in MLL-driven AML by reverting histone modifications, and high levels of histone demethylase G9a can inhibit self-renewal of CSCs in glioma (Tao et al., 2014). This demonstrates that chromatin component integrity is a crucial regulator of cancer cell differentiation.

Finally, non-coding RNAs like miRNAs were also found to regulate stemness in CSCs and their loss of regulation can cause tumorigenesis. miR-34a can inhibit functional properties of CSCs by targeting stemness factors like NOTCH, MYC, BCL-2, and CD44 and is found to be downregulated in various cancers (Li et al., 2021). piRNA-823’s upregulation in multiple myeloma stem cells, on the other hand, can lead to the activation of DNMT3b and induce stem-like properties (Ai et al., 2019).

Epithelial-to-mesenchymal transition (EMT) is a process that can be found at the initiation of cancer metastasis that results in cells losing cell-cell adhesion and polarity with increased migratory and invasive properties. Its activation was found to give cell CSC properties, and miR-200 family miRNAs and miR-205 can repress EMT by inhibiting transcription factors ZEB1 and ZEB2. Thus, their inhibition will cause increased metastasis which was seen in high-grade breast cancers (Mercurio, 2008). CSCs also possess a characteristic of asymmetric cell division that allows them to have self-renewal properties, and this is suppressed by miR-146a in colorectal cancer (Hwang et al., 2014).

Challenges to effectively treating tumours with CSCs lies within the advantages of CSCs against common cancer therapies. Their self-renewing ability allows them to reconstitute after chemotherapy and even select subclones with drug resistance (Kreso & Dick, 2014). CSCs have also been found with a greater number of efflux transporters including ABC transporters to maintain an efflux of drug from the cells(Wu et al., 2008).

The study of the epigenetic of CSCs has provided insights for epigenetic modifying drugs, within which DNMT and HDAC inhibitors have been widely studied and put into clinical trials. DNMT inhibitors including azacytidine and decitabine act as analogues of cytosine. They prevent DNMT from functioning by incorporating into DNA and lead to DNMT degradation after covalent bonding (Juttermann et al., 1994). DNMT inhibitors can even better sustain DNA methylation in lower doses and re-express silenced genes in leukemic and epithelial tumour cells (Tsai et al., 2012).

Histone deacetylases (HDACs) inhibitors target deregulated gene silencing caused by histone acetylation in cancer cells, and have been approved to treat cutaneous T-cell lymphoma (Piekarz et al., 2009). They can also induce CSC differentiation and thus re-sensitise them to chemotherapies (Debeb et al., 2012). Other drugs target histone methylation or demethylation like histone methyltransferase(HMT) inhibitors and histone demethylase(HDM) inhibitors(Toh et al., 2017). As regulators of CSC stemness, miRNAs are also under research as targets for therapy (Shukla & Meeran, 2014).

Overall, with the growing understanding of epigenetic mechanisms in cancer development, better therapeutic strategies using epigenetic approaches are being designed. CSC targeted approaches can focus on epigenetic modulators and increase the sensitivity of cancer cells to other therapies or restore chromatin remodelling by tumour suppressor proteins. miRNA re-expression and mediation also has the potential to inhibit cancer cell stemness and result in loss of pluripotency. Future applications of epigenetic drugs combined with immune- or chemo-therapies also show promising potential and are yet to be verified by ongoing trials. 

References:

Ai, L., Mu, S., Sun, C., Fan, F., Yan, H., Qin, Y., Cui, G., Wang, Y., Guo, T., Mei, H., Wang, H., & Hu, Y. (2019). Myeloid-derived suppressor cells endow stem-like qualities to multiple myeloma cells by inducing piRNA-823 expression and DNMT3B activation. Molecular Cancer, 18(1). https://doi.org/10.1186/s12943-019-1011-5 

Chaffer, C. L., Marjanovic, N. D., Lee, T., Bell, G., Kleer, C. G., Reinhardt, F., D’Alessio, A. C., Young, R. A., & Weinberg, R. A. (2013). Poised Chromatin at the ZEB1 Promoter Enables Breast Cancer Cell Plasticity and Enhances Tumorigenicity. Cell, 154(1), 61–74. https://doi.org/10.1016/j.cell.2013.06.005 

