Advances in pancreatic cancer epigenetics: From the mechanism to the clinic

Abstract

Pancreatic cancer (PC) is a highly malignant digestive system cancer that is difficult to diagnose early and is highly resistant to conventional treatments. Recent studies on epigenetics have provided new insights into the early diagnosis of PC and treatments including immunotherapy. Epigenetic changes can alter gene expression without altering the DNA sequence, involving mechanisms such as DNA methylation, histone modification, and the abnormal expression of noncoding RNAs. These mechanisms play crucial roles in the occurrence, development, and therapeutic response of tumors. This review summarizes progress in epigenetic research on PC and discusses the epigenetic characteristics, underlying mechanisms, early diagnosis, prognostic evaluation, treatment strategies, and future challenges for PC. Strategies targeting epigenetic changes may restore the expression of tumor suppressor genes and regulate the immune microenvironment and may be combined with immune checkpoint inhibitors as a new approach for the precise treatment of PC. However, there are challenges associated with the clinical application of epigenetic therapy, such as the development of resistance and adverse side effects, and challenges in target selection, so new epigenetic drugs and combination treatment strategies are needed. With the development of precision medicine, the integration of epigenetics and clinical medicine research has yielded broadly applicable findings. Interdisciplinary cooperation and advancements in clinical trials will further promote the application of epigenetics in PC research, providing more effective treatment options for patients. In the future, the combined application of epigenetic therapy and immunotherapy, the design of personalized treatment plans, and the development of technologies such as liquid biopsy will likely change the treatment landscape of PC.

Key Words: Pancreatic cancer; Epigenetics; Diagnosis; Treatment; Prognosis

Core Tip: Pancreatic cancer (PC) is a highly malignant cancer that is difficult to diagnose early and is resistant to conventional treatments. Epigenetic modifications, including DNA methylation, histone modification, and noncoding RNA expression, regulate gene expression without altering DNA sequences and play key roles in tumor development and treatment responses. This review discusses the epigenetic mechanisms, early diagnosis, prognosis, and treatment strategies for PC. Epigenetics, combined with immunotherapies, offers new treatment possibilities, but challenges remain, including resistance and side effects. The integration of epigenetics and precision medicine holds great potential for improving PC treatment and outcomes.

INTRODUCTION

Pancreatic cancer (PC) is a highly lethal malignancy with a survival rate that is far lower than that of other types of cancer. According to global cancer statistics, PC has become the seventh leading cause of cancer-related death worldwide, with a five-year survival rate of less than 10%[1]. These data reflect the difficulty in early diagnosis, the unclear course of the disease, and its rapid progression. Clinically, the treatment of PC typically relies on surgical resection, chemotherapy, and radiotherapy. PC patients with localized tumors that are amenable to complete surgical resection have the best prognosis. Surgery is typically an option for patients with early-stage PC or those with borderline resectable tumors. However, despite advancements in imaging techniques, most patients are diagnosed at an advanced stage, when the tumor has spread beyond the pancreas, making surgical intervention less feasible. As a result, the indications for surgery are limited, and the clinical outcomes of these patients are poor[2]. Therefore, methods for the early diagnosis and precise treatment of PC are urgently needed.

In recent years, epigenetic research, an important approach for studying gene expression regulation, has led to significant progress in cancer research. The term epigenetics refers to changes in gene expression that do not depend on alterations in the DNA sequence but are regulated through mechanisms such as DNA methylation, histone modification, and noncoding RNA (ncRNA) regulation[3]. These epigenetic changes play crucial roles in the initiation, development, and metastasis of PC and provide new directions for early diagnosis, prognostic evaluation, and improvement of treatment strategies[4].

Epigenetic research on PC has garnered increasing attention, especially the study of DNA methylation in PC. DNA methylation is an important epigenetic modification that profoundly affects tumor development by regulating gene silencing or activation. In PC, studies have revealed that specific genes, such as p16 and BRCA1, exhibit abnormal methylation patterns in tumor cells, other genes commonly affected include RASSF1A, CDKN2A, and APC, which are also associated with tumor progression and a poor prognosis. The methylation of p16 and BRCA1 is especially relevant due to the roles of these factors in cell cycle regulation and DNA repair, as these processes that are critical for maintaining genomic stability and preventing cancer development[5-8]. In addition, abnormal histone modifications are also a significant feature of epigenetic changes in PC. Research on histone deacetylation and demethylation has shown that these modifications play important roles in cell proliferation and the inhibition of apoptosis in PC[9,10]. Abnormal expression of ncRNAs, particularly miRNAs and long noncoding RNAs (lncRNAs), is also a significant aspect of epigenetic research in PC. miRNAs, as small RNA molecules, regulate gene expression by binding to the mRNAs of target genes and play roles in the invasion and metastasis of PC. Studies have shown that the expression of miR-21, miR-155, and other miRNAs is significantly elevated in PC and is closely associated with tumor invasiveness and metastatic potential[11,12]. In addition, the role of lncRNAs in PC has gradually been recognized. Some lncRNAs influence the biological behavior of tumors by regulating chromatin remodeling or interacting directly with epigenetic regulators[13,14].

Despite significant progress in epigenetic research on PC, many challenges remain in clinical translation. Epigenetic changes could serve as potential early diagnostic biomarkers, but effectively utilizing these epigenetic features for clinical detection and early screening remains one of the major challenges in research. Additionally, the application of epigenetic therapeutic strategies, such as the use of DNA demethylating agents and histone deacetylase (HDACs) inhibitors, has shown some efficacy in clinical studies. These agents target specific epigenetic alterations and have shown promise in overcoming drug resistance and improving treatment outcomes; however, their effectiveness and safety in clinical practice still need further validation[15,16].

Given the current challenge of poor prognosis for PC patients, the importance of early diagnosis and more effective treatment methods is self-evident. Epigenetic research provides a new perspective for addressing this issue. A deeper understanding of the epigenetic mechanisms of PC and the exploration of potential biomarkers will not only improve early diagnosis but also reveal new pathways for the precise treatment of PC. Therefore, the clinical application of epigenetics, especially in early screening, prognostic evaluation, and treatment response monitoring, holds great clinical value. In this review, we revisit the latest developments in epigenetic research on PC, with a focus on the roles of DNA methylation, histone modifications, and ncRNAs, as well as the potential applications of epigenetics in the early diagnosis, prognostic evaluation, and treatment of PC.

