Medicine, Faculty of
Other UBC
Urologic Sciences, Department of
DSpace
Vancouver Prostate Centre
BC Cancer Research Centre. Department of Experimental Therapeutics
Ni, Yuchao
Shi, Mingchen
Liu, Liangliang
Lin, Dong
Zeng, Hao
Ong, Christopher J.
Wang, Yuzhuo, Ph.D.
G9a, also named EHMT2, is a histone 3 lysine 9 (H3K9) methyltransferase responsible for catalyzing H3K9 mono- and dimethylation (H3K9me1 and H3K9me2). G9a contributes to various aspects of embryonic development and tissue differentiation through epigenetic regulation. Furthermore, the aberrant expression of G9a is frequently observed in various tumors, particularly in prostate cancer, where it contributes to cancer pathogenesis and progression. This review highlights the critical role of G9a in multiple cancer-related processes, such as epigenetic dysregulation, tumor suppressor gene silencing, cancer lineage plasticity, hypoxia adaption, and cancer progression. Despite the increased research on G9a in prostate cancer, there are still significant gaps, particularly in understanding its interactions within the tumor microenvironment and its broader epigenetic effects. Furthermore, this review discusses the recent advancements in G9a inhibitors, including the development of dual-target inhibitors that target G9a along with other epigenetic factors such as EZH2 and HDAC. It aims to bring together the existing knowledge, identify gaps in the current research, and suggest future directions for research and treatment strategies.
Citation: Ni, Y.; Shi, M.; Liu, L.; Lin,D.; Zeng, H.; Ong, C.; Wang, Y. G9a inCancer: Mechanisms, TherapeuticAdvancements, and ClinicalImplications. Cancers 2024, 16, 2175.https://doi.org/10.3390/cancers16122175Academic Editor: Craig N RobsonReceived: 19 April 2024Revised: 5 June 2024Accepted: 6 June 2024Published: 8 June 2024Copyright: © 2024 by the authors.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).cancersReviewG9a in Cancer: Mechanisms, Therapeutic Advancements, andClinical ImplicationsYuchao Ni 1,2,3, Mingchen Shi 2,3, Liangliang Liu 2,3, Dong Lin 2,3 , Hao Zeng 1,*, Christopher Ong 2,3,*and Yuzhuo Wang 2,3,41 Department of Urology, West China Hospital, Sichuan University, Chengdu 610041, China;yni@prostatecentre.com2 Vancouver Prostate Centre, Vancouver, BC V6H 3Z6, Canada; ashi@prostatecentre.com (M.S.);lliu@prostatecentre.com (L.L.); dlin@prostatecentre.com (D.L.); ywang@bccrc.ca (Y.W.)3 Department of Urologic Sciences, University of British Columbia, Vancouver, BC V5Z 1M9, Canada4 Department of Experimental Therapeutics, BC Cancer, Vancouver, BC V5Z 1L3, Canada* Correspondence: kucaizeng@163.com (H.Z.); chris.ong@ubc.ca (C.O.)Simple Summary: In this review, we have summarized the crucial role of G9a in cellular biologyand disease. By synthesizing recent research discoveries, we delve into the functions of G9a ingene expression regulation, cell proliferation, and biological development. Additionally, we exploreits potential involvement in cancer and discuss its prospects as a therapeutic target specifically inprostate cancer. In summary, this review provides a comprehensive perspective on G9a, enhancesunderstanding of the significance of G9a in biology and cancer, and offers insights into future researchand drug development.Abstract: G9a, also named EHMT2, is a histone 3 lysine 9 (H3K9) methyltransferase responsible forcatalyzing H3K9 mono- and dimethylation (H3K9me1 and H3K9me2). G9a contributes to variousaspects of embryonic development and tissue differentiation through epigenetic regulation. Fur-thermore, the aberrant expression of G9a is frequently observed in various tumors, particularly inprostate cancer, where it contributes to cancer pathogenesis and progression. This review highlightsthe critical role of G9a in multiple cancer-related processes, such as epigenetic dysregulation, tumorsuppressor gene silencing, cancer lineage plasticity, hypoxia adaption, and cancer progression. De-spite the increased research on G9a in prostate cancer, there are still significant gaps, particularlyin understanding its interactions within the tumor microenvironment and its broader epigeneticeffects. Furthermore, this review discusses the recent advancements in G9a inhibitors, includingthe development of dual-target inhibitors that target G9a along with other epigenetic factors suchas EZH2 and HDAC. It aims to bring together the existing knowledge, identify gaps in the currentresearch, and suggest future directions for research and treatment strategies.Keywords: G9a; EHMT2; histone methyltransferase; prostate cancer; epigenetic inhibitor1. IntroductionEpigenetic modifications represent the regulation of gene expression without alteringthe DNA sequence. They contribute to various biological processes and disease states,including cancer [1]. Aberrant epigenetic reprogramming associated with profound tran-scriptional changes has been consistently identified across cancer types, correlated withtumor progression and poor prognosis [2–4]. Among the known epigenetic alterations, his-tone post-translational modifications emerge as crucial players in orchestrating chromatinstatus and its corresponding transcriptional networks [5].In particular, histone methylation has gained extensive attention as the subject ofa thorough investigation. Its significance has been revealed in multiple biological andCancers 2024, 16, 2175. https://doi.org/10.3390/cancers16122175 https://www.mdpi.com/journal/cancersCancers 2024, 16, 2175 2 of 17molecular processes, including cell cycle regulation, gene expression regulation, and tu-morigenesis [6,7]. Histone methylation is precisely balanced by the function of histonemethyltransferases (HMTs, writers) and demethylases (erasers) [8]. Notably, among theseregulatory players, G9a, an HMT catalyzing mono- and dimethylation at histone 3 lysine 9(H3K9me1 and H3K9me2), has been evidenced to hold a pivotal role in carcinogenesis andaggressiveness across cancer types [9,10].Despite recent reviews on G9a primarily focusing on its structure and function, aswell as its relationship with stemness, drug resistance, and roles in pediatric and adultbrain tumors, significant progress has also been made in studying G9a in the context ofprostate cancer and the tumor microenvironment [11–13]. Herein, in this review, we offercomprehensive insights into the roles of G9a in biology and the current research landscapethat highlights the involvement of G9a in tumorigenesis, progression, and cancer stemnessin different types of cancer. We place particular emphasis on the recent findings in the fieldof prostate cancer. Furthermore, we aim to conclude the information on G9a inhibitors,pinpoint the existing gaps in the prostate cancer research field, and shed light on futureresearch directions.2. Molecular Structure and Function of G9a in Epigenetic