Debeb, B. G., Lacerda, L., Xu, W., Larson, R., Solley, T., Atkinson, R., Sulman, E. P., Ueno, N. T., Krishnamurthy, S., Reuben, J. M., Buchholz, T. A., & Woodward, W. A. (2012). Histone Deacetylase Inhibitors Stimulate Dedifferentiation of Human Breast Cancer Cells Through WNT/β-Catenin Signaling. STEM CELLS, 30(11), 2366–2377. https://doi.org/10.1002/stem.1219 

Genomic and Epigenomic Landscapes of Adult De Novo Acute Myeloid Leukemia. (2013). New England Journal of Medicine, 368(22), 2059–2074. https://doi.org/10.1056/nejmoa1301689 

Hwang, W.-L., Jiang, J.-K., Yang, S.-H., Huang, T.-S., Lan, H.-Y., Teng, H.-W., Yang, C.-Y., Tsai, Y.-P., Lin, C.-H., Wang, H.-W., & Yang, M.-H. (2014). Erratum: MicroRNA-146a directs the symmetric division of Snail-dominant colorectal cancer stem cells. Nature Cell Biology, 16(4), 383–383. https://doi.org/10.1038/ncb2951 

Jin, B., Ernst, J., Tiedemann, R. L., Xu, H., Sureshchandra, S., Kellis, M., Dalton, S., Liu, C., Choi, J.-H., & Robertson, K. D. (2012). Linking DNA Methyltransferases to Epigenetic Marks and Nucleosome Structure Genome-wide in Human Tumor Cells. Cell Reports, 2(5), 1411–1424. https://doi.org/10.1016/j.celrep.2012.10.017 

Juttermann, R., Li, E., & Jaenisch, R. (1994). Toxicity of 5-aza-2′-deoxycytidine to mammalian cells is mediated primarily by covalent trapping of DNA methyltransferase rather than DNA demethylation. Proceedings of the National Academy of Sciences, 91(25), 11797–11801. https://doi.org/10.1073/pnas.91.25.11797 

Kreso, A., & Dick, J. E. (2014). Evolution of the Cancer Stem Cell Model. Cell Stem Cell, 14(3), 275–291. https://doi.org/10.1016/j.stem.2014.02.006 

Lee, R. S., & Roberts, C. W. (2013). Linking the SWI/SNF complex to prostate cancer. Nature Genetics, 45(11), 1268–1269. https://doi.org/10.1038/ng.2805 

Li, W. (J., Wang, Y., Liu, R., Kasinski, A. L., Shen, H., Slack, F. J., & Tang, D. G. (2021). MicroRNA-34a: Potent Tumor Suppressor, Cancer Stem Cell Inhibitor, and Potential Anticancer Therapeutic. Frontiers in Cell and Developmental Biology, 9. https://doi.org/10.3389/fcell.2021.640587 

Mazumdar, C., Shen, Y., Xavy, S., Zhao, F., Reinisch, A., Li, R., Corces, M. R., Flynn, R. A., Buenrostro, J. D., Chan, S. M., Thomas, D., Koenig, J. L., Hong, W.-J., Chang, H. Y., & Majeti, R. (2015). Leukemia-Associated Cohesin Mutants Dominantly Enforce Stem Cell Programs and Impair Human Hematopoietic Progenitor Differentiation. Cell Stem Cell, 17(6), 675–688. https://doi.org/10.1016/j.stem.2015.09.017 

Mercurio, A. (2008). Faculty Opinions recommendation of The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature. https://doi.org/10.3410/f.1104825.560908 

O’Flaherty, J. D., Barr, M., Fennell, D., Richard, D., Reynolds, J., O’Leary, J., & O’Byrne, K. (2012). The Cancer Stem-Cell Hypothesis: Its Emerging Role in Lung Cancer Biology and Its Relevance for Future Therapy. Journal of Thoracic Oncology, 7(12), 1880–1890. https://doi.org/10.1097/jto.0b013e31826bfbc6 

