The tumor microenvironment (TME) is a complex ecosystem composed of the extracellular matrix, vascular and lymphatic networks, adipocytes, immune cells, and cancer-associated fibroblasts, all of them collectively induce tumor initiation and metastatic dissemination (1). As pivotal stromal components, CAFs—also referred to as activated fibroblasts—play essential roles in tumor progression. CAF-derived exosomes facilitate tumor growth and contribute to therapeutic resistance.

Epigenetic modification refers to heritable changes in gene function that occur without mutation in the DNA sequence itself, primarily mediated through chemical modifications of DNA, RNA, or relative proteins (2). Broadly defined, epigenetic mechanisms include DNA methylation, histone post-translational modifications, ATP-dependent chromatin remodeling, regulatory non-coding RNAs, and RNA chemical modifications. These mechanisms regulate cellular function and tissue architecture through targeted molecular alterations.

Epigenetic regulation plays crucial roles in diverse biological processes, including embryonic development, tissue repair, aging, and physiological responses to environmental stimuli (3). Dysregulation of epigenetic processes will induce a hallmark of numerous human diseases, particularly tumor (4).

Accumulating evidence shows that epigenetic modification reshapes both the transcriptome of malignant cells and the functional identity of stromal cells, particularly CAFs as tumors evolving (5). Epigenetic alterations promote carcinogenesis by enhancing cell proliferation, facilitating invasion and metastasis, and metabolism reprogramming (6). Furthermore, epigenetic modification regulate extracellular matrix deposition and tissue stiffness, contributing to the formation of a fibrotic and tumor-supportive stromal environment.

Origin and heterogeneity of CAFs

Fibroblasts were first described by Virchow in 1858. They originate from mesenchymal cells and are widely distributed throughout connective tissues. Fibroblasts play essential roles in maintaining extracellular matrix homeostasis, preserving tissue architecture, promoting wound repair, and regulating immune responses (7).

In physiological conditions, fibroblasts keep in a quiescent state characterized by minimal intercellular communication, limited collagen synthesis, low cytokine secretion, and restricted proliferative activity (8). However, during wound healing, inflammation, or fibrotic processes, fibroblasts can transform into myofibroblasts characterized by increased expression of α-smooth muscle actin (α-SMA) (4). Activated fibroblasts exhibit enhanced contractility and an increased capacity for extracellular matrix production and remodeling, thereby promoting tissue repair and structure (9). Following the completion of tissue repair, these cells typically undergo apoptosis or revert to a quiescent fibroblast phenotype (10).

In the tumor microenvironment, CAFs represent the dominant stromal cell population. CAFs originate from various cells, including resident tissue fibroblasts, bone marrow–derived mesenchymal stem cells, epithelial cells undergoing epithelial–mesenchymal transition (EMT), endothelial cells undergoing endothelial–mesenchymal transition (EndoMT), pericytes, adipocytes, smooth muscle cells, and macrophages (11). During tumor progression, numerous signals—including TGF-β, LIF, IL-1, TNF-α, IL-6, activation of the JAK/STAT3 pathway, hypoxia-induced HIF-1 stabilization, Notch signaling, reactive oxygen species, and cytotoxic therapies—can induce fibroblast activation and their transformation into CAFs (12). These factors can promote extracellular matrix deposition, fibrotic remodeling, angiogenesis, and the recruitment of tumor-associated immune cells, creating a microenvironment supporting tumor growth and invasion.

Recent studies have also suggested that cancer stem cells (CSCs) may contribute to the CAF population, indicating that tumors can reprogram their own stem-like cells into stromal fibroblasts (13). CAFs exhibit substantial functional heterogeneity for cellular diversity, which contributes to the complexity of tumor–stromal interactions (see Figures 1, 2).

The origin of CAFs. The origination of CAFs is presented in this figure, including resident tissue fibroblasts, adipocytes, cancer stem cells, smooth muscle cells, bone marrow-derived mesenchymal stem cells, macrophages, pericytes, endothelial cells, epithelial cells. The whole CAFs group can be divided into three subtypes: myCAFs, apCAFs, and iCAFs. Created with Figdraw.com.