ModificationG9a, alternatively known as KTM1C, EHMT2, or BAT8, was first identified by Milnerin 1993 [14]. As a nuclear lysine methyltransferase, G9a belongs to the SET HMT family. Itexists in the form of a homodimer or a heterodimer with another HMT G9a-like protein(GLP) [15]. G9a, together with GLP, forms a dimer complex that is crucial for catalyzinghistone markers H3K9me1 and H3K9me2. Notably, overexpression of G9a, and not ofits related protein GLP, has often been associated with a more aggressive phenotypein cancer [16]. H3K9 methylation is dynamically regulated by various enzymes and isinvolved in multiple biological processes, including development, differentiation, andresponse to environmental signals. This modification plays a critical role in regulatinggene expression by contributing to the formation of heterochromatin and the repressionof specific genomic regions [10]. Since H3K9 methylation primarily contributes to genesilencing, G9a was generally regarded as an epigenetic repressor [17]. However, certainstudies have reported that G9a could generate the trimethylation of H3K9 (H3K9me3) inlong-time incubation in vitro [18,19]. Given that the blocking effect of the hydroxyl group atY1067 in the SET domain of G9a hinders the alignment of the dimethylamine in H3K9me2and prevents the formation of H3K9me3, G9a is considered as the major H3K9me1 andH3K9me2 methyltransferase [20,21].Structurally, G9a comprises several domains critical for its function: an activationdomain, a glutamate-rich region, nuclear localization signals, a cysteine-rich region, sevenankyrin repeats, and a SET domain responsible for its methyltransferase activity from the N-terminus to the C-terminus (Figure 1). The SET domain, named after Su(Var)3-9, Enhancer-of-zeste, and Trithorax, is a common shared structural feature among all six members of theH3K9 methyltransferase family [10]. The SET domain contains an evolutionarily conservedsequence of 130 to 140 amino acids and contributes to the catalytic activity of G9a [22]. Wuet al. reported the indispensability of the tyrosine residue Y1067 for the catalytic functionof G9a as an HMT [21]. Similar to that of other H3K9 methyltransferases, the SET domainwithin G9a also encompasses pre-SET and post-SET domains [10]. The pre-SET domainfunctions as a structural stabilizer that establishes multiple interactions with the surfaces ofcore-SET domains; the post-SET domain facilitates the formation of hydrophobic channels.A total of 6 contiguous ankyrin repeats, each consisting of 33 amino acids, form amodule in G9a that is capable of binding mono- and dimethyl lysine, thereby mediatingprotein–protein interactions [23,24]. The specificity of the ankyrin repeat domain of G9a iscomparable to that observed in other H3K9 methylation binding protein modules, includingthe chromodomain (of SUV3H1/H2) and the tudor domain (of SETDB1, MET-2), whichcould specifically bind lysines methylated at histone tail residues [25–27]. This domainCancers 2024, 16, 2175 3 of 17could operate as a scaffold, facilitating the recruitment of other corepressors through therecognition of H3K9 methylation markers [23].Cancers 2024, 16, x FOR PEER REVIEW 3 of 18 domain could operate as a scaffold, facilitating the recruitment of other corepressors through the recognition of H3K9 methylation markers [23]. Figure 1. The schematic domains of G9a. Glu: glutamate; NLS: nuclear localization signals; Cys: cysteine. H3K9: histone 3 lysine 9. 3. Embryonic Development and Tissue Differentiation Embryonic development and tissue differentiation are intricately regulated by epige-netic mechanisms, among which the histone methyltransferase G9a plays a pivotal role. The enzymatic activity of G9a, particularly its methylation of histone H3 on lysine 9 (H3K9me1 and H3K9me2), is crucial for the transcriptional repression of genes involved in developmental processes across various organisms [28,29]. Studies in mice have demonstrated that depletion of G9a drastically reduced H3K9 methylation in embryonic stem (ES) cells. Consequently, these G9a-deficient ES cells displayed severe growth inhi-bition and early lethality [28]. Surprisingly, G9a depletion in ES cells led to a significant increase in methylated H3 lysine 4 (H3K4), a mark associated with gene activation, re-gardless of H3K9, suggesting a complex regulatory network balancing gene expression during development [29]. Further research has elucidated the pivotal role of G9a in bone formation and im-mune cell differentiation. For example, the interaction of G9a with RUNX2 is essential for normal osteoblast development; its regulatory influence on the Twist gene is vital for os-teogenesis [30,31]. Specifically, Hisashi et al. reported that G9a could endogenously bind to RUNX2-related regions; depletion of G9a in osteoblasts hindered the binding of RUNX2 to the osteocalcin promoters, resulting in the abnormal formation of cranial bones [30]. In addition, Higashihori et al. demonstrated that G9a regulated osteogenesis via Twist gene repression. They found that Twist1 and Twist2 regulatory regions contained substantial H3K9me2 catalyzed by G9a in primary osteogenic mesenchyme from calvaria. Moreover, pharmacological inhibition of G9a decreased several osteogenic markers and prevented skeletal differentiation in vivo [31]. The significance of G9a in embryonic development parallels its role in cancer biology [16]. In cancer, aberrant G9a activity can lead to improper gene silencing, similar to its role in silencing developmental genes. However, in the context of cancer, this results in the repression of tumor suppressor genes and the activation of pathways that promote cancer cell proliferation, survival, and metastasis. Understanding the function of G9a in develop-mental biology, particularly its ability to dictate cell fate and maintain cellular identity through epigenetic modifications, informs its potential to drive oncogenesis when dysreg-ulated. G9a could also contribute to the T helper (Th) cell differentiation. Depletion of G9a in Th precursor cells was observed to impair their ability to develop into Th2 cells under exposure to chronic infection. G9a depletion might elevate the IL-17A expression via the loss of H3K9me2 in the IL-17A locus, thereby mitigating the lineage plasticity capacity of Th precursor cells [32]. Understanding the dual roles of G9a in promoting normal development and its ab-errant expressions contributing to cancer allows researchers and clinicians to gain a better appreciation of the potential for targeting G9a in cancer therapy. Figure 1. The schematic domains of 9a. Glu: glutamate; NLS: nuclear localization signals; Cys:cysteine. H3K9: histone 3 lysine 9.3. Embryonic Development and Tissue DifferentiationEmbryonic development and tissue differentiation are intricately regulated by