Epigenetic regulation of TGF-β pathway and its role in radiation response

Abstract

Purpose

Transforming growth factor (TGF-β) plays a dual role in tumor progression as well as a pivotal role in radiation response. TGF-β-related epigenetic regulations, including DNA methylation, histone modifications (including methylation, acetylation, phosphorylation, ubiquitination), chromatin remodeling and non-coding RNA regulation, have been found to affect the occurrence and development of tumors as well as their radiation response in multiple dimensions. Due to the significance of radiotherapy in tumor treatment and the essential roles of TGF-β signaling in radiation response, it is important to better understand the role of epigenetic regulation mechanisms mediated by TGF-β signaling pathways in radiation-induced targeted and non-targeted effects.

Conclusions

By revealing the epigenetic mechanism related to TGF-β-mediated radiation response, summarizing the existing relevant adjuvant strategies for radiotherapy based on TGF-β signaling, and discovering potential therapeutic targets, we hope to provide a new perspective for improving clinical treatment.

Keywords:

• Transforming growth factor (TGF-β)
• ionizing radiation
• methylation
• histone modification
• non-coding RNA

Introduction

As we all know, cancer is one of the diseases with high morbidity and mortality worldwide, and effective targeted tumor treatment has always been the goal of both researchers and clinicians (Najafi et al. 2021). In recent years, more and more studies have shown that epigenetic reprogramming dysfunction affects the development of many types of cancer (Greer and Yang 2014). Epigenetics mainly describes biochemical changes in DNA or chromatin, i.e. changing gene expression patterns without changing the DNA sequence. Most epigenetic changes are mediated by DNA methylation regulators, histone modifiers, and non-coding RNAs such as long non-coding RNAs (lncRNAs) and microRNAs (miRNAs)(Suriyamurthy et al. 2019). At present, epigenetics has been found to play a key role in autoimmune diseases, cancer, congenital diseases, mental retardation, endocrine diseases and neuropsychiatric diseases (Tzika et al. 2018; Zhang et al. 2020).

Transforming growth factor-β (TGF-β) is a key factor in cancer progression, and its role is mainly manifested in inhibiting tumor growth at an early stage by inducing apoptosis and cell cycle arrest. However, it promotes advanced-stage tumor progression by regulating genomic instability, epithelial-mesenchymal transition (EMT), angiogenesis, immune evasion, cell motility and metastasis. Disturbance of TGF-β/Smad signaling often leads to epigenetic variation in its target genes and the subsequent phenotypic changes. Radiotherapy is a conventional therapy, in which the change of TGF-β expression induced by ionizing radiation exposure plays an important role in tumor killing and normal tissue damage repair. Overexpression of TGF-β often leads to different side effects of radiotherapy, including vascular injury and fibrosis (Russell et al. 2015; Dadrich et al. 2016). Therefore, understanding the role of the epigenetic mechanism of the TGF-β pathway in the radiation response may contribute to effective targeted tumor therapy.

Therefore, in this review, we first focus on epigenetics-related advances in the TGF-β pathway, and then we underscore the epigenetic regulatory mechanisms of TGF-β in response to radiation. Finally, we summarize the existing adjuvant strategies for radiotherapy targeting the TGF-β signaling pathway and propose potential research directions for future application in clinical therapy.

A brief overview of the TGF-β pathway

The TGF-β superfamily includes TGF-β, bone morphogenetic protein (BMP), activin and related proteins, which regulate body function and tissue differentiation by affecting cell proliferation, differentiation and migration (Zhang 2009; Farhood et al. 2020). The TGF-β signal mainly includes three kinds of ligands: TGF-β1, TGF-β2 and TGF-β3, which have three kinds of receptors: the transmembrane receptor serine/threonine kinase TβRI, TβRII and β-glycan TβRIII (Ahmadi et al. 2019). The TGF-β signaling pathway mainly mediates tumor progression in two ways: canonical (Smad dependent) and non-canonical (Smad independent) pathways (Derynck and Zhang 2003).

Canonical pathway

The classical TGF-β pathway mainly involves the binding of dimer to the membrane receptor serine kinase. In short, TGF-β ligand binding triggers the assembly of TβRI and TβRII complexes (Vander Ark et al. 2018). In the complex, type II receptor subunit phosphorylates type I at multiple serine and threonine residues located at the N-terminal of the type I receptor kinase domain (Shi and Massagué 2003). Therefore, type I receptor kinase is activated and phosphorylated Smad2/3 (receptor Smads, R-Smads) is recruited for signal transduction (Macias et al. 2015). Smad4 (Common Smad, Co-Smad) forms a trimeric complex with phosphorylated R-Smads, which is then transferred to the nucleus to interact with DNA or transcription factors (Feng and Derynck 2005).

Smads is the downstream effector of TGF-β signal transduction. Both R-Smads and Co-Smad proteins consist of two highly conserved domains, Mad homology 1 (MH1) and Mad homology 2 (MH2). The C-terminal Ser-X-Ser (SXS) motif in the R-Smads MH2 domain is the phosphorylation site of TβRI, which can lead to its activation (Tzavlaki and Moustakas 2020). There is an L3 loop in the C-terminal MH2 domain of both R-Smads and Co-Smad, which is essential for the interaction of Smad-TβRs and the formation of trimer complexes (Lo Roger et al. 1998). At the same time, TGF-β often interacts with other pathways in a Smad-dependent manner. For example, the Smad3/Smad4 complex directly interacts with PKA regulatory subunits to activate PKA signaling pathway (L. Zhang et al. 2004); the interaction between Smad factor and RAS response element binding protein 1 (RREB1) also provides a molecular link between RAS and TGF-β signaling pathways (Su et al. 2020). In addition, inhibitory Smad (I-Smad), Smad7, negatively regulates TGF-β signal transduction through multiple mechanisms (de Ceuninck van Capelle et al. 2020). Smad7 protein can competitively bind R-Smads through the first α-helix (α1) and L3 loop in the C-terminal MH2 domain, interfering with the formation of trimer complex (Murayama et al. 2020). On the other hand, Smad7 promotes the binding to TβRI by interacting with E3 ubiquitin ligases such as Smurf1 and Smurf2 (Asano et al. 2004). Finally, Smad7 can act on the downstream of TβRs and reduce the activation of Smad2 and Smad3 to inhibit TGF-β signal transduction (Humeres et al. 2022).