The comprehensive analysis how CAFs epigenetic modification influence tumor progression. The factors inducing epigenetic modification in CAFs include: inflammation, hypoxia, and cancer cells contact and so on. The forms of modification include DNA, RNA, histone modification. CAFs undergoing modification produce many signal factors, such as IL-1, IL-6, TGF-β, TNF-α and so on. These factors will get involved in many biological activities, including produce more acid, remodeling ECM, immune suppression and so on. Created with Figdraw.com.

After introducing CAF’s origination and substyles, following content will illustrate what epigenetic modification arises in CAFs and the mechanism how these alterations affects CAFs biological action.

DNA methylation in CAFs

DNA methylation is a covalent epigenetic modification that occurs at cytosine residues within CpG islands locating in gene promoter regions (14). Hypermethylation of promoter CpG islands typically results in chromatin condensation and transcriptional repression. DNA methylation patterns are established and maintained by DNA methyltransferases (DNMTs) (15). Among DNMT family, DNMT1 maintains methylation patterns, DNMT3A and DNMT3B are responsible for establishing de novo methylation (16) (see Figure 3).

The process and outcome of DNA methylation. The factors influencing DNA methylation modification include smoking, drinking, cancer cells, and aging. This is a reversible process, which DNMT accounts for catalyzing DNA methylation and TET catalyzes adverse process. After modifying, the CpG islands present hypermethylation and whole genome present hypomethylation. This modification result in chromatin construction alteration and abnormal transcription. Created with Figdraw.com.

Although DNA methylation is generally considered a stable epigenetic modification, it keep in dynamic balance between methylation and demethylation. Demethylation.

has two patterns: one is passive demethylation arising during DNA replication when methylation is not maintained, another one is active demethylation catalyzed by ten–eleven translocation (TET) family of enzymes (17, 18). In general, promoter hypermethylation is associated with gene silencing and hypomethylation is linked to transcriptional activation (19).

The one dominant character of DNA modification is the coexistence of genome-wide hypomethylation and promoter-specific hypermethylation. Environmental factors such as smoking, alcohol, and aging can also induce DNA methylation (20–24). Genome-wide hypomethylation may contribute to genomic instability, promoter hypermethylation frequently silences tumor suppressor genes involved in cell cycle control, apoptosis, DNA repair, and angiogenesis (25). DNMTs are often overexpressed in various cancers and are associated with poor clinical prognosis (26). DNA methylation patterns have been detected in multiple malignant tumor, including colorectal cancer, breast cancer, gastric cancer, and lung adenocarcinomas (27–29).

Compared with normal fibroblasts (NFs), cancer-associated fibroblasts (CAFs) exhibit extensive methylation alterations. For instance, one study identified 1,772 differentially methylated CpG sites between CAFs and NFs, with approximately 60% showing hypermethylation in colon cancer (17). However, other studies report this pattern is not dominant in prostate cancer (30). It suggests DNA methylation is one of mechanisms influencing tumor progression.

DNA methylation is involved in fibroblast activation during fibrotic diseases. DNMT1-mediated methylation induces activation of hepatic stellate cells and contributes to liver fibrosis (2). The promoter 5′-UTR, and gene body of PPARγ contain numerous CpG islands, suggesting potential regulatory methylation sites. PPARγ interacts with the TGF-β/SMAD signaling pathway, playing a key role in fibrosis (31). Another evidence is TGF-β stimulation has been shown to reduce global DNA methylation levels in pulmonary fibrosis (32). Tumor cells can also secrete TGF-β and CXCL12, which activate CXCR4 signaling and stabilize the SMAD-dependent TGF-β pathway, thereby educating the differentiation of normal fibroblasts into CAFs (33).