EPIGENETIC CHARACTERISTICS OF PC

PC is a highly malignant cancer, and its characteristic epigenetic changes play key roles not only in the initiation and progression of PC but also in the identification of new biomarkers for early diagnosis, prognostic evaluation, and therapeutic intervention. Epigenetic alterations in PC involve multiple processes, including DNA methylation, histone modification, and the expression of ncRNAs. These changes may be closely related to tumor cell proliferation, invasion, metastasis, and resistance to treatment. The main characteristics of the epigenetics of PC are described below (Table 1).

Table 1 Epigenetic characteristics of pancreatic cancer.

Epigenetic mechanism	Key changes	Genes affected	Role in pancreatic cancer
DNA methylation	Hypermethylation of tumor suppressor genes, particularly in the promoter regions	p16, BRCA1, RASSF1A, CDKN2A, APC	DNA hypermethylation in the promoter regions of tumor suppressor genes (e.g., p16 and BRCA1) silences their expression, allowing for uncontrolled cell division, which leads to cancer initiation and progression. Methylation of CDKN2A and RASSF1A contributes to tumor resistance and poor prognosis
Histone modifications	Histone acetylation (activation) and deacetylation (silencing)	p21, p53, H3K9me, H3K27me, HDACs	Histone acetylation (activation) and deacetylation (silencing) affect the accessibility of DNA. In pancreatic cancer, HDACs are overexpressed, leading to silencing of tumor suppressor genes like p21 and p53, which promotes cell cycle dysregulation and tumor progression
Noncoding RNAs (miRNAs and lncRNAs)	Dysregulated expression of miRNAs and lncRNAs that impact tumor behavior	miR-21, miR-155, HOTAIR, MALAT1	Upregulation of miRNAs (e.g., miR-21 and miR-155) and lncRNAs (e.g., MALAT1 and HOTAIR) suppress tumor suppressor genes, promoting cancer cell proliferation, invasiveness, and metastasis by modulating gene expression
Chromatin remodeling	ATP-dependent chromatin remodeling complexes change chromatin structure, affecting gene transcription	SWI/SNF complex, BRG1	ATP-dependent chromatin remodeling complexes like SWI/SNF and BRG1 modify chromatin structure, making genes more or less accessible for transcription. In pancreatic cancer, the dysfunction or downregulation of these complexes leads to silencing of tumor suppressor genes, promoting cancer progression and metastasis

lncRNA: Long noncoding RNA.

DNA methylation

DNA methylation is one of the core features of epigenetic changes in PC. The term DNA methylation refers to the addition of a methyl group to cytosine residues of genomic DNA, especially the methylation of CpG islands, which often leads to the silencing of tumor suppressor genes. In PC, abnormal DNA methylation is widespread, and significant methylation changes occur in several key tumor suppressor genes. Studies have shown that genes such as p16, BRCA1, and RASSF1A are frequently hypermethylated in PC patients and that the silencing of these genes is often closely associated with the onset and progression of PC[5,8,10].

In particular, the p16 gene is highly methylated in PC and is associated with a poor patient prognosis. Methylation of the p16 gene inhibits the function of the protein it encodes, leading to a loss of control over the cell cycle, which promotes abnormal cell proliferation[17-19]. In addition, methylation of the BRCA1 gene is associated with the invasiveness of PC and resistance to chemotherapy. These findings suggest that DNA methylation not only plays a key role in the initiation of PC but also potentially influences the treatment response[20-23].

Histone modifications

Histone modifications constitute another widely occurring type of epigenetic change in PC[4]. Histones regulate chromatin structure and gene expression through modifications such as acetylation, methylation, and phosphorylation. In PC cells, histone deacetylation and demethylation are particularly common. These changes lead to the silencing of tumor suppressor genes and the activation of oncogenes, thereby promoting the initiation and progression of PC[24-27].

For example, the upregulation of HDAC is closely associated with the invasiveness, tumor growth, and treatment resistance of PC[28-30]. In clinical studies of PC, the use of HDAC inhibitors has been considered a potential therapeutic strategy, capable of reactivating tumor suppressor genes and inhibiting the proliferation of tumor cells[30]. In addition, the acetylation of histones H3K9 and H3K27 is associated with chemotherapy resistance in PC, suggesting that histone modifications may play important regulatory roles in the treatment of PC[31].

The role of ncRNAs

ncRNAs, including miRNAs and lncRNAs, play important roles in the initiation, metastasis, and treatment response of PC by interacting with target mRNAs or chromatin remodeling factors. miRNAs are small, ncRNA molecules that bind to the 3' untranslated region of target gene mRNAs, inhibiting their translation or promoting their degradation. In PC, the abnormal expression of miRNAs is widespread, with many studies showing significant upregulation of the expression of miR-21, miR-155, and other miRNAs. These miRNAs are closely associated with tumor invasiveness, metastatic potential, and chemotherapy resistance[12,32].

For example, miR-21 is considered an "oncogenic" miRNA, and its high expression in PC is closely associated with tumor infiltration and metastasis[12,33,34]. Studies have shown that miR-21 promotes the proliferation, invasiveness, and metastasis of PC cells by inhibiting the expression of target genes such as PTEN and PDCD4[12,33,34]. Additionally, miR-155, an immune-related miRNA, is highly expressed in PC and is associated with immune evasion and poor patient prognosis[35,36].

The role of lncRNAs in PC has also garnered increasing attention. lncRNAs regulate gene expression and chromatin structure by interacting with chromatin remodeling factors, playing crucial roles in the initiation and progression of PC. Studies have shown that the expression of lncRNA-HOTAIR and lncRNA-MALAT1 is elevated in PC cells and that these lncRNAs are closely associated with the invasiveness, metastasis, and poor prognosis of PC[37,38].