Piekarz, R. L., Frye, R., Turner, M., Wright, J. J., Allen, S. L., Kirschbaum, M. H., Zain, J., Prince, H. M., Leonard, J. P., Geskin, L. J., Reeder, C., Joske, D., Figg, W. D., Gardner, E. R., Steinberg, S. M., Jaffe, E. S., Stetler-Stevenson, M., Lade, S., Fojo, A. T., & Bates, S. E. (2009). Phase II Multi-Institutional Trial of the Histone Deacetylase Inhibitor Romidepsin As Monotherapy for Patients With Cutaneous T-Cell Lymphoma. Journal of Clinical Oncology, 27(32), 5410–5417. https://doi.org/10.1200/jco.2008.21.6150 

Russler-Germain, D. A., Spencer, D. H., Young, M. A., Lamprecht, T. L., Miller, C. A., Fulton, R., Meyer, M. R., Erdmann-Gilmore, P., Townsend, R. R., Wilson, R. K., & Ley, T. J. (2014). The R882H DNMT3A Mutation Associated with AML Dominantly Inhibits Wild-Type DNMT3A by Blocking Its Ability to Form Active Tetramers. Cancer Cell, 25(4), 442–454. https://doi.org/10.1016/j.ccr.2014.02.010 

Shi, J., Whyte, W. A., Zepeda-Mendoza, C. J., Milazzo, J. P., Shen, C., Roe, J.-S., Minder, J. L., Mercan, F., Wang, E., Eckersley-Maslin, M. A., Campbell, A. E., Kawaoka, S., Shareef, S., Zhu, Z., Kendall, J., Muhar, M., Haslinger, C., Yu, M., Roeder, R. G., … Vakoc, C. R. (2013). Role of SWI/SNF in acute leukemia maintenance and enhancer-mediated Myc regulation. Genes & Development, 27(24), 2648–2662. https://doi.org/10.1101/gad.232710.113 

Shukla, S., & Meeran, S. M. (2014). Epigenetics of cancer stem cells: Pathways and therapeutics. Biochimica Et Biophysica Acta (BBA) – General Subjects, 1840(12), 3494–3502. https://doi.org/10.1016/j.bbagen.2014.09.017 

Tao, H., Li, H., Su, Y., Feng, D., Wang, X., Zhang, C., Ma, H., & Hu, Q. (2014). Histone methyltransferase G9a and H3K9 dimethylation inhibit the self-renewal of glioma cancer stem cells. Molecular and Cellular Biochemistry, 394(1-2), 23–30. https://doi.org/10.1007/s11010-014-2077-4 

Toh, T. B., Lim, J. J., & Chow, E. K.-H. (2017). Epigenetics in cancer stem cells. Molecular Cancer, 16(1). https://doi.org/10.1186/s12943-017-0596-9 

Tsai, H.-C., Li, H., Neste, L. V., Cai, Y., Robert, C., Rassool, F. V., Shin, J. J., Harbom, K. M., Beaty, R., Pappou, E., Harris, J., Yen, R.-W. C., Ahuja, N., Brock, M. V., Stearns, V., Feller-Kopman, D., Lin, Y.-C., Welm, A. L., Issa, J.-P., … Zahnow, C. A. (2012). Abstract 995: Transient low doses of DNA demethylating agents exert durable antitumor effects on hematological and epithelial tumor cells. Molecular and Cellular Biology. https://doi.org/10.1158/1538-7445.am2012-995 

Wilson, B. G., & Roberts, C. W. (2011). SWI/SNF nucleosome remodellers and cancer. Nature Reviews Cancer, 11(7), 481–492. https://doi.org/10.1038/nrc3068 

Wu, C.-P., Calcagno, A., & Ambudkar, S. (2008). Reversal of ABC Drug Transporter-Mediated Multidrug Resistance in Cancer Cells: Evaluation of Current Strategies. Current Molecular Pharmacology, 1(2), 93–105. https://doi.org/10.2174/1874467210801020093 

Wu, J. I. (2011). Diverse functions of ATP-dependent chromatin remodeling complexes in development and cancer. Acta Biochimica Et Biophysica Sinica, 44(1), 54–69. https://doi.org/10.1093/abbs/gmr099 

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out /  Change )

Google photo

You are commenting using your Google account. Log Out /  Change )

Twitter picture

You are commenting using your Twitter account. Log Out /  Change )

Facebook photo

You are commenting using your Facebook account. Log Out /  Change )

Connecting to %s