Non-canonical pathways

In addition to the canonical pathway, TGF-β can also mobilize some other intracellular pathways (Zhang 2017). The non-canonical signaling pathway is mainly mediated by activation of phosphatidylinositol 3′-kinase (PI3K)-protein kinase B (AKT), nuclear factor kappa B (NF-kB), small G protein (Rac and Rho) and mitogen-activated protein kinase members (MAPK, including ERK, JNK and p38) (Cammareri et al. 2017; Yuan et al. 2020). Through phosphorylation or direct interaction, TGF-β receptors activate and regulate non-canonical signaling pathways, and finally lead to genetic regulation (Zhang 2009). For example, after activating kinase TAK1, the TGF-β receptor initiates NLRP3 inflammatory body-related signaling pathway in LX-2 cells by activating NF-kB (Kang et al. 2022); The binding of anti-inflammatory transcription factor PPARα to TAK1 can inhibit TGF-β signal transduction (Subramanian et al. 2018); TGF-β induces down-regulation of RhoE which belongs to the RhoA family to activate the signal transduction of RhoA/ROCK leading to EMT (Nishizuka et al. 2019).

The dual role of TGF-β in tumor

TGF-β plays a dual role in tumor progression. TGF-β acts as an inhibitor to induce growth arrest in various cell types, including epithelial cells, endothelial cells, hematopoietic cells, nerve cells and some mesenchymal cells (Maliekal et al. 2004; Zemann et al. 2010; Cammareri et al. 2017). The expression of cyclin D and CDK down-regulated the expression of TGF-β in the G1 phase (Zhang et al. 2009). The TGF-β/Smad signaling pathway is activated and mediates apoptosis through direct or indirect interaction with AKT, JNK and p39 (Pardali and Moustakas 2007; Massagué 2008). Some studies have also shown that reactive oxygen species (ROS) are closely related to TGF-β-induced apoptosis (Al-Khayal et al. 2017). In later stages, TGF-β continues to be produced by which EMT (Feng et al. 2022), cell metastasis (Yoon et al. 2021), angiogenesis(Chen et al. 2021), and immune escape (Huang et al. 2019) are induced through increasing neovascularization and extracellular matrix production, upregulating peritumoral proteases, or inhibiting immune surveillance mechanisms in cancer hosts to promote cancer progression (Mortezaee 2021).

Epigenetic regulatory mechanisms play an important role in tumor progression

Epigenetic aberration is a common feature of cancer, characterized by hypermethylation of specific promoter regions and global DNA hypomethylation, and/or either loss or gain of histone acetylation (Liang and Weisenberger 2017). Remodeling of chromatin is critical for nucleosome localization, chromatin accessibility to transcription factors, and regulation of gene expression. Silencing of tumor suppressor genes and activation of oncogenes are hallmarks of epigenetic variation.

Epigenetic modification is very complex and plays an important role in the occurrence and development of tumors by linking different genes or proteins. For example, palmitate environments in tumors promote the expression of lysine acetyltransferase 2a (KAT2a) and promote metastatic growth by increasing p65 acetylation to produce metastatic NF-κB signal transduction (Altea-Manzano et al. 2023). SWI/SNF complex causes metastasis and progression of lung cancer through SMARCA4 deletion (Concepcion et al. 2022). Lactate secreted by CAF (cancer associated fibroblast) also can regulate histone acetylation, up-regulate PLIN2, then establishing a metabolic-epigenetic regulatory loop and affecting the growth and metastasis of prostate cancer (Ippolito et al. 2022). On the other hand, H4K20me2 acetylation can directly regulate the relative chromatin occupancy of BRCA1 and 53BP1, thus affecting the mechanism of DNA repair (Tang et al. 2013). H3K27me3 mediates the up-regulation of TWIST1 and inhibition of SLFN11 in SCLC, which affects the efficiency of DNA damage repair and leads to chemotherapy resistance (Gardner et al. 2017). TGF-β is one of the most important targets for epigenetic regulation and plays a role in the modulation of cellular radiation response by participating in the epigenetic variation related to radiation response (Suriyamurthy et al. 2019).

The TGF-β pathway is an important target for epigenetic regulation

DNA methylation

As the most common epigenetic phenomenon, DNA methylation refers to the process by which cytosine of CpG double nucleotides is catalyzed by DNA methyltransferases (DNMTs) with S-adenosylmethionine (SAM) as the methyl donor and obtains methyl group chemical modification through covalent bonding. DNA methylation plays an important role in cancer progression by regulating the TGF-β signaling pathway. Zheng et al. found that RUNX3 and TGF-β levels were decreased in metastatic renal cancer tissues as a result of their CpG methylation (Zheng et al. 2018). Interestingly, database-based analysis of immune cells from breast cancer found that RUNX3 gene methylation was negatively correlated with TGF-β1 expression, while the situation was exactly the opposite for RUNX1(Gao and Zhou 2021). In conclusion, altering TGF-β1 expression through RUNX methylation can regulate the TGF-β signaling pathway to influence cancer progression. For non-small-cell lung carcinoma (NSCLC), -459CpG methylation inhibits RNF111 transcriptional expression by interfering with the recruitment of Sp1 to the RNF111 promoter, thereby inhibiting TGF-β/Smad signaling activation and Snail expression, increasing E-cadherin expression to prevent invasion (Chen et al. 2015). Other studies have also demonstrated that decreased TβRII expression in NSCLC is highly correlated with CpG methylation (Zhang et al. 2004; Pardali and Moustakas 2007). In the microenvironment of gastric cancer, high expression and hypomethylation of TGF-β2 can induce high levels of infiltration of multiple immune cells and expression of cytokines, which are positively correlated with EMT (Han et al. 2022). In addition, different N6-methyladenosine (m6A) reader proteins, YTHDF3 and IGF2BP1, were shown to associate with JUN and JUNB mRNA, respectively to mediate the m6A-dependent regulation of JUN protein translation and JUNB mRNA stability, thus promoting TGF-β-mediated EMT (Suphakhong et al. 2022). However, methyltransferase METTL3 deletion can down-regulate m6A and inhibit Snail expression, ultimately preventing TGF-β1-induced EMT (Lin et al. 2019; Li et al. 2023).

Histone modification

Different histone modifications, including methylation, acetylation, phosphorylation and ubiquitination, occur under the action of different histone modification enzymes. To some extent, histone modification leads to transcriptional activation or gene silencing, which regulates gene expression and even leads to tumorigenesis.