Chromatin remodeling

Chromatin remodeling is another important aspect of epigenetics and refers to the alteration of the chromatin structure by ATP-dependent chromatin remodeling complexes, thereby regulating gene transcriptional activity. In PC, abnormal expression of chromatin remodeling factors, such as the SWI/SNF complex, is associated with tumor progression, metastasis, and treatment resistance[39-42]. For example, BRG1, a core factor of the SWI/SNF complex, is downregulated in PC, leading to chromatin compaction and the inhibition of tumor suppressor gene expression, which may be one of the key mechanisms of PC initiation[43,44].

Epigenetic interaction networks

Epigenetic changes in PC do not occur in isolation but are regulated through complex molecular networks that collectively influence the biological behavior of tumor cells. Changes in DNA methylation, histone modifications, and ncRNAs interact with each other to form a sophisticated regulatory network that drives the initiation, metastasis, and drug resistance of PC. Epigenetic modifications not only affect the expression of individual genes but also may influence the transcriptional programs of the entire genome through global changes in chromatin[45]. Therefore, understanding the epigenetic network not only reveals the molecular mechanisms of PC but also provides a new theoretical basis for the development of precision therapies.

IN-DEPTH UNDERSTANDING OF EPIGENETIC MECHANISMS

The epigenetic mechanisms of PC involve not only the individual effects of DNA methylation, histone modifications, or ncRNAs but also the complex interactions and regulatory networks that collectively determine the biological behavior of tumor cells. Epigenetic modifications participate in the initiation, progression, metastasis, and treatment response of PC by regulating gene expression, modulating chromatin states, and influencing the composition and function of the tumor microenvironment (TME). A deeper understanding of these epigenetic mechanisms will help researchers elucidate the molecular mechanisms of PC and provide new targeted therapeutic strategies for clinical application.

Epigenetic regulatory networks and the initiation of PC

The onset of PC is often accompanied by epigenetic changes, which play crucial roles in the silencing of tumor suppressor genes and the activation of oncogenes. DNA methylation is an important mechanism of epigenetic regulation. Methylation of CpG islands in the promoter region of genes usually leads to the silencing of tumor suppressor genes. In PC cells, the methylation-induced silencing of tumor suppressor genes such as p16, BRCA1, and RASSF1A is often an early marker of tumor initiation, and these altered methylation patterns can accumulate throughout tumor progression[5,8,10].

For example, methylation of the p16INK4a gene is typically one of the early epigenetic changes in PC. p16INK4a inhibits CDK4/6-dependent RB phosphorylation, thereby preventing excessive progression of the cell cycle. Its methylation-induced silencing leads to uncontrolled cell cycle progression, providing the foundation for tumor initiation[17-19]. Additionally, methylation of the BRCA1 gene is closely associated with radiation resistance in PC, further proving the impact of epigenetic changes on tumor progression[20-23].

Interaction between epigenetics and the TME

The TME plays crucial roles in the initiation and metastasis of PC, and epigenetic modifications play a key role in shaping this microenvironment. Epigenetic changes not only affect the tumor cells themselves but also alter intercellular communication and immune responses within the TME by regulating the expression of tumor-associated genes. For example, cancer-associated fibroblasts (CAFs) play a critical role in the progression of PC and pancreatic neuroendocrine tumors (PanNETs)[46-49]. Studies have shown that epigenetic modifications influence the function of CAFs, affecting the secretion of cytokines and extracellular matrix components and their regulatory effects on immune cells; however, the understanding of the impact of epigenetic modifications on CAFs in PanNETs is still limited[49-52].

The immune evasion mechanisms within the tumor immune microenvironment are also influenced by epigenetics. PC cells resist immune surveillance through epigenetic changes that silence immunosuppressive genes or activate immune evasion pathways. For example, epigenetic modifications can regulate the expression of immune checkpoint molecules such as PD-1 and PD-L1, providing a potential mechanism for immune evasion[53,54]. However, in pancreatic neuroendocrine neoplasms, immunohistochemical analysis revealed low immune infiltration (CD3/CD8/CD4, macrophages, NK cells) and reduced HLA-I expression in most tumors. Poorly differentiated neuroendocrine carcinomas (PanNECs) exhibited higher PD-1/PD-L1 expression, increased lymphocyte/macrophage infiltration, and HLA-I downregulation compared to well-differentiated tumors (PanNETs). Multivariate Cox analysis identified tumor grade, stage, and PD-1 expression as independent prognostic factors. While PanNETs displayed an immunologically "cold" phenotype, PanNECs expressed adaptive immune resistance markers, suggesting that PD-1/PD-L1 inhibitors may be effective against aggressive PanNEC but show limited efficacy in PanNET treatment[55,56]. Therefore, epigenetic changes promote the immune evasion and metastasis of tumors not only by directly affecting the behavior of tumor cells but also by altering immune cells and stromal components within the TME[9,57,58].

Epigenetic mechanisms of gene silencing and activation

The epigenetic mechanisms of PC are reflected not only in gene silencing but also in the activation of oncogenes. In epigenetics, gene silencing is typically achieved through mechanisms such as DNA methylation, histone deacetylation, and demethylation, whereas gene activation is accomplished primarily through demethylation and histone acetylation. Through the regulation of these epigenetic mechanisms, PC cells can lose the normal control of gene expression at multiple levels, thereby promoting tumor initiation and metastasis.

Histone modification is an important type of epigenetic mechanisms, with changes in histone acetylation and methylation playing key roles in PC. Histone acetylation is usually associated with gene activation, whereas deacetylation often leads to gene silencing. In PC cells, the upregulation of HDACs leads to histone deacetylation, which suppresses the expression of tumor suppressor genes such as p21 and p53 and promotes the activation of oncogenes[59,60]. These findings suggest that histone modifications have a profound impact on the regulation of gene expression.

The relationship between epigenetics and drug resistance

Epigenetic changes also play an important role in the treatment of PC, particularly in the development of drug resistance. PC cells often exhibit resistance to chemotherapy or radiotherapy, which is closely related to changes in their epigenetic mechanisms. Studies have shown that PC cells may evade the effects of drugs during chemotherapy through altered DNA methylation, histone modification, or regulation by ncRNAs[61-63].