In pancreatic ductal adenocarcinoma (PDAC), interactions between tumor cells and CAFs induce methylation of the SOCS1 promoter, leading to its downregulation combining with activating STAT3 signaling and promoting tumor progression (34). Additionally, the tumor microenvironment often keeps in high levels of lactic acid condition, which induces demethylation of CXCR4 and increases levels of 5-hydroxymethylcytosine (5hmC), ultimately enhancing CAF-mediated chemokine production and tumor invasiveness (34–36). And DNA methylation also regulates the expression of extracellular matrix–related genes, including COL1A1, COL1A2, and components of the TGF-β/SMAD signaling pathway (37, 38).

Notably, cancer cells can secrete TGF-β, thereby inducing the transformation of NFs into CAFs. During this process, CAFs undergo DNA methylation changes that subsequently enhance TGF-β transcription, forming a positive feedback loop between cancer cells and CAFs.

Epigenetic modifications of histone in CAFs

Nuclear DNA and histone organize into nucleosomes which is a highly ordered structure. The histone forms the core structural unit of chromatin, which consists of four types of core histone proteins with two copies of each type, including H2A, H2B, H3, and H4. Approximately 147 base pairs of DNA are wrapped around this histone complex (39, 40). Histones undergo extensive post-translational covalent modifications. Acetylation, methylation, and phosphorylation dynamically remodel chromatin architecture by loosening or tightening chromatin, regulating transcription factors, and recruiting regulatory proteins that regulate gene expression (41–43). These modifications alter histone charges, disrupting electrostatic interactions with DNA, and engage in complex crosstalk, collectively influencing transcriptional programs (30, 44).

Histone acetylation in CAFs

Histone acetylation is a reversible process that is catalyzed by histone acetyltransferases (HATs), removed by histone deacetylases (HDACs), and interpreted by bromodomain and extraterminal (BET) proteins that recognize acetylated histone marks (45). The major HAT families include p300/CBP, GNAT, MYST, p160, PCAF, and TAFII230 (46). HDACs are classified into two main groups: Zn2+-dependent deacetylases and NAD+-dependent sirtuins. The BET family consists of four members—BRD2, BRD3, BRD4, and BRDT. Among them, BRD4 can specifically binds to acetylated histones and promotes transcriptional activation (47).

The dynamic balance between histone acetylation and deacetylation in CAFs plays a critical role in cancer development and progression. The reader protein BRD4 is upregulated in advanced pancreatic cancer and correlates with increased enrichment of H3K27ac (48). In gastric cancer, BRD4 and H3K27ac co-occupy the enhancer region of SAA1, thereby promoting CAF activation and metastatic potential; pharmacological inhibition of BRD4 can reverse this phenotype (49). Similarly, in breast cancer-associated CAFs, EP300-mediated H3K27 acetylation upregulates genes involved in collagen synthesis (50).

Acetylation of lysine residues neutralizes their positive charge, leading to chromatin relaxation and enhanced accessibility of DNA to the transcription. In contrast, HDACs remove acetyl groups from histones, promoting chromatin condensation and transcription repression. Increasing evidence suggests that HDACs play complex roles in fibrosis: some HDACs promote fibrotic processes (51). However, HDACs do not uniformly promote tumor progression. A study find different HDAC inhibitors may result in opposite effects within pancreatic ductal adenocarcinoma (PDAC) mouse models. Certain HDAC family members are associated with immunosuppression, and inhibition of these HDACs can alleviate immunosuppressive conditions and enhance T-cell activity (46) (see Figure 4).

How histone modification effect CAFs. The histone modification in this figure include two sections. One is histone acetylation, another one is methylation. Histone acetylation occurs in histone lysine residues by HATs, it neutralizes positive charges. The adverse reaction is catalyzed by HDACs. The histone acetylation relax chromatin structure, which contributing to genetic transcription. BRD4 recognize acetylated histone and bind it, following promoting transcription. Created with Figdraw.com.

Histone methylation

Histone methylation is mediated by two major enzyme families: histone methyltransferases (HMTs) and histone demethylases (HDMs). HMTs catalyze the transfer of methyl groups from S-adenosylmethionine (SAM) to histones and HDMs remove these modifications. Histone methylation predominantly occurs in lysine and arginine residues located on the N-terminal tails of histones (52). Unlike acetylation, histone methylation does not alter histone charges (53).