Methylation

Mutations or altered expression of histone methyl modifiers and methyl-binding proteins are associated with increased incidence of many cancers (Albert and Helin 2010). For example, H3K27me3 methyltransferase is upregulated in a number of cancers, including prostate cancer (Genao et al. 2002), breast cancer (Kleer et al. 2003) and lymphoma (Visser et al. 2001). Down-regulation of NSD2, which regulates the histone methyltransferase of histone3 lysine36(H3K36me2) dimethylation, can significantly reduce the expression levels of TGF-β1, TβRI, phosphorylated Smad2 and Smad3 in cervical cancer cells and inhibit tumor metastasis (Zhu et al. 2019). The significant down-regulation of H3K79me3 and the increased expression of axon guide proteins semaphorin 3 C (SEMA3C) and PD-L1 involved in immune system suppression are caused by the up-regulation of TGF-β1 in lung cancer (Evanno et al. 2017). Zhang et al also found that the H3K9/H3K27 double demethylase JHDM1D/KDM7A is critical for TGF-β-induced transcription of RHOJ in breast cancer cells (Zhang et al. 2021). Among them, KDM7A seems to be a direct transcriptional target of TGF-β signaling (Liu et al. 2019). The Smad2/Smad4 complex binds to the KDM7A promoter to mediate TGF-β-induced KDM7A transcription and regulate EMT and cell migration. Another histone methyltransferase, SUV39H1, regulates telomere length upon upregulation of TGF-β while affecting telomere-terminal histone methylation levels, ultimately leading to telomere variation that affects tumor progression (Mishra et al. 2022).

Acetylation

Histone acetyltransferase (HAT) and histone deacetylase (HDACS) antagonize each other to maintain a dynamic equilibrium of histone acetylation levels and participate in the regulation of gene expression (Grunstein 1997). p300/CBP is an important member of a group of acetyltransferases, whose abnormal expression often leads to a series of effects, such as inducing tumor cell proliferation and metastasis (Asaduzzaman et al. 2017; Boudreau et al. 2017). Reducing the protein levels of p300/CBP and p-Smad2/3 in the nucleus can hinder the proliferation and metastasis of esophageal carcinoma cells (Wang et al. 2020). Another HAT member, GCN5, also plays a key role in regulating EMT in breast cancer downstream of the TGF-β/Smad signaling pathway (Zhao et al. 2018). Among HDACs family members, the increase of HDAC1 activity is mediated by post-translational modification, and independently mediates the activation of TGF-β-Smad2/3 signaling and CAF status in breast cancer (Mezawa et al. 2023). HDAC1-mediated global histone deacetylation and acquisition of enhancers labeled with specific histone H3 lysine27 acetylation (H3K27AC) are critical for TGF-β-induced EMT (Qiao et al. 2020). HDAC4 silencing blocks TGF-β signal transduction and reduces the expression of Sox2 and Nanog to inhibit the acetylation of STAT1 and hinder tumor progression (Kaowinn et al. 2018). In NSCLC, TGF-β1 upregulates the expression of HDAC6, and HDAC6 inhibits the expression of H3K27ac on the transmembrane protein TMEM100 promoter by deacetylation, which leads to endothelial cell metastasis and activation of Wnt/β-catenin signaling pathway (Wang et al. 2022). In addition, lysine 102 in Smad7 promotes the migration and invasion of prostate cancer cells by binding to regulatory regions in c-Jun and HDAC6 to enhance the induction of c-Jun and HDAC6 by TGF-β(Thakur et al. 2020). H3K27AC simultaneously regulates carcinogenesis and alters the TGF-β3 and Wnt/β-catenin pathways, and its deacetylation has been shown to reverse and up-regulate tumor suppressor gene expression in uterine leiomyoma (UL)(Carbajo‐garcía et al. 2022).

Phosphorylation

Phosphorylation occurs on all histones and plays a different role on each histone, influencing tumor progression by regulating cell proliferation, differentiation and apoptosis. For example, in non-canonical pathways, Ras-PI3K pathway activation promotes osteosarcoma development by upregulating histone H2AT120 phosphorylation (Xu and Yu 2019), or by PKA regulating H1.4S35 phosphorylation (Zhang et al. 2019). In pancreatic cancer, activation of the Ras-MAPK pathway results in increased phosphorylation of histone H3 kinase, mitogen and stress-activated kinase (MSK1), and histone H3(H3S10) at serine 10 to regulate tumor transformation (Espino et al. 2009). Phosphorylation of H3S10 mediated by JNK and PI3K/AKT pathways may also promote lung carcinogenesis (Ibuki et al. 2014). However, the role of canonical pathway-mediated histone phosphorylation in tumor regulation seems to be unclear and needs to be further explored.

Ubiquitination

Ubiquitination refers to the covalent binding of ubiquitin to target proteins catalyzed by a series of enzymes, in which E3 ubiquitin ligase (E3s) plays an important role, and its regulation is related to cancer development (Senft et al. 2018; Sinha et al. 2021). According to the specific structural motif, E3s can be divided into E3s with HECT domain, RING domain, and U-box domain (Nakayama and Nakayama 2006). In the HECT domain E3s, Smad ubiquitin regulators 1 (Smurf1) and Smurf2 negatively regulate TGF-β/BMP signaling (Fu et al. 2019). Smurf2 promotes pancreatic cancer cell proliferation, migration and invasion through the TGF-β-induced non-canonical PI3K/AKT pathway (Sinha et al. 2021). RING domain E3s affect TGF-β signaling at different nodes. One of the family members of the RING, TRIM37, is upregulated in renal cell carcinoma (RCC), promoting EMT directly mediated by ubiquitination-H2A modification through activation of TGF-β1 signaling (Miao et al. 2021). Overexpression of another family member, RNF20, can significantly reverse TGF-β-induced activation of fibrotic proteins in hepatocytes (LX-2) and significantly inhibit the progression of liver fibrosis through ubiquitination of H2B (Chen et al. 2021). In addition, overexpression of KDM2B, a member of the polycomb repressive complex-1 (PRC1), specifically recognizes regulatory regions of Cdh1, miR-200a, and CGN genes and induces their regulated H2AK119 monoubiquitination as a component of the PRC1 complex, at the same time, enhances TGF-β-induced cell morphological transformation, thereby inhibiting the EMT, migration and invasion of lung and pancreatic cancer cells (Wanna-Udom et al. 2021). On the other hand, ubiquitin C-terminal hydrolase-L1 (Uch-L1) with deubiquitinating activity interacts with Smad2 and Smad3 to promote the signal transduction of DAF-7/TGF-β and promote the hypoxic survival rate of lung cancer cells (Nagata et al. 2020).