For example, HDAC inhibitors can reverse the drug resistance of PC cells by reactivating silenced tumor suppressor genes, thereby increasing the effectiveness of chemotherapy drugs[64]. In addition, epigenetic regulation is not limited to the PC cells themselves but may also influence tumor sensitivity to immunotherapy by modulating the immune response within the TME. Changes in epigenetic mechanisms provide new biomarkers and potential targets for drug resistance in PC, and future treatments may overcome these challenges by targeting epigenetic alterations.

CLINICAL APPLICATION POTENTIAL OF EPIGENETIC MARKERS IN PC

The early diagnosis and precise treatment of PC have long been major clinical challenges. With advancements in molecular biology and epigenetic research, epigenetic markers have shown great potential in early diagnosis, prognostic evaluation, and treatment response monitoring for PC. These markers, through various detection techniques, such as liquid biopsy and genomic analysis, not only reveal the molecular characteristics of the tumor but also serve as a basis for personalized treatment. The following section discusses several emerging epigenetic markers and their potential applications in PC.

Epigenetic markers in early diagnosis

The bottleneck in the early diagnosis of PC lies in the lack of highly sensitive and specific biomarkers. Epigenetic markers, such as DNA methylation and ncRNAs, have become important targets for liquid biopsy because of their dynamic nature and detectability in the early stages of tumor development (Table 2).

Table 2 Epigenetic markers for early diagnosis in pancreatic cancer.

Epigenetic marker	Description	Stage sensitivity (%)	Sensitivity (%)
BNC1 methylation	Methylation of BNC1 in blood differentiates early-stage pancreatic cancer from healthy individuals	Stage I: 62.5%, Stage II: 65%, Stage III/IV: 100%	100% in Stage I, 88.9% in Stage IIA, 100% in Stage IIB
ADAMTS1 methylation	Methylation of ADAMTS1 in blood has high sensitivity and specificity for early diagnosis of pancreatic cancer	Stage I/II: 87.2%, Stage I/II: 95.8%	87.2% sensitivity, 95.8% specificity
GATA4 methylation	GATA4 methylation is associated with early diagnosis and prognosis of pancreatic cancer	High sensitivity and specificity for early diagnosis	High specificity for early detection
SFRP1 methylation	SFRP1 methylation is predictive of malignancy risk in pancreatic cancer and detected in pancreatic juice/blood	High sensitivity in pancreatic juice, predictive of malignancy risk	Widely detected in blood samples
miR-181b	miR-181b expression levels correlate with pancreatic cancer stages	MiR-196a, MiR-210 combined with CA19-9 improves accuracy	Improved staging accuracy with miR-196a and miR-210 detection
miR-196a	miR-196a expression levels correlate with pancreatic cancer stages, combined detection with CA19-9 improves staging accuracy	MiR-196a, MiR-210 combined with CA19-9 improves accuracy	Improved staging accuracy with miR-196a and miR-210 detection
miR-210	miR-210 expression levels correlate with pancreatic cancer stages, combined detection with CA19-9 improves staging accuracy	HOTAIR correlates with early diagnosis, found in saliva and blood	Effective early detection in pancreatic cancer patients
HOTAIR (lncRNA)	Overexpression of HOTAIR correlates with early onset and malignancy of pancreatic tumors	miR-210 and miR-196a combined with CA19-9 improve accuracy	Effective for early detection and staging

lncRNA: Long noncoding RNA.

DNA methylation markers: Clinical studies indicate that blood-based BNC1 and ADAMTS1 methylation effectively differentiate early-stage (stage I/II) PC patients from healthy individuals. The percentage of cells with BNC1 methylation increases with disease progression, reaching 100% in advanced stages (stage III/IV). Combining these biomarkers achieves near-perfect sensitivity (100%) in stages I and IIB, with overall sensitivity and specificity of 97.4% and 91.6%, respectively[65-67]. ADAMTS1 demonstrates higher standalone performance (87.2% sensitivity, 95.8% specificity) than BNC1 (64.1% sensitivity). Additionally, GATA4 hypermethylation and overexpression, along with SFRP1 methylation in pancreatic juice or blood, serve as robust biomarkers for early diagnosis and malignancy risk prediction[68-71].

ncRNA markers: In addition to DNA methylation markers, certain ncRNAs (miRNAs and lncRNAs) have also become important biomarkers for early diagnosis. miR-181b, miR-196a, and miR-210: The expression of miR-196a and miR-210 in serum is closely associated with the stage of PC, and their combined detection with CA19-9 can further improve the accuracy of staging[72]. The abnormal expression of HOTAIR (HOX transcript antisense RNA) in PC has been confirmed. Studies have shown that the overexpression of HOTAIR is associated with the early onset of tumors and their malignancy, and its levels in patients’ saliva and blood can serve as effective biomarkers for early diagnosis[73-75].

Epigenetic markers in prognosis evaluation

Epigenetic markers not only are important in the early diagnosis of PC but also reflect tumor heterogeneity and biological behavior, providing a basis for prognostic stratification. By evaluating changes in epigenetic markers, physicians can predict the treatment response, tumor recurrence, and other aspects, thereby developing more personalized treatment plans (Table 3).

Table 3 Epigenetic markers in prognosis evaluation.

Epigenetic marker	Description	Association with prognosis	Potential for use
MET hypomethylation	Hypomethylation of MET and ITGA2 correlates with high gene expression, associated with lower survival rates	High expression correlated with low survival rates	Useful in prognostic evaluation, treatment monitoring
ITGA2 hypomethylation	Methylation of the SFRP1 promoter serves as a prognostic and predictive biomarker in pancreatic cancer	Predicted poor prognosis and potential recurrence	Useful as a blood biomarker for stage III or IV pancreatic cancer
CDKN2A methylation	Methylation of CDKN2A promotes tumor initiation and metastasis, correlating with poor prognosis	Strong correlation with cell cycle dysregulation, leading to metastasis	Can be used for monitoring prognosis and recurrence
SFRP1 methylation	Methylation of SFRP1 has high specificity for advanced pancreatic cancer, correlating with poor prognosis	A predictive blood biomarker for stage III or IV pancreatic cancer	Potential blood biomarker for pancreatic cancer diagnosis
NPTX2 methylation	NPTX2 methylation in cfDNA correlates with poor prognosis and overall survival prediction	Associated with poor prognosis and treatment response	Non-invasive blood biomarker for prognosis and monitoring
HOTAIR (lncRNA)	HOTAIR overexpression in advanced cancer stages correlates with poor survival and advanced tumor stages	High expression linked to low survival, particularly in late-stage cancer	Useful for determining survival potential in late cancer stages
circFARP1	High expression of circFARP1 in serum correlates with lower survival rates in pancreatic cancer	High levels in serum linked to poor survival rates	Potential prognostic marker in serum for advanced PDAC
miR-196a	High expression of miR-196a is associated with lower survival rates and poor prognosis	Associated with poor prognosis, affects metastasis	Associated with better survival outcomes, useful in treatment
miR-210	High expression of miR-210 correlates with improved survival rates in pancreatic cancer patients	Correlated with improved survival rates in pancreatic cancer	May help predict the prognosis and overall survival
miR-124	Low expression of miR-124 is associated with metastasis and poor prognosis	Low miR-124 expression correlated with metastasis and reduced survival	Potential prognostic marker for cancer invasiveness