Histone lysine methyltransferases (HMTs) catalyze lysine methylation, histone lysine demethylases function as “erasers” that remove methyl groups (54). Arginine methylation is mediated by protein arginine methyltransferases (PRMTs), including PRMT1, PRMT3, PRMT4/CARM1, and PRMT5 (55). In general, Lysine methylation represents a relatively stable and complex epigenetic modification occuring on histones H3 and H4. Histone H3 contains several lysine residues that can undergo methylation. H3K4 and H3K36 methylation are generally associated with transcription activation, whereas H3K9, H3K27, and H3K20 methylation are typically linked to transcription repression. In contrast, H3K79 methylation is associated with transcription activation (52).

Methylated histones can regulate the expression of inflammatory mediators and activate signaling pathways. Nuclear factor-κB (NF-κB) is a classical mediator of inflammatory responses, and histone methylation plays an important role in NF-κB-dependent inflammation (56). Histone methylation can also regulate fibrosis-related genes, thereby influencing extracellular matrix accumulation (57). Transforming growth factor-β1 (TGF-β1) is a well-recognized pathogenic factor in fibrosis across multiple organs (58). The TGF-β1/Snail1 axis can recruit PRMT1 and PRMT4 to promote arginine methylation, following driving CAF activation and fibronectin production.

Among histone methylation marks, H3K27 methylation is one of the most extensively studied modifications and is predominantly enriched in promoter and enhancer regions. This modification generally represses transcription and participates in cellular differentiation and cancer progression. The methylation status of H3K27 is dynamically regulated by the opposing activities of HMTs and HDMs. These epigenetic modification are closely associated with chromatin structure, cellular function, inflammation, and tumor progression (59). Specifically, the histone methyltransferase enhancer of zeste homolog 2 (EZH2) mediates H3K27 methylation, leading to chromatin compaction and transcriptional silencing, thereby influencing inflammation and cancer progression (60).

EZH2 is a critical regulator of cancer progression. The tumor microenvironment frequently exhibits hypoxic conditions, which induce the upregulation of hypoxia-inducible factor-α (HIF-α) (61). EZH2 expression can be regulated by HIF-α and contributes to tumor angiogenesis, metastasis, and invasion. To maintain active differentiation states, DNA repair capacity, and cellular plasticity, histones often undergo demethylation. The key enzymes responsible for this process include JMJD3 and UTX, which convert H3K27me2 and H3K27me3 to monomethylated H3K27 (H3K27me1). In actively transcribed genes, enrichment of H3K27me1 antagonizes EZH2-mediated transcriptional repression, thereby influencing cellular fate decisions (62).

In addition, upregulation of nicotinamide N-methyltransferase (NNMT) drives CAFs to produce biomarkers, cytokines, and pro-tumorigenic extracellular matrix components. Depletion of NNMT increases histone methylation marks such as H3K4me3 and H3K27me3, whereas NNMT overexpression decreases these methylation marks at target gene (15) (see Figure 5).

The positive feedback loop of TGF-β. Cancer cells secrete TGF-β factor, which can make CAFs occur DNA and histone modification. The modification promoting CAFs producing this factor combining with the upregulation of COL1A1 and COL1A2. Derived-CAFs TGF-β promote tumor cells progressing. And DNA modification can repress the expression of 5-UTR, PPARγ, SOCS1. Created with Figdraw.com.

RNA modifications in CAFs

RNA molecules function not only as targets but also as key mediators of epigenetic regulation, including messenger RNA (mRNA), microRNA (miRNA), and long non-coding RNA (lncRNA). To date, more than 150 RNA chemical modifications have been identified, among which N6-methyladenosine (m6A), 5-methylcytosine (m5C), and N7-methylguanosine (m7G) are the most extensively studied (63, 64). These modifications regulate RNA stability, splicing, localization, and translation efficiency, thereby influencing gene expression and cellular functions.