Chromatin remodeling

Chromatin remodeling is a dynamic process in which chromatin structures change between the condensed state and transcriptional accessible state. The SWI/SNF chromatin remodeling complex component, DPF3A, a short subtype of DPF3, specifically interacts with Smad nuclear-interacting protein (SNIP1) to form a complex with Smad4 and p300 HAT, inhibition of p300 HAT activity by the release of SNIP1 leads to increased local histone acetylation and activation of cell movement-related genes, promoting migration of kidney cancer cells (Cui et al. 2022). Recruitment of ARID1A, a subunit of SWI/SNF chromatin remodeling complex, can inhibit TβR2, KLF4 and FoxQ1 and induce BMP6, which hinders the morphological reprogramming of mammary epithelial cells (Jdeed et al. 2022). In breast cancer cells, the chromatin remodeling protein BRM binds directly to the promoter region of the Claudins gene by interacting with Sp1 and activates transcription by regulating histone modifications to inhibit TGF-β-induced migration and invasion (Yang, Liu, Fang, et al. 2019). In prostate cancer cells, the expression of chromatin remodeling proteins BRG1 and Elvol3 is upregulated with androgen and TGF-β responses. BRG1 interacts with and is recruited to the Elovl3 promoter to activate transcription by the retinoic acid receptor-associated orphan receptor, and it also interacts with p300 HAT to transactivate Elovl3 to promote migration and invasion (Yang, Liu, Li, et al. 2019). In lung cancer cells, BRG1 recruits histone H3K9 demethylase KDM3A, and after removing dimethyl H3K9 from the target gene promoter, interacts with Sp1 to activate potential TGF-β binding protein 2 (LTBP2) transcription to promote proliferation and migration (Li et al. 2019). Chromatin remodeling factor Pontin recruited LEF1 as a coactivator of LEF1 and upregulated TβR2 expression, activating TGF-β/Smad signaling to promote glioma formation (Zhou et al. 2022).

Non-coding RNA regulation

Non-coding RNA is a class of RNA that does not code proteins, in which lncRNA, miRNA, and siRNA play a role in regulating gene expression. Some studies on the epigenetic mechanisms of non-coding RNA-mediated TGF-β signaling pathways in tumor progression are shown in Table 1. LncRNA coordinates DNA methylation, chromatin structure remodeling, and histone modification in epigenetic regulation, and some studies have found that many lncRNAs are involved in the development and progression of tumors through the regulation of TGF-β signaling. Zhang et al. found that activation of the TGF-β/Smad pathway after binding of lncRNA PVT1 to TGF-β accelerates glioma proliferation, migration and invasion, while enrichment of p53 in the lncRNA PVT1 promoter region prevents its occurrence (Li et al. 2022). LncRNA ANCR is essential for TGF-β1-induced EMT. TGF-β1 downregulates ANCR expression by increasing the HDAC3 enrichment on the ANCR promoter region and reducing H3 and H4 acetylation of ANCR initiator genes. Ectopic expression of ANCR partially attenuates TGF-β1-induced EMT (Li et al. 2017). MiRNAs have also been found to be important in the regulation of TGF-β signaling. For example, TGF-β-induced miR-23a can target its downstream effector HMGN2, which alters chromatin structure by interfering with the binding of the linker histone H1 to nucleosomes to regulate transcription and induce EMT in lung cancer cells (Saito et al. 2013). MiR-132 targets TGF-β2 to enhance the expression of dexamethasone (DEX)-induced wave protein and E-cadherin, leading to reduced clonal formation and migration of pancreatic cancer cells (Abukiwan et al. 2019). TGF-β1 high expression decreases miR-200b/c while increasing miR-221, makes DNA methyltransferase DNMT3B stable and induces miR-200s promoter DNA methylation, thereby establishing and activating CAF and acting on malignant transformation of breast cancer (Tang et al. 2019).

Table 1. Non-coding RNA-mediated TGF-β signaling regulation in tumor progression.

Non-coding RNA	The process of action	Tumor	Influence	Reference
lncRNA PVT1	p53 suppresses lncRNA PVT1 and subsequently inactivates TGF-β/Smad pathway.	glioma	inhibits proliferation, migration and invasion	(Li et al. 2022)
lncRNA ANCR	TGF-β1 downregulates lncRNA ANCR to influence RUNX2 expression.	breast cancer	promotes EMT	(Li et al. 2017)
lncRNA GASL1	Downregulates TGF-β1 expression.	NSCLC	inhibits tumor growth	(Su and Yuan 2018)
lncRNA CASC7	Inhibits miR-26, ASPN and TGF-β, activates the Smad/XIAP pathway.	endometrial cancer	promotes invasion	(Zhang et al. 2022)
lncRNA ATB	Regulates miR-204-3p/TβR2 axis to change the expression of EMT markers.	ovarian cancer	promotes proliferation	(Yuan et al. 2022)
lncRNA MUF	TGF-β1 upregulates lncRNA MUF to promote the expression of Snail1	glioblastoma	promotes invasion	(Shree et al. 2022)
miR-21	TGF-β1 upregulates miR-21	NSCLC	promotes EMT	(Camerlingo et al. 2019)
miR-23a	Targets HMGN2 to upregulate TGF-β.	lung cancer	promotes EMT	(Saito et al. 2013)
miR-29b	Activates the TGF-β1 signaling pathway and p53-mediated apoptotic pathway.	hepatocarcinoma	inhibits proliferation and induces apoptosis	(Liu et al. 2021)
miR-34a	Targets Smad4 to downregulate TGF-β signaling.	nasopharyngeal carcinoma	inhibits EMT, migration and invasion	(Huang et al. 2018)
novel-miR56	TGF-β1/TβRII upregulates miR-56 to target PRAS40.	glioblastoma	promotes proliferation and inhibit autophagy	(Xie et al. 2020)
miR-132	Upregulates TGF-β2 expression.	pancreatic cancer	inhibits EMT and cancer progression	(Abukiwan et al. 2019)
miR-145	Downregulates TGF-β signaling.	pancreatic cancer	inhibits EMT and tumor growth	(Chen et al. 2020)
miR-182	Targets BRCA1 and FOXO3 to upregulate TGF-β signaling.	oropharyngeal cancer	promotes HR	(Liu et al. 2018; Farhood et al. 2020)
miR-195/miR-497	Targets Smurf2 to regulate TβR1 ubiquitination.	lung cancer	inhibits proliferation	(Chae et al. 2019)
miR-200b/c/miR-221	Upregulates TGF-β1 to activate CAF.	breast cancer	promotes malignant transformation	(Tang et al. 2019)