epi-genetic mechanisms, among which the histone methyltransferase G9a plays a pivotalrole. The enzymatic activity of G9a, particularly its methylation of histone H3 on lysine 9(H3K9me1 and H3K9me2), is crucial for the transcriptional repression of genes involved indevelopmental processes across various organisms [28,29]. Studies in mice have demon-strated that depletion of G9a drastically reduced H3K9 methylation in embryonic stem(ES) cells. Consequently, these G9a-deficient ES cells displayed severe growth inhibitionand early lethality [28]. Surprisingly, G9a depletion in ES cells led to a significant increasein methylated H3 lysine 4 (H3K4), a mark associated with gene activation, regardlessof H3K9, suggesting a complex regulatory network balancing gene expression duringdevelopment [29].Further research has elucidated the pivotal role of G9a in bone formation and immunecell differentiation. For example, the interaction of G9a with RUNX2 is essential fornormal osteoblast development; its regulatory influence on the Twist gene is vital forosteogenesis [30,31]. Specifically, Hisashi et al. reported that G9a could endogenously bindto RUNX2-related regions; depletion of G9a in osteoblasts hindered the binding of RUNX2to the osteocalcin promoters, resulting in the abnormal formation of cranial bones [30]. Inaddition, Higashihori et al. demonstrated that G9a regulated osteogenesis via Twist generepression. They found that Twist1 and Twist2 regulatory regions contained substantialH3K9me2 catalyzed by G9a in primary osteogenic mesenchyme from calvaria. Moreover,phar acological inhibition of G9a decreased several osteogenic markers and preventedskeletal differentiation in vivo [31].The signific nce of G9a in embryonic development parallels its role in cancer biol-ogy [16]. In cancer, aberrant G9a activity can lead to improp r gene sile ing, similar toits role in silencing developmental ge es. H wever, in th cont xt of cancer, this resultsin th repression f tumor suppressor g n s a d the activa i n of pathways that promotecancer cell prolife ation, survival, and metastasis. Understanding t e functi n of G9ain developmental biology, particularly its ability to dictate cell fate and ma tain cellu-lar ident ty through epigenetic modifications, informs its potential to drive oncog esiswhen dysregula ed.G9a could also contribute to the T helper (Th) cell differentiation. Depletion of G9ain Th pre ursor cells was obs rved to impair their ability to develop into Th2 cells underexposure to chronic infection. G9a depletion might eleva e the IL-17A expression via theloss of H3K9me2 in the IL-17A locus, thereby mitigating the lineage plasticity capacity ofTh precursor c lls [32].Understanding the dual roles of G9a in promoting normal development and its aber-rant expressions contributing to cancer allows researchers and clinicians to gain a betterappreciation of the potential for targeting G9a in cancer therapy.Cancers 2024, 16, 2175 4 of 174. Pivotal Role of G9a in CancersBeyond its fundamental role in embryonic development, G9a has been recognizedas an upregulated player in various cancers, including breast, lung, and liver tumors.There is a growing consensus on the significant impact of G9a-induced aberrant epigeneticreprogramming on tumorigenesis, disease progression, autophagy, apoptosis, lineageplasticity, and hypoxia adaptation (Figure 2). Understanding how G9a contributes to cancerdevelopment and progression is critical for advancing targeted epigenetic therapies aimedat enhancing patient outcomes.Cancers 2024, 16, x FOR PEER REVIEW 4 of 18 4. Pivotal role of G9a in Cancers Beyond its fundamental role in embryonic development, G9a has been recognized as an upregulated player in various cancers, including breast, lung, and liver tumors. There is a growing consensus on the significant impact of G9a-induced aberrant epigenetic re-programming on tumorigenesis, disease progression, autophagy, apoptosis, lineage plas-ticity, and hypoxia adaptation (Figure 2). Understanding how G9a contributes to cancer development and p ogression s critical for adv ncing tar t d epigen tic therapies aimed at enhancing patient utcomes. Figure 2. The graphic abstract summarizing four different aspects of G9a in cancers. A, G9a can methylate both histone and non-histone substrates, dysregulating the expression of tumor suppres-sor genes; B, G9a could impact various pathways in tumors, promoting tumor proliferation and progression; C, G9a plays dual roles in cancer lineage plasticity across various conditions and tumor types; D, G9a plays a crucial role in tumor adaptation to hypoxia, promoting tumor formation and progression. The arrows”↑” and “↓” mean the upregulation and downregulation of genes or path-ways. 4.1. Dysregulation of Tumor Suppressor Genes The inactivation of tumor suppressor genes is a pivotal step in cancer development. G9a-mediated H3K9 methylation plays a central role in the repression of tumor suppres-sor genes, promoting carcinogenesis. G9a overexpression leads to increased H3K9 meth-ylation, which in turn attracts proteins such as heterochromatin protein 1 (HP-1), promot-ing the formation of compact, transcriptionally inactive chromatin structures [10,33]. This silencing mechanism contributes to the downregulations of tumor suppressor genes across various cancer types, facilitating tumor growth and progression. Notably, Fran-cesco et al. demonstrated that G9a under hypoxic conditions in breast cancer could down-regulate key genes, including ARNTL and CEACAM7, highlighting common pathways cancer cells utilize to evade growth control [34]. Similarly, in liver cancer, overexpression of G9a is linked to the silencing of RARRES3, promoting tumor aggressiveness via the miR-1/G9a/RARRES3 signaling axis [35]. Additionally, a study conducted by Lee et al. showed that G9a significantly downregulated the expression of the tumor suppressor gene RUNX3 in gastric cancers [36]. They found that upregulated G9a significantly Figure 2. The graphic abstract summarizing four different aspec s of G9a in cancers. A, G9acan methylate bot histone and non-hist strates, dysregulating the expression of tumorsuppressor genes; B, G9a could impact va s pathways in tumors, p omoting tumor prolife ationand progression; C, G9a plays les in cancer lineage plasticity across various c nditi s andtumor types; D, G9a plays a crucial rol i tumor adaptation to hypoxia, promoting tumor for ationand progression. The arrows”↑” and “↓” e t e upregulation and ownregulation of genesor pathways.4.1. Dysregulation of Tumor Suppressor GenesThe inactivation of tumor suppressor genes is a pivotal step in cancer development.G9a-mediated H3K9 methyla ion plays a central role i the repressi n of umor suppr ssorgenes, promoting carcinogenesis. G9a overexpression leads to increased H3K9 methylation,which