The cancer cells in a tumour mass are highly heterogeneous, consisting of different populations of cells (Meacham and Morrison, 2013, Vogelstein et al., 2013). Cancer stem cells (CSCs) are one such subpopulation of cancer cells that influence tumour initiation, maintenance, and recurrence (Ayob & Ramasamy, 2018). They possess the ability to self-renew and differentiate into multiple cell types, which further contributes to the heterogeneous nature of tumours (Walcher et al., 2020). “Cancer stemness” refers to the stem-cell-like features possessed by this population of cancer cells, including activation of stem-cell-like gene expression patterns (Miranda et al., 2019).

CSCs are not very actively dividing cells and constitute a small fraction of the tumour mass (Gulaia et al., 2018). This makes the CSCs resistant to chemo or radiation therapy (Kim et al., 2009, Phi et al., 2018). The distinct transcriptome of CSCs influences their response to cancer treatments and also helps with evading the host immune system (Comertpay et al., 2021, Jiang et al., 2021). This includes increased expression of DNA damage repair factors and activation of immunosuppressive cellular signalling via Signal Transducer and Activator of Transcription 3 (STAT3) and Nuclear Factor-κB (NF-κB) pathways (Lei & Lee, 2021). Additionally, the heterogeneous tumour microenvironment, which comprises both cancerous and non-cancerous cells, impacts the CSCs transcriptome and their epigenetic landscape (Anderson & Simon, 2020). Growing evidence suggests that epigenetic changes taking place during cancer progression regulate cancer stemness. Therefore, one needs to focus beyond the genetic and biochemical aspects of CSCs and explore the underlying epigenetic mechanisms of CSCs formation and maintenance.

The three major epigenetic pathways regulating cancer stemness include DNA methylation, post-translational modifications of histones and expression of non-coding RNAs. Alteration in one of these interdependent pathways can have a feed-forward effect leading to formation, stable maintenance and propagation of CSCs.

DNA methylation occurs on the carbon‐5 position of the cytosine ring and is catalysed by DNA methyltransferases (e.g. DNMT1, DNMT3a and DNMT3b). Under normal conditions, increased DNA methylation in the gene promoters is repressive to transcription and regulates gene expression. Cancer cells have a peculiar change in their DNA methylation profiles: global DNA (Mazloumi et al., 2022) hypomethylation and site-specific DNA hypermethylation (Huang et al., 2020, Mazloumi et al., 2022). These changes are shared by cancer stem cells and are a consequence of differential DNMT recruitment and DNMT protein levels in these cells (Pathania et al., 2015, Verona et al., 2022, Zagorac et al., 2016). Global DNA hypomethylation is associated with the activation of transposable elements and other repetitive elements leading to increased genomic instability (Ma, Babarinde, Zhou, & Hutchins, 2022). Such an increase in genomic instability leads to increased heterogeneity in tumour cells and in rare instances can contribute to the generation of cancer stem cells (Liang et al., 2010). DNA hypomethylation also occurs in the pluripotency gene promoters leading to upregulation of stem cell-related genes, such as OCT4, NANOG, and SOX2, and promotes the stemness and survival of CSCs (Chiou et al., 2010, Liu et al., 2020, Shi et al., 2013). On the other hand, DNA hypermethylation of tumour suppressor genes enhances the tumorigenicity of CSCs (Ohm et al., 2007). DNA methylation targeting therapeutic interventions inhibiting the catalytic activity of DNMTs, help restore the expression of silenced tumour suppressor genes and induce apoptosis (Notarstefano et al., 2021, Pollyea et al., 2018, Wu et al., 2019). For a more in-depth review on the role of DNA methylation in cancer stem cells please refer to (Klutstein et al., 2016, Mazloumi et al., 2022, Nishiyama and Nakanishi, 2021, Wainwright and Scaffidi, 2017).

Post-translational modifications of the histone tails such as acetylation, methylation, ubiquitination and phosphorylation are linked to the regulation of stem cell properties and the maintenance of cancer stem cells by altering the chromatin accessibility to transcriptional machinery (Audia and Campbell, 2016, Völker-Albert et al., 2020). These altered histone modifications are not limited to the cancer stem cells but also extend to the cells in tumour microenvironment, providing CSCs with drug resistance and helping them escape the host immune system (Jin & Jeong, 2023).

The imbalanced activity of histone acetyltransferases (e.g. EP300, TIP60, GCN5) and histone deacetylases (e.g. HDACs and Sirtuins) in CSCs leads to site-specific changes in the levels of histone acetylation and promotes cancer stemness (Liu, Li, Wu, & Cho, 2017). Fusion proteins of MOZ (monocytic leukaemic zinc-finger protein) help maintain leukaemia stem cells by exploiting the histone acetyltransferase activity of MOZ protein (Yang & Ullah, 2007). SIRT1, a histone deacetylase, regulates the tumorigenicity and expression of pluripotency genes in colorectal cancer stem cells (Chen et al., 2014). Cancer stem cells show increased expression of HDACs and sensitivity towards HDAC inhibitors such as Vorinostat, Romidepsin, SAHA and Belinostat (Kumar et al., 2015, Nalls et al., 2011).

Bivalent chromatin regions, which contain both the active (H3K4me3) and repressive (H3K27me3) histone marks, play an essential role during embryonic development and differentiation (Kumar, Cinghu, Oldfield, Yang, & Jothi, 2021). Cancer cells show disruption in these bivalent chromatin regions and show increased levels of DNA methylation (Dunican et al., 2020, Kumar et al., 2021). Counterintuitively, this increase in DNA methylation at these bivalent chromatin regions correlates with an increased expression of multiple developmental genes, partially rendering stem cell-like properties to cancer stem cells (Dunican et al., 2020, French and Pauklin, 2020, Yamazaki et al., 2013). This highlights how the interdependent epigenetic factors are affected during cancer stem cell progression. Enhancer of zeste homologue 2 (EZH2), a subunit of the polycomb repressive complex 2, is a histone methyltransferase and regulates the levels of H3K27me3. EZH2 inhibitor treatment leads to loss of self-renewal properties and tumour initiation for glioblastoma multiforme CSCs via suppression of c-myc in these cells (Suvà et al., 2009). For a more in-depth review, please refer to (Audia and Campbell, 2016, Jin and Jeong, 2023, Völker-Albert et al., 2020).

In addition to these factors, non-coding RNAs (ncRNAs) such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) have been implicated in the regulation of cancer stemness (Lei & Lee, 2021). Studies have shown that miRNAs can regulate CSCs by controlling the expression of genes involved in stem cell self-renewal, differentiation, and apoptosis (Khan et al., 2019, Yoshida et al., 2021). In addition to directly regulating CSCs, miRNAs also affect the tumour microenvironment, which affects CSCs properties. For instance, miRNAs can regulate the expression of cytokines, growth factors, and other signalling molecules that support the survival and growth of CSCs (Asirvatham et al., 2009, Kedmi et al., 2015). For a more in-depth review, please refer to (Rajabi et al., 2022, Schwerdtfeger et al., 2021).

The role of these epigenetic factors extends beyond regulating the pluripotency and tumour suppressor gene expression in CSCs. A wide range of epigenetic factors is known to influence the signalling pathways in cancer cells and contribute towards cancer stemness (Matsui, 2016). Some of the most widely affected pathways in cancer stem cells include the Wnt/β-catenin pathway, Hedgehog (Hh) pathway, Notch pathway, and TGFβ/BMP pathway (refer to Fig. 1). In this chapter, we will discuss how epigenetic regulators affect these signalling pathways and promote CSC formation and maintenance.