TGF-β-mediated epigenetic regulation involved in radiation response

External irradiation

At present, low LET radiation is the most widely used irradiation method in the field of radiotherapy. The effect of TGF-β on the radiation damage of the intestine, heart and kidney after low LET radiation has been widely studied (Haydont et al. 2007; Boerma et al. 2013; Hritzo et al. 2021). More importantly, TGF-β often causes a series of sequelae after radiotherapy. Excessive activation of TGF-β caused by ionizing radiation often causes EMT and cell migration, (Al-Assar et al. 2014; Carl et al. 2016; Qu et al. 2022). Fibrosis is another common side effect induced by TGF-β after radiotherapy (Saitoh 2022). In radiation-induced skin fibrosis, radiation induces CpG dinucleotide demethylation in exon 1 of the zinc transporter ZIP9, leading to recruitment of the transcription factor Sp1 and increased ZIP9 expression, then protein kinase B (PKB) can activate the TGF-β signaling pathway in human fibroblasts, and further promote fibrosis (Qiu et al. 2020). Ryu et al found that the natural dithiol compound, α-LA, synthesized in mitochondria, can decrease the transcriptional activity of NF-κB by inhibiting TGF-β1-mediated upregulation of p300/CBP activity and histone acetyltransferase activity, to prevent radiation-induced fibrosis (RIF)(Ryu et al. 2016). In addition, in terms of radiosensitivity, overexpression of m6A methylase WTAP can promote TGF-β expression and mRNA stability, while reducing radiosensitivity in gastric cancer (GC) tissues, to enhance chemoradiotherapy resistance and promote cell migration and EMT (Liu and Da 2022). Meanwhile, TGF-β also plays an important role in the phosphorylation of key enzymes in the DNA damage response (DDR) following radiation and inhibition of TGF-β reduces ATM (Kirshner et al. 2006; Wang et al. 2013), γH2AX, and p53 phosphorylation (Wiegman et al. 2007; Bouquet et al. 2011), thereby exerting distinct effects on various types of cancer. Some epigenetics studies of non-coding RNAs mediating TGF-β modulation in radiation response in tumor progression are shown in Table 2. Radiation-induced lncRNA RP11 overexpression promotes various common collagen expressions by downregulating miR-29a and modulating human fibroblast activity after TGF-β1 or radiation treatment (Yang et al. 2020). TGF-β/Smad signal transduction significantly decreased miR-29 transcription and enhanced the expression of type I collagen induced by ionizing radiation, suggesting that miR-29 may be an important regulator of RIF (Yano et al. 2018). The results of Lu et al found that miR-3591-5p/USP33/PPM1A plays a key role in radiation-induced EMT. Radiation-upregulated miR-3591-5p inhibited USP33 transcription and increased PPM1A (a Smad2/3 phosphatase) ubiquitination, ultimately inhibiting TGF-β signaling (Lu et al. 2018).

Table 2. Non-coding RNA-mediated TGF-β signaling regulation in radiation-induced targeted effects.

Non-coding RNA	Expression after exposure	Targets	Studied materials	Influence	Reference
lncRNA RP11	increased	Cooperates with TGF-β1 targeting miR-29a.	human lung fibroblasts cells HFL1 and WI-38	promotes RIFs	(Yang et al. 2020)
miR-21	increased	Targets Big-h3 to downregulate TGF-β signaling.	5-to-6-week-old male BALB/c mice	carcinogenesis	(Liu et al. 2011)
miR-21	increased	Upregulates TGF-β1 and Smad3 expression.	murine lung alveolar type II epithelial cells RLE-6TN	promotes RILI	(Wang et al. 2021)
miR-125a	increased	Downregulates TGF-β expression.	human NSCLC cells A549	mitigates pneumonia	(Quan et al. 2016)
miR-140	decreased	Increases α-SMA, Smad3, and fibronectin expression.	murine fibroblasts cells L-929	promotes RILF	(Duru et al. 2016)
miR-146a-5p	increased	Downregulates α-SMA and collagen 1 expression.	human HSC cells LX2	inhibits RIFs	(Yuan et al. 2019)
miR-193a	increased	Targets E2F6/c-Myc and ARHGEF15/ABL2 to downregulate TGF-β2/TβRIII signaling.	human pancreatic cancer cells SW1990 and PANC-1	accelerates proliferation and metastasis	(Fang et al. 2018)
miR-199a-3p	–	Targets AK4 to downregulate Smad4 expression.	human esophageal cancer cells Kyse30 and Kyse150	promotes radiation resistance	(Zang et al. 2018)
miR-663a	decreased	Upregulates TGF-β1 expression.	human NSCLC cells A549 and NCI-H1299, human SCLC cells NCI-H446, and human bronchial epithelium cells Beas-2B	promotes EMT	(Qu et al. 2022)
miR-3591-5p	increased	Targets USP33/PPM1A to upregulate TGF-β signaling	human NSCLC cells A549	enhances EMT	(Lu et al. 2018)

In recent years, with the extensive development of proton and heavy ions radiotherapy, different TGF-β expression and its epigenetic effects caused by different types of high LET radiation are paid more and more attention (Table 3). In the comparison of carbon ions with X-rays, Ran et al found that at the same dose, carbon ions induced higher expression of HMGB1 and decreased the expression of immunosuppressive factors IL-6 and TGF-β, so as to better induce immune effect (Ran et al. 2021). In another comparative study of iron and boron ions, the authors found that γ-H2AX foci and Smad7 foci were detected in radiation-induced micronuclei, suggesting that Smad7 is related to DNA repair and genomic instability induced by high LET particles (Wang et al. 2013). In addition, after comparing fractionated proton radiation with acute proton radiation, it was found that acute proton irradiation up-regulated β-catenin and AKT pathway by increasing proliferation marker phosphate histones, reducing DNA damage repair factors and resulting in an increased risk of cancer (Suman et al. 2019). However, due to the complexity of influencing factors, such as LET, radiation dose and dose rate, the epigenetic mechanism correlated with TGF-β induced by different radiation types still needs to be further studied in external irradiation.

Table 3. Effects of different radiation types on epigenetic mechanism regulated by TGF-β.