in turn attracts proteins such as heterochromatin protein 1 (HP-1), promoting theformation of compact, transcriptionally inactive chromatin structures [10,33]. This silencingmechanism contributes to the downregulations of tumor suppressor genes across variouscancer types, facilitating tumor growth and progression. Notably, Francesco et al. demon-strated that G9a under hypoxic conditions in br ast cancer could downregulate key genes,in luding ARNTL and CEACAM7, highlighting co mon pathways cancer cell utilize tevade growth control [34]. Similarly, in liver cancer, overexpression of G9a is linked to theCancers 2024, 16, 2175 5 of 17silencing of RARRES3, promoting tumor aggressiveness via the miR-1/G9a/RARRES3 sig-naling axis [35]. Additionally, a study conducted by Lee et al. showed that G9a significantlydownregulated the expression of the tumor suppressor gene RUNX3 in gastric cancers [36].They found that upregulated G9a significantly inhibited the nuclear localization of RUNX3and attenuated the RUNX3 expression during tumor progression. Similarly, overexpressedG9a is found in cholangiocarcinoma (CCA) as a silencer to the tumor suppressor gene,LATS2, mediating tumor development [37]. Ma et al. reported that inhibition of G9a inCCA significantly reduced the level of H3K9me2 and restored the expression of LATS2,leading to YAP inhibition.Moreover, the capacity of G9a extends beyond histone methylation to directly methy-late non-histone proteins, such as p53, further illustrating its broad role in cancers. Huanget al. found that G9a specifically dimethylated p53 at Lys373, resulting in the inactivationof p53 [38]. These data suggested that G9a has the potential regulations directly on genesthemselves, regardless of its catalytic function of histone methylation. In subsequent stud-ies, additional targets have been identified to serve as direct substrates for G9a methylation,including transcription factors and related transcription co-regulatory factors, such as p53,ERα, CEBP, HDAC1, and HIF1α, further illustrating the extensive influence of G9a incancer biology [38–42].4.2. Promote Tumor Proliferation and ProgressionElevated G9a expression is strongly associated with an aggressive cancer phenotype,significantly influencing disease progression and metastasis through its epigenetic reg-ulatory functions. G9a exerts a profound impact on cancer biology by modulating keypathways crucial for tumor growth and dissemination, such as epithelial–mesenchymaltransition (EMT), mTOR, and Wnt/β-catenin signaling.In lung cancer, numerous studies underscore the pivotal role of G9a in promotingaggressiveness through its impact on the EMT pathway [43–48]. Chen et al. demonstratedthat G9a knockdown not only ablated H3K9me2 expression but also suppressed the EMT-related gene Ep-CAM by decreasing the recruitment of the transcriptional cofactors HP1,DNMT1, and HDAC1 to the promoter region [44]. Similarly, Hu et al. found that G9aand HDAC jointly regulated Snail-induced E-cadherin suppression to enhance migrationand invasion in lung and liver cancers [47,49]. In breast cancer, the involvement of G9ain the EMT pathway has been reported to contribute to heightened cancer growth andmetastatic potential. For example, Wozniak et al. highlighted the facilitative role of G9a inenhancing the EMT pathway through silencing desmocollin 3 (DSC3) and MASPIN viaH3K9 methylation [50]. In addition, G9a could regulate Snail during EMT in breast cancer,leading to the suppression of E-cadherin expression through promoter methylation. Donget al. reported that in vivo and in vitro knocking out of G9a in breast cancer could rescueE-cadherin expression and concurrently inhibit cancer proliferation and progression [51].Additionally, this G9a–Snail–E-cadherin axis was also reported in the head and necksquamous cell carcinoma (HNSCC). Liu et al. demonstrated that the G9a protein could beessential for the induction of EMT in HNSCC through the G9a–Snail–E-cadherin axis topromote tumor cell migration and tumorphere formation [52].Beyond EMT, several other pivotal pathways have been investigated to be regulated byG9a to promote tumor cell proliferation in various cancers [44,48,53]. For instance, Zhanget al. observed overexpression of G9a in 43.2% of non-small cell lung cancer (NSCLC)tissues. They found that G9a suppression could dramatically repress NSCLC tumor growththrough Wnt signaling pathway inhibition [44]. Also, Sun et al. reported that elevated G9aexpression was correlated with focal adhesion kinase activation via NF-κB signaling [48].Another study exploring the role of G9a in NSCLC uncovered its contribution to tumor cellgrowth and invasion through the repression of CASP1 [46]. In gastric cancer, Yin et al. foundthat knockdown of G9a or treatment with G9a inhibitor BIX01294 significantly reducedcell growth by inducing cell cycle arrest and autophagy. Mechanically, mTOR expressionwas linked to promoter methylation and enrichment for H3K9me1 and H3K9me2. G9aCancers 2024, 16, 2175 6 of 17knockdown decreased the H3K9 methylation level at the mTOR promoter in gastric cancercells, leading to inhibited tumor growth. Notably, they demonstrated an apparent decreaseonly in the H3K9me1 but not in the H3K9me2 level after G9a knockdown [53].However, there remain other vital mechanisms associated with G9a in the processof tumor proliferation and progression. In gastric cancer, Kim et al. reported that G9ainhibition might lead to autophagic cell death under the endoplasmic reticulum (ER)stress via the HDAC–G9a and STAT3–G9a axes [54,55]. Simultaneously, Zhang et al.uncovered the association between G9a and the p21-Bim signal in breast cancer. Theyfound that G9a inhibition by GA001, a potent G9a antagonist, significantly inhibited theMCF7 cell proliferation and survival by inducing autophagy and apoptosis dependent onthe activation of AMPK [56]. Furthermore, Nakatsuka et al. demonstrated that G9a allowedDNA-damaged hepatocytes to escape apoptosis by suppressing Bcl-G [57]. They proposedthe mechanism that DNA damage stimuli recruited G9a to the p53-responsive elementof the Bcl-G gene. G9a enriched in the p53-responsive region significantly decreased theexpression of the Bcl-G gene; therefore, it facilitated the evasion of p53-induced apoptosis,which might contribute to the tumor initiation and progression.4.3. Cancer Lineage PlasticityAccumulating evidence indicated that cancer cells have the ability to switch theircellular phenotype through lineage plasticity, transitioning themselves into a more undif-ferentiated, pluripotent, embryo-like, stem-like state. This adaptive mechanism facilitatestumor proliferation, drug resistance, and metastasis but remains unaddressed by conven-tional therapeutic measures in clinical practice. In concordance with its role in embryonicdevelopment, G9a is expected