Increasing evidence indicates that RNA epigenetic modifications play critical roles in tumor progression and in regulating the phenotypes of cancer-associated fibroblasts (CAFs). Bi et al. (65) reported that Helicobacter pylori-induced hypermethylation of miR-149 in CAFs enhances IL-6 secretion through activation of the COX-2/PGE₂ signaling pathway, promoting epithelial–mesenchymal transition (EMT) and stem-like characteristics in gastric cancer cells.

Among the various RNA modifications, m6A and m5C have attracted particular attention because of their significant roles in regulating CAF activation and tumor-promoting functions (see Figure 6).

The m6A modification and m5C modification in CAFs. Two modifications can promote pri-RNA exporting from nucleus. The enzymes group accounting for m6A modification consist of writer proteins, reader proteins, and eraser proteins. The m6A modification and m5C modification can stabilize RNA structure and protect them from degradation and promote translation. The m6A modification can also recruit ribosomes to induce translation. Created with Figdraw.com.

N6-methyladenosine (m6A) in CAFs

m6A represents the most prevalent internal modification in eukaryotic mRNAs and plays a critical role in regulating RNA splicing, half-life, and translation efficiency. This modification is installed by the METTL3–METTL14 methyltransferase complex, removed by the demethylases FTO and ALKBH5, and recognized primarily by YTHDF family reader proteins (66, 67).

The m6A modification is dynamically regulated by three groups of proteins:

Writers: methyltransferases such as METTL3 and METTL14 that install m6A marks.

Erasers: demethylases such as FTO and ALKBH5 that remove m6A modifications.

-Readers: RNA-binding proteins, including YTHDF family members, that recognize m6A-modified transcripts and mediate downstream biological effects (68).

Through the coordinated actions of these regulatory proteins, m6A modification influences multiple aspects of RNA metabolism, including RNA stability, splicing, nuclear export, translation efficiency, and degradation (69). Following methylation, mRNAs are recognized by m6A-binding proteins that interact with RNA-processing factors, following altering RNA secondary structures and influencing splicing patterns. These modifications can also facilitate nuclear export by recruiting export receptors and enhance translation by promoting ribosome recruitment (70). In addition, m6A modification regulates microRNA processing by recruiting DGCR8 to facilitate the maturation of primary miRNAs (pri-miRNAs) (71, 72).

Recent studies have demonstrated that m6A RNA methylation participates in fibroblast activation and fibrotic diseases. The m6A modification has been shown to regulate the activation of hepatic stellate cells, contributing to liver fibrosis (2). In this process, METTL3 and METTL14 increase m6A modification within the 5′-UTR of TGF-β mRNA, while the reader protein YTHDF1 enhances TGF-β translation efficiency, thereby promoting fibroblast activation.

Furthermore, m6A modification is closely associated with metabolic reprogramming in cancer. METTL3-mediated m6A modification can increase the expression of glycolytic enzymes and enhance aerobic glycolysis in tumor cells (68). Under hypoxic conditions in the tumor microenvironment, the levels of HIF-1α increase and participate in promoting tumor development. METTL3-induced m6A modification further enhances HIF-1α expression and promotes glycolytic metabolism.

The m6A-dependent regulation of glycolytic enzyme transcripts has been reported in numerous malignancies, including colon, lung, pancreatic, renal, cervical, and gastric cancers, as well as osteosarcoma (73–82). Lactic acid–mediated histone H3K18 lactylation has been shown to regulate m6A modification of downstream targets by increasing the transcription of related enzymes (83). In addition, metabolic by-products in the tumor microenvironment may influence epigenetic modifications. Lactic acid–mediated histone lactylation at H3K18 can increase the transcription of m6A-related enzymes, thereby regulating downstream RNA methylation processes (84).

Within the tumor microenvironment, elevated glucose consumption leads to increased lactic acid production, creating an acidic environment that contributes to immune suppression. Under these conditions, macrophages frequently undergo M2 polarization, while the survival and activity of T cells and natural killer (NK) cells are suppressed. For instance, m6A-mediated stabilization of circQSOX1 has been reported to facilitate lactic acid accumulation and promote the recruitment of regulatory T cells (Tregs), thereby contributing to tumor immune evasion (85) (see Figure 7).