Type of radiation	Parameter	Dose	Studied materials	Influence	Reference
Carbon	Energy: 80 MeV/u LET: 50 keV/μm	2Gy	human NSCLC cells A549 and NCI-H1299, human SCLC cells NCI-H446, and human bronchial epithelium cells Beas-2B	Decreases miR-663 and increases TGF-β1 to promote EMT	(Qu et al. 2022)
Carbon;X-rays	Energy: 80 MeV/u;100keV LET: 30 keV/μm	2-6Gy (2Gy/min;1Gy/min)	human LUAD cells A549, LUSC cells NCI-H520 and mouse Lewis lung cancer cells LLC	Promotes HMGB1, IL-10, and TGF-β in a time-dependent manner to induce immune effects	(Ran et al. 2021)
Proton	Energy: 100 MeV/n	1.88Gy/(0.47Gy x 4d)	6-to-8-week-old female Apc^Min/+ mice	Upregulates β-catenin and Akt pathways with increased phospho-histone H3 to increase DNA damage	(Suman et al. 2019)
Iron; Boron	Energy: 600 MeV/u;5.6MeV/u LET: 180 keV/μm; 200 keV/μm	1Gy(0.5Gy/min;1Gy/min)	human skin fibroblast cells 82-6, human prostate cancer cells PC3 and human esophageal epithelial cells	Compared to Fe particles, the frequency of MN carrying γH2AX and Smad7 foci induced by B particles was lower	(Wang, Saha, and Cucinotta 2013)
Alpha	Source:^241Am	1.36-54.4cGy(1.36cGy/min)	human NSCLC cells A549, human breast cancer cells MCF-7, mucoepidermoid pulmonary carcinoma NCI-H292, human normal lung cell fibroblasts WI26VA4, and human normal liver cells WRL68	Low dose of alpha (1.36 cGy) particles increases TGF-β1, phosphorylation of connexin 43 and survivin but decreases caveolin-1 to promote tumor growth	(Rajan and Pandey 2021)
Gamma-rays	Source:^60Co	8Gy(3.64Gy/min)	human NSCLC cells A549	Increases miR3591-5p to target USP33/PPM1A, and upregulates Smad2/3 to enhance EMT	(Lu et al. 2018)
Gamma-rays	Source:^137Cs	2.5-10Gy	human bladder carcinoma cells T24 and UMUC-3	HDAC6 knockdown decreases CXCL1 and TGF-β2 to repress invasion and migration	(Tsai et al. 2023)
Gamma-rays	Source:^137Cs	2.6Gy/min	human breast cancer cells MCF7, T47D	Histone methyltransferase G9a deletion induces Smad protein phosphorylation by increasing the expression of BMP5, which ultimately improves radiation response	(Jin et al. 2022)

Internal irradiation

In recent years, targeted radionuclide therapy (TRT) provides a new direction for tumor therapy. Short-distance and high-energy radiation promotes double-strand DNA breaks, leading to cell death and reducing toxicity to surrounding non-cancerous tissues. In addition, when the 225Ac-labeled minigastrin analog 225Ac-PP-F11 was used for targeted alpha therapy (TAT), it was found that it promoted both MAPK pathway phosphorylation and HDAC phosphorylation, and HDAC inhibitors combined with it could enhance radiosensitivity (Qin et al. 2023). However, another study using 225Ac found that tubulointerstitial damage induced by α particles, resulting in up-regulated expression of TGF-β, could lead to renal parenchyma damage and loss of renal function (Jaggi et al. 2005). It can be seen that the damage of surrounding normal tissue caused by TAT treatment is still the focus of our continuous attention. In addition, it has been shown that the combination of PI3K inhibitor and apigenin increases the radiosensitivity of radioactive iodine in the treatment of thyroid cancer, suggesting that TGF-β may affect the therapeutic effect of radioactive iodine on thyroid cancer through PI3K (Lakshmanan et al. 2015). Moreover, the combination of external irradiation and Smad responsive promoter (SMAD-NIS-MSCs)-mediated I-131 therapy induced by TGF-β1 could significantly improve the tumor growth delay and survival time of treated mice, which proved that TGF-β plays an important role in internal irradiation therapy (Schug et al. 2019). Although there are still some problems in TRT, for example, the short-range emission of alpha particles can limit the cytotoxic effects on cancerous lesions and the surrounding tumor microenvironment, and it may also damage the surrounding normal tissue, however, this emerging treatment still has great potential and exploration space (Parker et al. 2018).

Non-targeted effects

Cells are also affected in a non-targeted manner, known as radiation-induced bystander effects (RIBE), in which irradiated cells release a variety of factors to induce changes in non-irradiated cells that increase radiation toxicity (Bryant et al. 2019; Khodamoradi et al. 2020). TGF-β signaling can be released from targeted cells and freely diffused in the media, acting in concert with other factors on unirradiated cells, causing micronucleus formation and accelerating cell metastasis (Shao et al. 2008; Gu et al. 2022). TGF-β plays a key role in the DDR of bystander cells. TGF-β signal significantly up-regulated γ-H2AX foci formation in glioma bystander cells induced by RAD3-related (ATR)-dependent radiation (Burdak-Rothkamm et al. 2007). In addition, in RIBE, TGF-β signaling is also associated with a variety of non-coding RNAs (Table 4). Hu et al used lncRNA microarray to determine the lncRNA and mRNA expression ­patterns in bystander cells and found that the differentially expressed lncRNAs were closely related to cell proliferation, transformation and migration. Knockdown of lnc-ABCA12-5, lnc-THEMIS-2 or lnc-HPN-AS1 inhibited TGF-β1-induced EMT to different degrees (Hu et al. 2018). Some studies have shown that TGF-β induces oxidative stress by upregulating miR-21. Evaluation of bystander mechanisms showed that miR-21 inhibits superoxide dismutase 2 (SOD2) activity and improves the defense ratio of superoxide to antioxidants (Tian et al. 2015). Further studies revealed that TGF-β is the most important miR-21 stimulator in bystander cells (Xu et al. 2014), and high expression of TGF-β receptors appears to inhibit SOD2 and increase ROS levels through the TGF-β-miR-21-ROS pathway (Jiang et al. 2014). Compared with miR-21, miR-663 inhibits TGF-β to reduce DNA damage and micronuclei formation in bystander cells. Interestingly, it has been shown that TGF-β1 downregulation in bystander cells is due to miR-663 induction after cell exposure to TGF-β1 released by irradiated cells (Hu et al. 2014). At the same time, other studies have also found that the unique miRNA content in senescence-associated exosomes (SA EXO) mediates the TGF-β pathway to play an important role in the bystander effects of radiation-induced senescence (Lee et al. 2023).

Table 4. Non-coding RNA-mediated TGF-β signaling regulation in radiation-induced non-targeted effects.