to be crucial to lineage plasticity within the field of cancer.Several studies have indeed substantiated this hypothesis, demonstrating that G9a couldmaintain the self-renewal ability of cancer cells and manifest cancer lineage plasticity asa stem-like phenomenon [52,58–64]. For instance, in NSCLC, Pangeni et al. reportedthat knocking down of G9a upregulated a series of genes (CDYL2, DPP4, SP5, FOXP1,STAMBPL1, and ROBO1) through hypomethylated H3K9, consequently sustaining tumor-initiating cells and facilitating cancer metastasis [58]. They found that G9a inhibition mightdownregulate the expression of cancer stem cell markers and defected sphere-formingcapacity, in vitro proliferation, and in vivo growth. Notably, the chromatin immunoprecipi-tation (ChIP) assay showed that G9a interacted with its target genes through H3K9me2,emphasizing the role of G9a in a novel therapeutic approach. Numerous studies havedelved into the role of G9a and cancer lineage plasticity across various tumors, exploringhow G9a regulates tumor stemness (e.g., CD133 expression). These investigations haverevealed that G9a may play distinct roles in different types of tumors. Recent reviews haveprovided detailed overviews and summaries of these findings, highlighting the diversefunctions of G9a in cancer stemness [11,12].The dual role of G9a in cancer introduces a novel perspective on the intricacies oflineage plasticity. While it sometimes serves as a suppressor of cancer lineage plasticity,G9a concurrently promotes cancer stem cell-like features in certain conditions. Indeed, thisduality not only highlights the diverse functions of G9a but also provides valuable insightsfor further exploration into its underlying mechanisms across different cancer types. Aprofound understanding of the functions of G9a holds the potential to uncover additionaltreatment strategies in cancer therapy in the future.4.4. Hypoxia AdaptionHypoxia is a common phenomenon in solid tumors, attributed to excessive oxygenconsumption due to the aggressively proliferating, overactive metabolism of tumors andabnormal formation of blood vessels supplying the tumor [65]. Cells perceive the change ofoxygen concentration through a family of transcription factors known as hypoxia-induciblefactors (HIF-1) [66,67]. A typical cellular response to hypoxic conditions is the maintenanceof oxygen homeostasis. Cancer cells initiate hypoxic molecular mechanisms, includingCancers 2024, 16, 2175 7 of 17activation of survival mechanisms, metabolic reprogramming, and angiogenesis during theprocess of hypoxia adaption [68]. Under hypoxia, G9a is demonstrated to be upregulatedto suppress a series of genes through H3K9 methylation to promote cancer survival [69].In breast cancer, Casciello et al. found that G9a was a key player in mediating thehypoxia-related response. Under hypoxic conditions, an increased level of G9a inhibitedseveral genes, including ARNTL, CEACAM7, and more to promote cancer cell survivaland tumorigenesis [34]. They further reported that G9a could repress CDH10 expressionthrough histone methylation, promoting tumor progression under hypoxia [65]. In gastriccancer, Kim further elucidated that G9a might promote cell survival under hypoxia via theSTAT3–G9a axis. Additionally, they employed an herbal formulation, SH003, to induce ERstress, inhibiting G9a activity and ultimately inducing autophagic cell death [55].5. Pharmacological Targeting of G9aGiven that G9a plays a pivotal role as an HMT in multiple tumorigenesis processes, in-cluding dysregulation of tumor suppressor genes, inactivation of transcription factors, andadaption to hypoxia tumor microenvironment (TME), it has been extensively investigatedas a promising therapeutic target in the past decades. Here, we summarize the currentprogress in the development of G9a inhibitors.To date, G9a inhibitors have broadly fallen into two groups [70,71]. The first cate-gory comprises substrate (H3 peptide) competitive inhibitors, including BIX-01294 [72],UNC0224 [73], UNC0321 [74], UNC0638 [75], UNC0642 [76], EML741 [77], and A-366 [78](Table 1). The second category encompasses the S-5′-Adenosyl-L-Methionine (SAM) co-factor competitive inhibitors, such as BIX-01338 [72], BRD9539 [79], and BRD4770 [79].Unfortunately, none of these inhibitors have progressed to clinical trials as of now. Con-sidering the superior selectivity and indicative lower toxicity of substrate competitiveinhibitors for G9a, recent research efforts have predominantly focused on the developmentof G9a inhibitors based on the BIX01294 scaffold (quinazoline-based) [80].BIX-01294, initially reported in 2007, is recognized as the earliest small-moleculeinhibitor specifically targeting G9a HMT activity and significantly reducing H3K9me2expression [72]. As reported by Kubicek et al., BIX-01294, which is structured as a diazepin-quinazolin-amine derivative, emerged as a highly selective compound selected from a poolof over 125,000 preselected candidates and demonstrated notable efficacy in inhibitingG9a catalytic function and the generation of H3K9me2 in several cell lines. Numerousstudies subsequently affirmed its effectiveness in inhibiting tumor growth by decreasingH3K9me2 expression levels [81–83]. However, the application of BIX-01294 is greatlylimited by the narrow gap between the effective concentration for inhibiting H3K9me2 andthe non-specific cytotoxic concentration [72,75]. Consequently, prompted by practical needs,UNC0638 was derived from the BIX-01294 structure through optimization. UNC0638met expectations and has been reported to effectively reduce tumor growth in in vitroexperiments across various cancer types [84–87].However, subsequent research revealed poor pharmacokinetics for UNC0638 inin vivo experiments [75]. To overcome this shortage, Liu and his colleagues further modi-fied UNC0637 and synthesized UNC0642 [76,88] (Figure 3). UNC0642 not only maintainshigh in vitro and cellular potency with low cell toxicity, like UNC0638, but also exhibits im-proved in vivo pharmacokinetic properties, rendering it suitable for animal studies [89,90].Notably, A-366, distinguished by its indole core-based structure in contrast to UNC0642, aquinazoline core-based inhibitor, has progressed into the preclinical stage for leukemia [91].This advancement is attributed to its heightened about ten-fold selectivity for G9a overGLP [78]. Moreover, several new G9a inhibitors have been developed in recent years,building on existing chemical structure cores or featuring entirely new structures, such asZZM-1220 and SDS-347 [92,93].Cancers 2024, 16, 2175 8 of 17Cancers 2024, 16, x FOR PEER REVIEW 8 of 18 conducted, encompassing both in vitro and in vivo settings, revealing its high selectivity for G9a and DNMT1. The study showed that CM-272 effectively inhibited the expression of H3K9me2 and 