PDAC: Pancreatic ductal adenocarcinoma; lncRNA: Long noncoding RNA.

DNA methylation prognostic markers: Hypomethylation of MET and ITGA2 is associated with high gene expression, and high gene expression is correlated with low survival rates[76]. Methylation-mediated silencing of the CDKN2A gene is closely associated with the prognosis of patients with PC. Methylation of this gene can lead to cell cycle dysregulation, thereby promoting tumor initiation and metastasis. Recently, its methylation status has also been found to be related to prognosis[77]. Methylation of the SFRP1 promoter can also serve as a prognostic and potential predictive blood biomarker for patients with stage III or IV pancreatic ductal adenocarcinoma (PDAC)[78]. NPTX2 is the most highly and frequently methylated gene in cfDNA samples from mPDAC patients. Elevated circulating NPTX2 methylation levels at the time of diagnosis are associated with poor prognosis and effective stratification of patients to predict overall survival. The dynamic changes in circulating NPTX2 methylation levels are correlated with disease progression and the treatment response, and they predict the development of metastatic PDAC better than CA19-9 levels[79].

ncRNA prognostic markers: HOTAIR is highly expressed in advanced cancer tissues, and due to its high expression, it can serve as a prognostic factor to determine a patient's survival potential in the late stages of cancer. Low expression of HOTAIR is associated with increased survival rates, whereas high expression of HOTAIR indicates a very low chance of survival and progression to advanced tumor stages[80,81]. Elevated serum circFARP1 levels are positively correlated with lower patient survival rates[82]. Additionally, high expression of miR-196a is associated with lower survival rates, whereas high expression of miR-210 is significantly correlated with improved survival. A multivariate survival analysis revealed that the expression patterns of miR-210 and miR-196a, lymph node metastasis, and comprehensive treatment are independent factors affecting overall survival. The overexpression of miR-196a and decreased expression of miR-210 are significantly associated with the overall survival time[83]. In miRNA research, in addition to miR-21 and miR-155, miR-124 has also been shown to have potential in the prognostic evaluation of PC. Low expression of miR-124 is associated with the invasiveness and metastasis of PC, and it is often downregulated in patients with advanced-stage tumors. Therefore, the detection of miR-124 can serve as a tool for predicting the prognosis of patients with PC[84].

Epigenetic markers in treatment response monitoring

With the development of precision medicine, personalized monitoring of the treatment response has received increasing attention. Epigenetic markers can not only be used for early diagnosis and prognostic evaluation of tumors but also provide important information for real-time monitoring of the treatment response (Table 4).

Table 4 Epigenetic markers in treatment response monitoring.

Epigenetic marker	Description	Impact on treatment response	Clinical relevance
SFRP1 methylation	Methylation of SFRP1 promoter is associated with poor treatment response and resistance to gemcitabine	Increased SFRP1 methylation linked to reduced sensitivity to chemotherapy drugs like gemcitabine	Potential biomarker for assessing chemotherapy resistance, especially in advanced PDAC
circFARP1	CircFARP1 is a CAF-specific circRNA positively correlated with gemcitabine resistance	Gemcitabine resistance observed in patients with high circFARP1 expression	Could guide personalized treatment strategies for patients showing gemcitabine resistance
circBIRC6	CircBIRC6, upregulated in CAF-derived EVs, correlates with oxaliplatin chemotherapy resistance	CircBIRC6 contributes to resistance against chemotherapy, especially oxaliplatin	Marker for predicting oxaliplatin resistance and helping in therapeutic planning
HOTAIR (lncRNA)	HOTAIR overexpression is linked to poor survival and chemotherapy resistance in advanced cancer stages	High expression correlates with poor response to therapy, particularly in advanced stages	Can be used to predict response to therapy in advanced pancreatic cancer
circFARP1 (hsa_circ_0002557)	High circFARP1 levels correlate with gemcitabine resistance and lower survival in advanced PDAC	CircFARP1 Levels predict resistance to gemcitabine and poor survival	Indicates poor prognosis, guiding clinicians in optimizing chemotherapy strategies
EZH2 (Histone Methyltransferase)	EZH2 overexpression in pancreatic cancer cells leads to chemotherapy resistance	EZH2 overexpression contributes to pancreatic cancer resistance to chemotherapy	Targeting EZH2 could enhance the efficacy of current chemotherapies.
HDAC1 & PRMT1	HDAC1 and PRMT1 are epigenetic regulators influencing drug resistance by modifying chromatin and gene expression	HDAC1 and PRMT1 modulation affect tumor cell resistance to chemotherapy and immunotherapy	Both are crucial in predicting and overcoming drug resistance in pancreatic cancer patients

PDAC: Pancreatic ductal adenocarcinoma; lncRNA: Long noncoding RNA.