Non-coding RNA	Expression in targeted/non-targeted cells	The process of action	Studied materials	Influence	Reference
miR-21	--/increased (1h), decreased (18h)	The activated TGF-β1 pathway regulates miR-21 to cause oxidative stress (1h) and induce cell cycle delay (18h).	human embryonic lung fibroblast cells MRC-5	mitigates RIBE	(Jiang et al. 2014)
miR-495	decreased/--	MiR-495 targets Sp1 indirectly to down-regulate eNOS and modulate TGF-β1.	human hepatocellular carcinoma cells HepG2, hepatic cells LO2, breast cancer cells ZR75-1 and embryonic kidney cells HEK293T	mitigates RIBE	(Fu et al. 2016)
miR-663	decreased/increased	The induction of TGF-β1 by reduced miR-663 in irradiated cells leads to increased level of miR-663 in bystander cells which suppresses TGF-β1.	human cervical cancer cells HeLa	mitigates RIBE	(Hu et al. 2014)

Some promising treatment strategies for clinical use

The existing preclinical studies on adjuvant drugs for radiotherapy

Radiation oncology is a conventional treatment that aims to maximize the protection of normal tissues while effectively killing tumors. The use of many inhibitors targeting the TGF-β signaling pathway has proven viable. The use of TGF-β1 receptor kinase inhibitors before irradiation reduced DNA damage response, H2AX and p53 phosphorylation, and induction of self-renewal signals Notch1 and CXCR4 to improve tumor treatment response (Hardee et al. 2012). TGF-β neutralizing antibodies improved the radiosensitivity of breast tumors, blocked γH2AX lesion formation and led to delayed tumor growth (Bouquet et al. 2011). Administration of neutralizing pan-TGF-β antibodies abrogated TGF-β1 upregulation and lung metastasis enhancement due to ionizing radiation or doxorubicin administration (Biswas et al. 2007). These results suggest that inhibition of TGF-β is an effective adjunct to radiotherapy for cancer.

More and more studies have shown that targeting epigenetic elements related to TGF-β signaling such as DNA methylation and histone modification is a new direction for tumor treatment (Zheng et al. 2018). The DNA methyltransferase inhibitor RG108 increases the radiosensitivity of EC cells, significantly enhancing X-ray-induced apoptosis and G2/M phase arrest. RNA-seq analysis showed that RG108 combined irradiation altered the expression of 121 genes in multiple pathways, including the TGF-β signaling pathway and Epstein-Barr virus infection pathway (Ou et al. 2018). Decitabine, a demethylating agent that has been clinically used to treat several cancers, greatly weakens the transcriptional response of hepatoma cells carcinoma (HCCs) to TGF-β and has been shown to induce expression of EMT-related transcription factors (e.g. Snail/2, Zeb1/2). The promoter of Snail is hypomethylated in human HCC with poor prognosis, i.e. associated with high Snail levels, high AFP levels, metastasis, and relapse. Therefore, epilogic medications for HCC should be carefully evaluated as they may activate tumor-promoting pathways (Bévant et al. 2021). The radiation protection agent MnTE-2-PyP has been shown to inhibit prostate cancer growth, migration and invasion, and inhibit the expression of three genes regulated by HIF-1β and CREB by altering p300DNA binding: TGF-β2, FGF-1 and PAI-1. Among them, MnTE-2-PyP reduced the binding of the p300 complex to a specific HRE motif in the promoter region of the PAI-1 gene, inhibited H3K9 acetylation, and thus inhibited the expression of PAI-1(Tong et al. 2016). However, since known epigenetic changes also regulate some other targets besides TGF-β, we need to consider many aspects when selecting targeted epigenetic inhibitors.

The achievements and challenges of clinical trials

In recent years, some clinical trials on targeting TGF-β have been carried out one after another. Oral administration of small molecular TβRI inhibitors has shown a good effect on tumors in current clinical trials. For example, LY3200882 alone or in combination with PD-L1 inhibitors or gemcitabine was well tolerated in Phase I clinical trials (NCT02937272), and its antineoplastic activity in cancer was initially observed (Yap et al. 2021). The combination of galuniserib and gemcitabine improved the overall survival rate of unresectable pancreatic cancer with less side effects (Melisi et al. 2018). However, there was no significant advantage in clinical efficacy and safety of galuniserib combined with radiotherapy and chemotherapy in malignant gliomas(NCT01220271) (Wick et al. 2020). In view of the availability of galuniserib, trials to evaluate galuniserib combined with durvalumab in the treatment of cancer are still ongoing (NCT02734160). In another prospective randomized trial of TGF-β blockers during radiotherapy, fresolimumab has been shown to promote a good systemic immune response and improve median overall survival in patients with metastatic breast cancer (NCT01401062) (Formenti et al. 2018). In addition, phase I clinical trials related to the bifunctional fusion protein formed by the fusion of monoclonal antibody against programmed death ligand 1 (PD-L1) and TGF-β have also proved that it can alleviate advanced solid tumors (NCT04551950, NCT02517398) (Strauss et al. 2018; Oaknin et al. 2024). Recently, in a clinical trial using TGF-β resistant chimeric receptor (CAR) T cells to target prostate specific membrane antigen (PSMA), cells were engineered with overexpression-dominant-negative TβRII (TGFβRDN) optimization to block TGF-β signaling (NCT03089203). The feasibility and safety of its clinical application were proved in 18 selected patients (Narayan et al. 2022). Unfortunately, there seems to be no clear conclusion about drugs targeting epigenetics, such as methylation inhibitors or acetylation inhibitors, in clinical trials that act on TGF-β.

Although, inhibition of TGF-β and its downstream targets has become an ideal treatment strategy. However, most clinical trials of TGF-β targeting remain at Phase I, the pharmacological blockade of TGF-β has not been translated into a successful treatment for humans. This may be due to the pleiotropic effect of TGF-β (Massagué and Sheppard 2023). Previous studies have shown that early life exposure to p40 enhances H3K4me1/3 by up-regulating the expression of methyltransferase setd1β, which leads to persistent production of TGF-β by intestinal epithelial cells, expands Tregs and protects the intestine from inflammation (Deng et al. 2021). Similarly, TGF-β plays a unique and critical role in pulmonary epithelial and interstitial cells (Saito et al. 2018). The absence of TβRII in mesenchyme destroys the morphogenesis of pulmonary branches by regulating Shh signal transduction, resulting in bronchial cystic malformation (Li et al. 2008). TGF-β1 deficient mice also showed systemic inflammation and systemic perivasculitis or interstitial pneumonia in the lungs (Shull et al. 1992). In addition, mice lacking the expression of TGF-β2 and TGF-β3 also developed defects in the central nervous system (Vogel et al. 2010). In addition, preclinical studies of genetic mouse models have shown that the signaling activity of TGF-β is essential for normal skin wound healing (Finnson et al. 2013; Kiritsi and Nyström 2018). On the other hand, TGF-β is essential for regulating the function of embryonic stem cells (David and Massagué 2018). For example, TGF-β activates Smads to promote embryonic stem cell differentiation (Mullen et al. 2011); in pluripotent cells, Activin A/Smad2/3 is regulated by PI3K/Akt and is used to maintain self-renewal by activating the target gene (Nanog) (Singh et al. 2012). The research on the protective effect of TGF-β on different organs and tissues is an aspect that cannot be ignored. So, we need to consider more carefully when formulating therapeutic strategies targeting TGF-β and its downstream targets in the future.