5mC, subsequently inducing IFN-responsive tumor apoptosis. Moreo-ver, CM-272 has undergone extensive validation for its noteworthy cytotoxicity across di-verse tumor types, including cholangiocarcinoma, hematologic malignancies, bladder cancer, and prostate cancer (PCa) [94–98]. Table 1. Substrate (H3 peptide) competitive inhibitors of G9a reported in the literature. Compound Core Application In Vivo IC50s BIX-01294 [72] Quinazoline No G9a = 1.7 μM; GLP = 0.9 μM UNC0224 [73] Quinazoline No G9a = 15 nM; GLP = 20–58 nM UNC0321 [74] Quinazoline No G9a = 6–9 nM; GLP = 15–23 nM UNC0638 [75] Quinazoline No G9a < 15 nM; GLP = 19 nM UNC0642 [76] Quinazoline Yes [76] G9a ≈ GLP < 2.5nM EML741 [77] Quinazoline No G9a ≈ GLP: 23 nM; DNMT1 = 3.1 μM A-366 [78] Indole Yes [91] G9a = 3.3 nM; GLP = 38 nM ZZM-1220 [92] Quinazoline No G9a = 458 nM; GLP = 924 nM Furthermore, Velez et al. recently developed a first-in-class G9a/GLP proteolysis tar-geting chimera (PROTAC) degrader 10 (MS8709) [99] and showed a promising therapeutic effect in vitro by inducing G9a and GLP degradation at 3 μM in several cell lines. Figure 3. Chemical structures of UNC0638 and UNC0642. 6. The Significance of G9a in Prostate Cancer 6.1. The Role of G9a as an HMT in Prostate Cancer The overexpression of G9a was shown to be correlated with poor survival outcomes in castration-resistant prostate cancer (CRPC) patients [100]. Besschetnova et al. demon-strated that the genetic alteration and expression of G9a were elevated in clinical CRPC samples [100]. Moreover, they found that the downregulation of G9a transcriptionally de-activated oncogenic programs, resulting in a significant decrease in cell proliferation and metastasis in LNCaP, PC3, and xenograft models. He subsequently pharmacologically in-hibited the enzymatic activity of G9a using UNC0642 and reverified this phenomenon. In previous studies, G9a was found to be able to methylate lysine 114 of LSD1, an H3K4 demethylase, leading to the activation of gene transcription in PCa cells. This activation was associated with the recognition and interaction of LSD1 with the chromatin-remodel-ing protein CHD1 [101]. This finding suggested that G9a might potentially establish a net-work with other epigenetic regulators in PCa, collectively influencing transcription through histone modification. AR inhibitors and androgen deprivation therapy (ADT) are crucial for the clinical treatment of prostate cancer. However, there is currently a lack of solid evidence indicating an interaction between G9a and AR, or even AR-V7. Although one study showed that H3K9 methylation can promote resistance to AR inhibitors in Figure 3. Chemical structures of UNC0638 and UNC0642.Initially, G9a was found to participate collaboratively with DNMT1 in gene silencingthrough epigenetic regulation. In 2016, José-Enériz et al. employed a novel, potent, selective,and reversible drug, CM-272, which significantly suppressed the activity of G9a andDNMT1 based on UNC0642 [94]. Comprehensive functional validation of CM-272 wasconducted, encompassing bo h in vit o and in vivo settings, revealing its high selectivityfo G9a and DNMT1. The study showed that CM-272 effectively inhibited the expressionof H3K9me2 and 5mC, subsequently inducing IFN-respons ve tumor apoptosis. Moreover,CM-272 has und rgone extensive va idation for its noteworthy cytotoxicity across diversetumor types, including cholangiocarcino a, hematologic malignancies, bladder cancer,and prostate cancer (PCa) [94–98].Table 1. Substrate (H3 peptide) competitive inhibitors of G9a rep rted in the literature.Compound Core Application In Vivo IC50sBIX-01294 [72] Quinazoline No G9a = 1.7 µM; GLP = 0.9 µMUNC0224 [73] Quinazoline No G9a = 15 nM; GLP = 20–58 nMUNC0321 [74] Quinazoline No G9a = 6–9 nM; GLP = 15–23 nMUNC0638 [75] Quinazoline No G9a < 15 nM; GLP = 19 nMUNC0642 [76] Quinazoline Yes [76] G9a ≈ GLP < 2.5nMEML741 [77] Quinazoline No G9a ≈ GLP: 23 nM; DNMT1 = 3.1 µMA-366 [78] Indole Yes [91] G9a = 3.3 nM; GLP = 38 nMZZM-1220 [92] Quinazoline No G9a = 458 nM; GLP = 924 nMFurthermore, Velez et al. recently developed a first-in-class G9a/GLP proteolysis tar-geting chimera (PROTAC) degrader 10 (MS8709) [99] and showed a promising therapeuticeffect in vitro by inducing G9a and GLP degradation at 3 µM in several cell lines.6. The Significance of G9a in Prostate Cancer6.1. The Role of G9a as an HMT in Prostate CancerThe overexpression of G9a was shown to be correlated with poor survival outcomesin castration-resistant prostate cancer (CRPC) patients [100]. Besschetnova et al. demon-strated that the genetic alteration and expression of G9a were elevated in clinical CRPCsamples [100]. Moreover, they found that the downregulation of G9a transcriptionallydeactivated oncogenic programs, resulting in a significant decrease in cell proliferationand metastasis in LNCaP, PC3, and xenograft models. He subsequently pharmacologicallyinhibited the enzymatic activity of G9a using UNC0642 and reverified this phenomenon.In previous studies, G9a was found to be able to methylate lysine 114 of LSD1, an H3K4demethylase, leading to the activation of gene transcription in PCa cells. This activationwas associated with the recognition and interaction of LSD1 with the chromatin-remodelingprotein CHD1 [101]. This finding suggested that G9a might potentially establish a networkwith other epigenetic regulators in PCa, collectively influencing transcription throughhistone modification. AR inhibitors and androgen deprivation therapy (ADT) are crucialfor the clinical treatment of prostate cancer. However, there is currently a lack of solidevidence indicating an interaction between G9a and AR, or even AR-V7. Although onestudy showed that H3K9 methylation can promote resistance to AR inhibitors in prostateCancers 2024, 16, 2175 9 of 17cancer cells and that de-methylation may potentially restore drug sensitivity, it remainsunclear whether there is a direct interaction between G9a and AR [102,103].6.2. G9a–PRC2 ComplexPolycomb repressive complex 2 (PRC2) consists of core subunits, including Enhancerof Zeste homolog 2 (EZH2), suppressor of Zeste 12 (SUZ12), and embryonic ectodermdevelopment (EED). This complex catalyzes mono-, di-, and trimethylation of histone 3lysine 27 (H3K27me1, H3K27me2, and H3K27me3), extensively involved in PCa diseaseprogression and drug resistance. As the catalytic component of the PRC2 complex, EZH2has drawn widespread attention. Multiple studies have suggested that knocking downEZH2 expression in prostate cancer effectively lowers the levels of H3K27me3, therebyhindering the growth and progression of PCa [104–107]. Consequently, the development ofEZH2 inhibitors has become a subject of significant interest among researchers, yieldingpromising outcomes.Moreover, with further investigation into the structure of the PRC2 