Studies have shown that tumors with high methylation of the SFRP1 promoter (phSFRP1) are more invasive and less sensitive to gemcitabine treatment. This knowledge may help guide personalized precision treatment for patients with stage IV PDAC. In the future, the detection of phSFRP1 in patients could be used to guide treatment decisions when planning stronger chemotherapy regimens[85]. The circRNA circFARP1 (hsa_circ_0002557) is a CAF-specific circRNA that is positively correlated with gemcitabine resistance and poor survival rates in patients with advanced PDAC[82]. CircBIRC6 is significantly upregulated in CAF-derived extracellular vesicles (EVs) and is positively correlated with oxaliplatin chemotherapy resistance. In vitro and in vivo functional assays have shown that circBIRC6 packaged in CAF-derived EVs enhances the resistance of PC cells and organoids to oxaliplatin by regulating nonhomologous end joining-dependent DNA repair[86]. In addition, epigenetic regulators such as HAT1 and PRMT1 are gradually attracting attention for their roles in PC treatment. EZH2, an important histone methyltransferase, is overexpressed and associated with the resistance of PC cells to chemotherapy drugs[87,88].

The application of epigenetic markers in the clinical management of PC has progressed from the bench to bedside. DNA methylation, ncRNA, and histone modification markers have shown high potential in early diagnosis, prognostic stratification, and treatment monitoring, but their clinical adoption still requires large-scale prospective studies for validation. In the future, the integration of multiomics technologies and artificial intelligence to create dynamic monitoring networks will be essential in achieving the goal of precision medicine.

EXPLORATION OF EPIGENETIC THERAPEUTIC STRATEGIES

Epigenetic research has become an important field in tumor therapy research. In recent years, researchers have gained a deeper understanding of the epigenetic mechanisms in malignant tumors such as PC, and epigenetic therapeutic strategies are being continuously explored. By targeting DNA methylation, histone modifications, and the abnormal expression of ncRNAs, epigenetic therapy provides new approaches for the precise treatment of PC. The current epigenetic therapeutic strategies for PC are discussed in detail below (Table 5).

Table 5 Epigenetic therapeutic strategies in pancreatic cancer.

Strategy	Targeted epigenetic mechanism	Therapeutic agents	Studies/efficacy
DNA demethylation	DNA methylation	5-Azacytidine (5-Aza), Decitabine	Used to restore silenced tumor suppressor genes. Clinical trials have shown limited success in PDAC
Histone deacetylation inhibition	Histone modification	Vorinostat, Romidepsin	Inhibits HDACs, leading to tumor suppressor gene activation and reduced tumor growth. Promising in early trials
miRNA therapy	Noncoding RNA	ExomiR-34a (miR-34a encapsulated in exosomes)	ExomiR-34a showed suppression of pancreatic cancer cell growth and induced apoptosis in preclinical trials
lncRNA modulation	Noncoding RNA	Antisense oligonucleotides, RNAi	Targets lncRNAs to reverse drug resistance and regulate oncogenes. Emerging as a promising approach
CRISPR/Cas13 for lncRNA	Noncoding RNA	CRISPR-Cas13	Modulates lncRNAs to target pancreatic cancer growth and metastasis. Still in preclinical stages
Combination therapy with immunotherapy	Epigenetic Modifications & Immunotherapy	HDAC inhibitors + PD-1/PD-L1 inhibitors	Epigenetic inhibitors combined with immune checkpoint inhibitors showed potential in enhancing immune responses

PDAC: Pancreatic ductal adenocarcinoma; lncRNA: Long noncoding RNA.

DNA methylation-based therapeutic strategies

DNA methylation was one of the first studied epigenetic modifications. Abnormal DNA methylation silences many tumor suppressor genes, promoting tumor initiation and progression. Therefore, restoring the normal expression of these genes is a key goal of epigenetic therapy. In response to the common DNA methylation abnormalities observed in PC, researchers have developed several therapeutic strategies targeting DNA demethylation.

The core strategy involves targeting key epigenetic regulatory factors, such as DNA methyltransferase 1 (DNMT1), which mediates the suppression of these programs in cancer cells. Two of the most important inhibitors or depleters are 5-azacytidine (5-Aza, azacytidine) and decitabine. Azacytidine is a classic DNA demethylation drug that inhibits the activity of DNA methyltransferases, thus restoring the expression of silenced tumor suppressor genes. Decitabine, another demethylating drug, has been extensively studied. It intercalates into DNA, irreversibly inhibits DNMT1, and induces genome-wide hypomethylation. In fact, DNMT1 has been extensively studied as a potential molecular target for treating PDAC, with numerous clinical studies conducted in patients with blood system disorders and other solid tumors. However, the results from these completed trials have been unsatisfactory[89-92].

A preclinical study of decitabine revealed pharmacodynamic effects of decitabine in less than 25% of treated patients' resected solid tumor tissues. Several hours after continuous intravenous infusion of decitabine, the plasma Cmax remained > 40 nM, which was sufficient to cause grade 3/4 myelosuppression (especially neutropenia), a result that was undoubtedly disappointing[89]. Decitabine and azacytidine are prodrugs that must undergo pyrimidine metabolism to deplete DNMT1. Therefore, tissue-specific differences in the expression of key pyrimidine metabolic enzymes have been shown to be the basis for the tissue-specific efficacy of these drugs. One key enzyme is cytidine deaminase (CDA), which rapidly metabolizes decitabine and azacytidine to their corresponding uridine analogs, which cannot deplete DNMT1. The naturally high expression of CDA in gastrointestinal tissues is the reason decitabine, azacytidine, and gemcitabine (a first-line treatment drug for PDAC, also a pyrimidine nucleoside analog) cannot be administered orally. CDA is also the reason the plasma half-life of decitabine, azacytidine, and gemcitabine after parenteral administration is approximately 15 minutes. Therefore, the addition of the CDA inhibitor tetrahydrouridine (THU) to oral decitabine results in pharmacokinetics comparable to those of continuous intravenous infusion of decitabine. Mild neutropenia was observed in the blood, and preliminary clinical trials confirmed that the combination of the CDA inhibitor THU and decitabine showed some efficacy in patients with advanced PC[93]. Recently, a decitabine derivative, guadecitabine (SGI-110), which is resistant to CDA, was indicated to have potential for further development[94].

Histone modification-based therapeutic strategies

Histone modifications constitute another important mechanism in epigenetics, where modifications such as acetylation, methylation, and phosphorylation can affect chromatin structure, thereby regulating gene expression. Abnormal histone modifications play crucial roles in the development of PC and other tumors, which has led to widespread interest in targeting histone modifications as a therapeutic strategy.