The future directions of drug development and treatment strategy

A study comparing changes in circulating miRNA profiles after total body irradiation (TBI) and total thoracic lung irradiation (WTLI) found that in TBI, ubiquitination-related pathways, especially histone H2A, were enriched, indicating that it had a greater impact on DNA damage repair mechanisms and apoptosis. However, in WTLI, profibrotic pathways including TGF-β and BMP signaling pathways were enriched. These results suggest that miRNAs can predict the occurrence of different types of radiation-induced damage (Rogers et al. 2021). The use of miRNA in clinical treatment can include many directions. First, before patients receive radiotherapy, the expression profile of radiation-related miRNAs in serum is evaluated to (i) predict each patient’s radiological response, (ii) determine individualized radiation doses to optimize treatment outcomes, and (iii) minimize acute and potential damage to normal tissues. Secondly, during radiotherapy, checking the expression profiles of radio-specific and dominant miRNAs in serum and changing the specific miRNA expression can help improve tumor radiosensitivity. Radiotherapy can be used in combination with other chemotherapy drugs, small molecule inhibitors and drugs that target specific miRNAs to activate or inhibit the expression of certain miRNAs and downstream target genes (Khalighfard et al. 2022). Finally, when radiotherapy is completed, regular testing of the expression of prognostic miRNAs in serum can help monitor the effectiveness of radiotherapy and reduce the risk of metastasis and cancer recurrence (Zhao et al. 2012).

Biomarkers have always played an important role in tumor diagnosis, treatment and prevention. Identification of predictive biomarkers that help identify patients susceptible to radiation-induced lung injury (RILI) can help administer the appropriate IR dose to the patient (Jin et al. 2020; Li et al. 2023). DJ-1 is a potential therapeutic target for the reversal of RIBE in esophageal squamous cell carcinoma and a useful biomarker for predicting the efficacy of radiotherapy (Gu et al. 2022). Altered DNA methylation is an interesting biomarker in cancer. The newly identified CRC metastasis biomarkers, including HLTF (Liu et al. 2018), IGFBP-3 (Cai et al. 2020), and INHBB, are highly dysregulated by methylation and strongly associated with metastasis (Yuan et al. 2020). Overexpression of WTAP in gastric cancer is strongly associated with TGF-β-induced EMT and can serve as a potential predictive biomarker for gastric cancer (Liu and Da 2022). Identifying such biomarkers in all types of cancer facilitates earlier identification and more effective treatment for better diagnosis and increased life expectancy (Gutierrez et al. 2021).

Conclusion

TGF-β, as a key factor, plays a dual role in the development of tumors and regulates the expression of upstream or downstream factors by acting on DNA methylation, histone modification, chromatin remodeling and non-coding RNA regulation, thereby promoting or inhibiting tumorigenesis and development. Meanwhile, the components of TGF-β signaling are also regulated by different epigenetic mechanisms, which exert a more refined regulation mode on this vital signaling pathway. During radiotherapy, due to the complexity of the tumor microenvironment, different histone modification sites and different non-coding RNAs involved in the epigenetic regulation of TGF-β signaling may have multiple roles in the modulation of tumor development. It has been demonstrated in several reports that radiation-induced hypomethylation or upregulation/downregulation of the expression of certain miRNAs can enhance tumor radiosensitivity by affecting TGF-β signaling, thereby killing tumor cells more efficiently (Zang et al. 2018; Liu and Da 2022). Several TGF-β inhibitors and demethylating agents have been developed for radiation sensitization (Yang et al. 2019; Bévant et al. 2021). However, understanding and identifying more methylated or non-coding RNA targets will facilitate the development of more effective targeted drugs and the identification of corresponding predictive biomarkers, which will be of great assistance to the development of adjunctive strategies for radiotherapy.

On the other hand, with the in-depth exploration of non-targeted effects, the radiation damage effects on the normal tissues are also the focus of attention. The effect of TGF-β secreted by irradiated cells on bystander cells to cause damage has been established (Shao et al. 2008; Zhang et al. 2019). Some radioprotective agents have been developed to reduce RIBE damage, for example, the DNA-binding methylproamine has been shown to repair oxidized DNA damage in RIBE (Burdak-Rothkamm et al. 2014). However, the specific mechanism of TGF-β-mediated epigenetic regulation in RIBE has not been fully elucidated. Therefore, it is of great significance for the protection of normal tissues to continue to mine the effective targets of methylation or non-coding RNAs in RIBE.

In conclusion, understanding the mechanism of TGF-β-mediated epigenetic regulation in radiation response, as well as exploring key upstream and downstream factors involved in the epigenetic regulation of TGF-β signaling will be extremely beneficial for the clinical application of both radiotherapy and radiation protection.

Acknowledgements

The authors thank the two reviewers for providing helpful comments and suggestions to improve this manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was funded by the National Natural Science Foundation of China under Grant No. 32071243, 82192882, 82192883 and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Notes on contributors

Yunan Ding

Yunan Ding, PG, is a postgraduate student in the School of Radiation Medicine and Protection at Soochow University.

Guangming Zhou

Guangming Zhou, PhD, is a professor in the School of Radiation Medicine and Protection at Soochow University. His interests include space radiation health risk assessment and protection, molecular mechanisms of radiation carcinogenesis and basic biomedical research related to particle radiation therapy for tumors.

Wentao Hu

Wentao Hu, PhD, is an associate professor in the School of Radiation Medicine and Protection at Soochow University. His interests include the molecular mechanisms of tumorigenesis caused by space radiation, non-coding RNA and radiobiological effects and the composite biological effects of multiple environmental factors in space.