complex, G9a wasfound to be able to bind with the PRC2 complex, forming a novel G9a–PALI1–PRC2 struc-ture and conducting dual H3K9 and H3K27 methylation epigenetic modifications [108,109].This suggests that the combined therapy of G9a and EZH2 may yield more significant clini-cal efficacy in prostate cancer. They separately used two inhibitors targeting G9a (UNC0642)and EZH2 (EPZ-6438) for in vitro and in vivo experiments in LNCaP and C4-2B, revealinga significant synergetic effect. Indeed, there remains a significant gap in understanding themechanism of G9a–PRC2 in PCa, underscoring the need for further research in this domain.6.3. G9a/GLP-DNMT1 ComplexThe tumor suppressor gene SPOP has been identified with a frequent mutation rate of6–15% in prostate cancer, and it is associated with ERG rearrangements, aberrant androgenreceptor (AR) activity, and globally elevated DNA methylation [110–112]. Zhang reportedthat in SPOP-mutant PCa cells, the aberrant upregulation of global DNA hypermethylationresulted from the inhibition of the SPOP-mediated polyubiquitination and degradation ofthe G9A/GLP-DNMT1 module [113]. This finding uncovered the mechanism by whichG9a regulates the epigenetic landscape in PCa through DNA methylation. Moreover, itprompted the exploration of the efficacy of dual-targeted therapy against G9a and DNMT1in PCa. CM-272, a dual inhibitor targeting G9a and DNMT1, has been reported by Filipato effectively inhibit the expression of DNMT1, G9a, and H3K9me2 in prostate cancer.Consequently, CM-272 effectively suppressed tumor growth in PCa cell lines DU145, PC3,and 3D PCa models, demonstrating the promising anti-tumor effects in PCa.7. Future Directions of G9a in Prostate Cancer7.1. ImmunotherapyThe integration of epigenetic inhibitors with immune checkpoint inhibitors (ICIs) hasgarnered attention in recent years, with an increasing number of studies delving into theunderlying mechanisms by which epigenetic regulation modulates the tumor immunemicroenvironment: (1) Epigenetic modification has the potential to dysregulate the normalantigen presentation of tumor cells to induce immune evasion. Research suggests thatepigenetic inhibitors can directly impact tumor cells by enhancing intracellular immunesignaling pathways [114,115]. This includes elevating the expression of cell surface MHCclass I molecules and restoring the cytotoxic effects of CD8+ T cells within the TME.(2) Epigenetic modification could influence the immunogenicity of tumor cells by alteringthe endogenous retrovirus (ERV) associated with sustained inflammatory stimulation.Studies have demonstrated that DNMT inhibitors and HDAC inhibitors can upregulatethe expression of interferon pathways related to ERV by re-modulating the epigeneticlandscape [97,116,117]. This induction creates a state in tumor cells similar to sustainedviral stimulation, continuously triggering immune cell activation and thereby enhancingthe efficacy of immunotherapy. (3) Beyond impacting tumor cells, epigenetic modificationCancers 2024, 16, 2175 10 of 17also regulates immune cells within the TME [118–120]. DNA methylation or histonemodification plays an indispensable role not only in the formation and differentiationof immune cells but also in the induction of T cell exhaustion. This occurs through thereduction of T cell effector molecules and the overexpression of co-inhibitory factors suchas PD-1, ultimately promoting tumor progression and drug resistance.The rapid advancement of epigenetic inhibitors in modulating TME has promptedgreat efforts in combining these inhibitors with ICIs, revealing promising clinical efficacyin certain tumor types [114,121]. For instance, in melanoma, Kato et al. discovered thatG9a played a crucial role in controlling tumor survival and progression by activating theWNT/β-catenin signaling pathway through stimulation of the microphthalmia-associatedtranscription factor (MITF). They observed that G9a contributed to the establishment of a‘cold’ TME by inhibiting DKK1, an antagonist of the canonical WNT pathway. Furthermore,the effectiveness of G9a inhibitors in transforming a ‘cold’ immune microenvironmentinto a ‘hot’ one has been confirmed in an in vivo syngeneic mouse melanoma model,highlighting the clinic efficacy of combination therapy involving G9a inhibitors and im-munotherapy [122]. In bladder cancer, Segovia et al. have confirmed the correlationbetween high G9a expression and poor survival in bladder cancer patients [97]. He verifiedthat targeting G9a and DNMT methyltransferase activity with a dual inhibitor, CM-272,can induce cancer cell apoptosis and immunogenic cell death. Moreover, he inferred thatepigenetic inhibitors have the capacity to convert cold immune tumors into hot immunetumors by modulating immune cells in the tumor microenvironment. This observationaligned with clinical findings that elevated G9a expression was correlated with poor efficacyof programmed cell death protein 1 inhibition in a cohort of patients with bladder cancer.These findings supported the potential opportunities of integrating immunotherapy withepigenetic inhibitors in the treatment of bladder cancer.While G9a, as a critical HMT in prostate cancer, has gained attention only in recentyears, it is reasonable to predict that G9a plays a crucial role in immune microenvironmentalregulation, referring to the observations in other cancers. Unfortunately, there is currentlylimited research on the involvement of G9a in ‘cold’ TME of prostate cancer. Futurestudies, such as whether focusing on G9a in mediating immune escape, exploring its role ininflammatory signals, or elucidating its mechanisms in promoting immune cell exhaustion,require further validation for their effectiveness in prostate cancer. This will establish asolid foundation for the future combined regimen of G9a inhibitors and ICI in prostatecancer treatment.7.2. Dual Inhibitor of G9a and EZH2Inspired by the aforementioned study, G9a has been confirmed as a substrate of thePRC2 complex in prostate cancer, interacting with EZH2 to carry out epigenetic modi-fication via the methylation of H3K9 and H3K27. The successful combination therapyemploying two individual drugs in vitro and in vivo has encouraged the ongoing develop-ment of dual-target inhibitors. Building upon this discovery, Shi et al. designed a noveldual-target inhibitor by combining the G9a inhibitor BIX-01294 and the EZH2 inhibitorTazematostat, effectively inhibiting the activity of both G9a and EZH2 [123]. It was demon-strated that in an in vitro experiment, Compound 10 exhibited anti-tumor growth effectsin RD and SW982 cells. In addition, Compound 11 demonstrated favorable pharmacoki-netic properties in the in vivo experiment. However, there is currently a lack of data toelucidate the efficacy of this dual-target inhibitor in prostate cancer. In this