HDAC inhibitors (HDAC inhibitors) promote the expression of tumor suppressor genes by inhibiting histone deacetylation, thereby suppressing tumor growth. Recent studies have shown promising results for vorinostat in phase I clinical trials for several solid tumors. A phase I trial evaluated the maximum tolerated dose (MTD) and safety of vorinostat combined with capecitabine and radiotherapy in patients with nonmetastatic PC. Twenty-one patients received increasing doses of vorinostat (100-400 mg/day) and a fixed dose of capecitabine (1000 mg/12 hours) combined with 30 Gy/10 fractions of radiotherapy. The results showed that the MTD of vorinostat was 400 mg, with dose-limiting toxicities, including gastrointestinal toxicity and thrombocytopenia. The most common adverse events were lymphopenia (76%) and nausea (14%). After treatment, the apparent diffusion coefficient of most tumors increased, and 90% of patients achieved stable disease. The median overall survival was 1.1 years, indicating that the combined treatment regimen was well tolerated and showed potential efficacy[95]. Another phase I clinical trial explored the antitumor effects of marizomib in combination with vorinostat. In vitro experiments revealed that marizomib and vorinostat had significant synergistic effects on tumor cell lines derived from non-small cell lung cancer, melanoma, and PC. In the clinical trial, 22 patients received the combination therapy, and no significant increase in toxicity was observed. The pharmacokinetics and pharmacodynamics of the drugs were not affected. Although no obvious treatment response was observed according to the RECIST criteria, 61% of assessable patients had stable disease and 39% experienced tumor shrinkage. Overall, the combination of marizomib and vorinostat showed strong synergistic antitumor activity in tumor cells and was well tolerated in patients[96].

Like vorinostat, romidepsin is an HDAC inhibitor with potential therapeutic effects on PC. Both can inhibit the activities of HDAC1, HDAC3, and other deacetylases, enhancing the efficacy of chemotherapy drugs. HDAC inhibitors not only inhibit cell proliferation in PC but also suppress tumor invasion and metastasis[30,97]. Another interesting HDAC inhibitor, belinostat, has been shown to significantly inhibit PC cell growth, induce apoptosis, and block the PI3K-mTOR signaling pathway. Belinostat also suppresses hypoxia-related signaling and, when combined with gemcitabine, significantly reduces the pancreatic tumor size. Therefore, belinostat may be an effective treatment for PC, especially when it is used in combination with other drugs[98].

PRMT inhibitors (protein arginine methyltransferase inhibitors) have also garnered attention. Basic research has explored the antitumor effects and mechanisms of combining anti-PD-L1 therapy with the type I PRMT inhibitor PT1001B in PDAC. The results showed that by inhibiting the expression of PD-L1 in tumor cells, PT1001B significantly enhanced the therapeutic effect of anti-PD-L1 monoclonal antibodies, reducing the tumor volume and weight. The combination treatment increased the number of tumor-infiltrating CD8+ T lymphocytes and reduced the number of PD-1+ leukocytes. PT1001B also enhanced the inhibitory effect of anti-PD-L1 therapy on tumor cell proliferation and promoted tumor cell apoptosis. PRMT1 downregulation was associated with PD-L1 downregulation. Studies suggest that PT1001B can enhance antitumor immunity and, when combined with anti-PD-L1 checkpoint inhibitors, may provide a new strategy to overcome PD-L1 resistance in PDAC[99].

ncRNA targeted therapy

ncRNAs (especially miRNAs and lncRNAs) play a key role in the epigenetic regulation of PC. In recent years, therapeutic strategies targeting miRNAs and lncRNAs have become an important direction in epigenetic therapy.

miRNA intervention therapy: By introducing specific miRNAs to restore the expression of tumor suppressor genes, progress has been made in clinical studies of various cancers. For example, the downregulation of miR-34a is closely associated with the progression of PC. Researchers have synthesized exosome-encapsulated miR-34a (exomiR-34a) and evaluated its anticancer effects on PC. The results showed that exomiR-34a efficiently penetrated the cell membrane, inhibited the expression of the target gene Bcl-2, significantly suppressed PC cell growth, and induced cell apoptosis. In mouse models, exomiR-34a also significantly inhibited tumor growth. Therefore, exomiR-34a is a promising PC therapeutic agent with the potential to become a novel anticancer drug[100]. Another study used an effective nanocarrier to deliver miR-34a and an siRNA for the treatment of PDAC. A biodegradable polyglutamic acid amine nanocarrier was used to codeliver the miRNA and siRNA to a PDAC mouse model. No toxicity was observed, and the drug accumulated at the tumor site, where it inhibited the oncogene MYC, thereby enhancing the antitumor effects[101].

lncRNA intervention therapy: Therapeutic strategies targeting lncRNAs are gradually emerging as a new direction. For example, antisense oligonucleotides and RNA interference technologies can specifically target lncRNAs to regulate relevant pathways or reverse drug resistance[102]. Additionally, new methods, such as the development of small-molecule compounds and RNA-targeted CRISPR-Cas13 technology, offer the potential to effectively modulate the function of lncRNAs, thus providing new therapeutic strategies[103]. Some preclinical studies have shown that these methods can effectively inhibit tumor growth and metastasis, demonstrating their potential for development for the treatment of PDAC[102]. In summary, lncRNAs, as new therapeutic targets in PC, may lead to breakthroughs in PDAC treatment, especially in addressing the issue of drug resistance that current therapies cannot effectively manage[104].

Although epigenetic therapeutic strategies have shown great potential in preclinical studies, their clinical application remains challenging. Currently, research on demethylation drugs, HDAC inhibitors, and miRNA therapies in PC is gradually progressing, but further clinical validation is needed. For example, the clinical application of HDAC inhibitors is limited by the side effects of the drugs and drug resistance; off-target effects have also been observed, and DNMT/HDAC inhibitors may activate oncogenes (such as MYC), which necessitates the development of highly selective drugs. Additionally, resistance issues, where cells become resistant to epigenetic drugs, must also be addressed. Therefore, reducing side effects and improving drug selectivity and efficacy are key areas of current research.