METTL family m6A modification METTL family can stabilize TGF-β mRNA, following activating fibrotic genes and induce fibrosis. In addition, tumor microenvironment keeps in a hypoxia condition, which induces CAFs enhance HIF-α level. The upregulation of HIF-α promote the synthesis of glycolytic enzymes, which accelerate glucose consumption and producing more acid. The acid environment suppress the recruitment of immune cells, such as NK cells and T cells. Created with Figdraw.com.

m5C RNA modification

The m5C RNA modification is widely distributed across multiple RNA species, including tRNA, rRNA, mRNA, and non-coding RNAs. Among these RNA types, tRNA and rRNA exhibit the high abundance of m5C modifications.

The enzymes for catalyzing m5C modifications mainly include members of the NOL1/NOP2/SUN (NSUN) family and tRNA aspartic acid methyltransferase 1 (TRDMT1). Different enzymes display substrate specificity: NSUN2 and NSUN4 modify mRNA, tRNA, and miRNA. NSUN6 and TRDMT1 mainly target tRNA. NSUN1 and NSUN5 primarily modify rRNA among these enzymes, NSUN2 plays a particularly important role in regulating m5C levels in mRNA (63).

Unlike DNA methylation and m6A RNA methylation, the existence of specific m5C demethylases (“erasers”) remains uncertain (86). However, m5C can undergo stepwise oxidative modification rather than direct demethylation. This process is catalyzed by enzymes such as ALKBH1 and the ten–eleven translocation (TET) family, generating the stable intermediates 5-hydroxymethylcytosine (hm5C), 5-formylcytosine (f5C), and 5-carboxylcytosine (ca5C) (87). These oxidative derivatives may influence RNA structure and function.

m5C modification plays essential roles in maintaining RNA stability and regulating translation. The m5C modification can stabilize RNA secondary structures and influence the stem–loop of tRNA molecules (88). In rRNA, m5C modification at position C2278 in 25S rRNA contributes to the stabilization of ribosome structure. Additionally, hypermethylated mRNAs can be stabilized through YBX1-dependent mechanisms (86, 89). NSUN2-mediated m5C modification also regulates the processing of vault RNA, facilitating its conversion into miRNA and protecting it from degradation (68, 87, 90).

Furthermore, m5C modifications can regulate mRNA nuclear export. The m5C reader protein ALYREF recognizes methylated RNA sequences and promotes the export of mRNA from the nucleus to the cytoplasm (91). In bladder cancer, ALYREF binding to the 3′-UTR of PKM2 mRNA stabilizes the transcript and promotes glycolysis and tumor growth (91).

That m5C RNA modification plays crucial roles in RNA stability, nuclear export, and translational regulation, following regulating various cellular fates. NSUN2-induced m5C methylation of the CNTTB1 mRNA can influence uveal melanoma cell proliferation and migration through blocking cycle G1 stage (92). The upregulation of m5C modified lncRNA NMR are associated with drug resistance, which may relate to the expression of MMP3 and MMP10. Undergoing m5C modification, epithelial differentiation is inhabited in pancreatic cancer (93).

In addition, the removal of m5C modification also regulate the stability of RNA.

The decrease of TET2 accelerate m5C accumulation in TSPAN13 mRNA, YBX1 specially recognize m5C sites and increase the expression of TSPAN13 transcription in acute myeloid leukemia (94) (see Figure 8).

The m5C modification. The enzymes catalyzing RNA m5C modification mainly include NOL1, NOL2, TRDMT1, SUN family. Among these enzymes, SUN family’s functions are complex and different numbers are responsible for catalyzing different RNA. NSUN2, NSUN4, NSUN6, TRDMT1 mainly catalyzing tRNA, NSUN1, NSUN5 mainly catalyzing rRNA, NSUN2, NSUN4 mainly catalyzing mRNA. The removal of m5C modification is not a directional process, which is transformed into hm5C RNA, f5C RNA, ca5C RNA through catalyzed by TET and ALKBH. Created with Figdraw.com.