regard, weanticipate forthcoming research findings and a more in-depth exploration of the mecha-nisms to comprehensively understand the role of G9a/EZH2 dual inhibitors in prostatecancer treatment.7.3. Dual Inhibitor of G9a and HDACIt is observed that G9a interacts with HDAC, thereby contributing to the aberrantglobal methylation status in cancers [47,49]. Mechanistically, H3K9 acetylation mightCancers 2024, 16, 2175 11 of 17impede the formation of H3K9 methylation. Hence, the sequential process involvingHDAC-mediated removal of acetylation on H3K9, followed by G9a-mediated methylationmodification, could dramatically lead to gene silencing. Numerous studies have revealedthat the simultaneous inhibition of G9a and HDAC effectively repressed tumor growthand metastasis. This not only highlights the potentiality of targeting them as therapeuticregimens but also emphasizes the need for the continued development of dual-targetinhibitors against G9a and HDAC.Overall, the development of dual-target inhibitors, especially in epigenetic inhibitorsfor cancer treatment, remains a large blur to explore. Great efforts have been made in inves-tigating the interactions between G9a and EZH2, as well as G9a and HDAC, demonstratingthe potential effectiveness of these combination inhibition strategies in anti-tumor therapy(Figure 4). Nonetheless, several challenges persist, particularly in specific cancer types suchas prostate cancer, where the actual efficacy of these inhibitors needs further validation.Cancers 2024, 16, x FOR PEER REVIEW 11 of 18 7.3. Dual Inhibitor of G9a and HDAC It is observed that G9a interacts with HDAC, thereby contributing to the aberrant global methylation status in cancers [47,49]. Mechanistically, H3K9 acetylation might im-pede the formation of H3K9 methylation. Hence, the sequential process involving HDAC-ediated removal of acetylation on H3K9, followed by G9a-mediated methylation modi-fication, could dra atically lead to gene silencing. Numerous studies have revealed that the simultane s inhibition of G9a and HDAC effectively represse tumor growth and metasta is. This n t only highlights the potentiality of targ ting th m as therapeutic regi-mens but lso emphasizes the need for t e c ntinued development of dual-target inhibi-tors against G9a and HDAC. Overall, the development of dual-target inhibitors, especially in epigenetic inhibitors for cancer treatm nt, re ai s a large blur to explore. Great efforts hav b en made in in-vestigating the i teractio betw en G9a and EZH2, s well as G9a and HDAC, demon-strating the potential effective ess of these combination inhibition strategies in anti-tumor therapy (Figure 4). Nonetheless, several challenges persist, particularly in specific cancer types such as prostate cancer, where the actual efficacy of these inhibitors needs further validation. Figure 4. Graphic abstract of the current research (blue, top left and bottom right) and prospective (red, top right and bottom left) of G9a in prostate cancer. Red “x” indicates the inhibition of the respective gene function through different methods. The illustration demonstrates that (A) G9a plays a crucial role in tumor immune microenvironment regulation; (B) G9a, cooperating with PALI1, SUZ12, EZH2, EED, forms the PRC2 complex to carry out the epigenetic modification in PCa; (C) G9a/DNMT1 complex maintains the epigenetic features of PCa through DNA methylation; (D) Figure 4. Graphic abstract of the current research (blue, top left and bottom right) and prospective(red, top right and bottom left) of G9a in prostate cancer. Red “x” indicates the inhibition of therespective gene function through different methods. The illustration demonstrates that (A) G9aplays a crucial role in tumor immune microenvironment regulation; (B) G9a, cooperating withPALI1, SUZ12, EZH2, EED, forms the PRC2 complex to carry out the epigenetic modification inPCa; (C) G9a/DNMT1 complex maintains the epigenetic features of PCa through DNA methylation;(D) G9a interacting with HDAC contributes to the aberrant global methylation status in cancers.(Created with BioRender.com accessed on 15 May 2024).Cancers 2024, 16, 2175 12 of 178. ConclusionsTaken together, this review has systematically explored the multifaceted roles of G9a inthe pathogenesis and progression of cancer, especially PCa. Through detailed examination,we have delineated the significant contributions of G9a to epigenetic dysregulation incancer, including the silencing of tumor suppressor genes, modulating critical pathwaysrelevant to tumor development, promoting the cancer lineage plasticity, and hypoxia adap-tion. Our review focused more attention on the field of PCa. The incidence of researchinvestigating the function of G9a in PCa has greatly increased over the past decade, owingto the development of more potent epigenetic inhibitors for the treatment of prostate can-cer. However, current research on G9a in prostate cancer reveals significant gaps in ourunderstanding, particularly regarding its interactions in the TME and its full epigeneticimpact. Exploring G9a inhibitors, especially in combination with immunotherapy, offerspromising avenues for PCa treatment research. In the future, more integrated multidisci-plinary approaches encompassing molecular biology, pharmacology, and clinical oncologywill be pivotal in uncovering the full therapeutic potential of targeting G9a in PCa. Wehope that this review serves as a valuable source for researchers and clinicians, guidingfuture investigations in the field of PCa or pan cancers and paving the way for knowledgeinto more effective treatment strategies.Author Contributions: Writing—original draft preparation, Y.N. and M.S.; writing—review andediting, Y.N., M.S., L.L. and D.L.; table and figure drawing, Y.N. and M.S.; supervision, C.O., H.Z.and Y.W.; funding acquisition, C.O., H.Z. and Y.W.; All authors have read and agreed to the publishedversion of the manuscript.Funding: This research was funded by the Canadian Institutes of Health Research (#153081, #173338,#180554, #186331) to Y.W., Terry Fox Research Institute (#1109) to Y.W. & C.O., and a CanadianCancer Society Breakthrough Team Grant generously supported by the Lotte & John Hecht MemorialFoundation (CCS grant #707683) (to Y.W.); a BC Cancer Foundation grant (to Y.W.), and financialsupport from the program of China Scholarships Council (to Y.N., No. 202206240124).Acknowledgments: The authors express their gratitude to all the investigators whose work hasenhanced our comprehension of the biochemistry, physiology, and tumor biology related to G9a. 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G9a
EHMT2
histone methyltransferase
prostate cancer
epigenetic inhibitor
G9a in Cancer: Mechanisms, Therapeutic Advancements, and Clinical Implications
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