CURRENT CHALLENGES AND FUTURE RESEARCH DIRECTIONS

Although epigenetics has shown great potential in the early diagnosis, treatment, and immunotherapy of PC, many challenges remain in its practical application. Future research should focus on addressing these challenges to further improve the efficacy and safety of epigenetic therapies. The following section discusses the major challenges currently faced and potential future research directions.

Current challenges

Drug resistance in clinical applications: Drug resistance is a key issue in the application of epigenetic therapies. Demethylating drugs such as azacytidine and decitabine have shown some preliminary effects in clinical studies of PC patients, but resistance often occurs[93]. The incidence of resistance is especially high when these drugs are administered in combination with immunotherapy, leading to a gradual decrease in treatment efficacy. Therefore, overcoming drug resistance in epigenetic therapy has become a major challenge in current research.

Adverse side effects and safety concerns: The side effects of epigenetic therapies, especially demethylating drugs and HDAC inhibitors, represent another significant challenge. For example, while HDAC inhibitors such as vorinostat can enhance immune responses, long-term use can lead to hematologic side effects such as anemia and leukopenia[95]. Therefore, improving the selectivity of epigenetic drugs and reducing damage to normal cells are urgent issues that need to be addressed.

Treatment target selection and precision: Although epigenetic therapies have shown good results in laboratory studies, the selection of treatment targets remains a clinical challenge. The epigenetic changes in PC are complex and diverse, with significant variability in epigenetic alterations among different patients. Therefore, developing precision treatment plans targeting specific epigenetic alterations will be a key focus of future research[105,106]. Additionally, targeting multiple epigenetic targets may be a way to increase treatment efficacy, but how to rationally combine different drugs remains a difficult research challenge.

Future research directions

Development of new epigenetic drugs: Future research should focus on the development of new epigenetic drugs, particularly novel demethylating drugs and HDAC inhibitors with increased selectivity and fewer side effects. Strategies to improve selectivity and reduce side effects include the design of small molecules that specifically target epigenetic enzymes with minimal off-target effects. Researchers can search for potential epigenetic regulatory molecules from natural products or develop new drugs through molecular design. For example, novel HDAC inhibitors such as trichostatin A have shown promising anticancer effects in preclinical trials and may become new treatment options in the future[107-109].

Combination therapy research: The combination of epigenetic therapy and immunotherapy is a key area for future research. Immune checkpoint inhibitors, such as PD-1 and CTLA-4 inhibitors, have become important tools for cancer treatment, but their efficacy remains limited for many patients with PC. Future studies could explore the combination of epigenetic drugs with immune checkpoint inhibitors, chemotherapy drugs, etc., to overcome tumor immune evasion mechanisms and enhance treatment efficacy. For example, the combination of demethylating drugs and immunotherapy has shown potential in enhancing immunotherapeutic effects in clinical trials[110,111]. In addition to PD-1 and CTLA-4, other factors in the TME, such as tumor-associated macrophages (TAMs), myeloid-derived suppressor cells, and regulatory T cells (Tregs), are potential targets for epigenetic-modified immunotherapy[112-114]. Current clinical trials (e.g., IPI-549, magrolimab, CAR-M) have shown preliminary efficacy, though further validation of safety and long-term outcomes is needed. Future directions involve multi-target combination therapies (e.g., TAM reprogramming combined with immune checkpoint blockade) and personalized epigenetic interventions to overcome immunosuppression and increase therapeutic efficacy[112].

Personalized epigenetic treatment plans: With the rapid development of precision medicine, future epigenetic treatments will focus more on individualized approaches. By analyzing a patient’s genomic and epigenetic characteristics, personalized treatment strategies can be designed for different patients. For example, identifying specific epigenetic biomarkers in PC patients through genomic sequencing can help clinicians select the most effective demethylating drugs or HDAC inhibitors[115-117].

Development of liquid biopsy technologies: Liquid biopsy, a noninvasive detection method, has garnered widespread attention in recent years. Future research could combine liquid biopsy technology with epigenetic methods to monitor epigenetic changes in tumors in real time, evaluate treatment effects, and monitor the onset of resistance. Epigenetic biomarkers in body fluids, such as blood and urine, can provide more precise guidance for the personalized treatment of PC and other tumors[118-121].

CONCLUSION

Recent advancements in the application of epigenetics in PC research have resulted in significant progress. PC is a highly malignant cancer that is difficult to diagnose early and is often diagnosed at advanced stages. Epigenetics provides new insights, offering potential interventions through the regulation of DNA methylation, histone modifications, and the expression of ncRNAs to influence tumor initiation, progression, and the treatment response. Epigenetic markers can serve as tools for the early diagnosis of PC and have also shown great potential in prognostic evaluation and immunotherapy.

Despite the progress made in epigenetic research on PC, many challenges remain to be overcome before clinical application. Drug resistance and related side effects remain urgent issues to be addressed.

Moreover, the epigenetic features of PC exhibit high heterogeneity, with significant differences in epigenetic changes among different patients. Therefore, the design of personalized treatment plans and development of precise epigenetic-targeted therapies are potential future research directions. Fully utilizing the potential of epigenetics while overcoming existing challenges is a key issue in the treatment of PC.

The deep integration of epigenetics and clinical medicine is a crucial direction for future PC treatment. Interdisciplinary collaboration will play a vital role in achieving this goal. These findings will accelerate the development of new epigenetic drugs and promote their clinical translation. Currently, the combined application of epigenetic therapy and immunotherapy has already shown tremendous potential in the treatment of PC. Immunotherapy, an emerging cancer treatment, has achieved some clinical success, but its efficacy remains limited for many patients with PC. By combining epigenetic regulation with immunotherapy, we can effectively improve the TME, increase immune cell activity, and thereby improve the effectiveness of immunotherapy.

In the future, with the development of precision medicine, epigenetics will be more widely applied in the personalized treatment of PC. Through comprehensive analysis of patients' genomic and epigenetic characteristics, more precise treatment plans can be formulated to improve treatment efficacy and reduce side effects. Additionally, the application of emerging technologies such as liquid biopsy will help in the real-time monitoring of patients' epigenetic changes, providing timely guidance for treatment adjustment by clinicians. In future research, clinical studies and the development of new therapeutic strategies will drive the widespread application of epigenetics in PC treatment, offering more effective treatment options for patients.