The Roles of Zinc Finger Proteins in Colorectal Cancer

Despite colorectal cancer remaining a leading worldwide cause of cancer-related death, there remains a paucity of effective treatments for advanced disease. The molecular mechanisms underlying the development of colorectal cancer include altered cell signaling and cell cycle regulation that may result from epigenetic modifications of gene expression and function. Acting as important transcriptional regulators of normal biological processes, zinc finger proteins also play key roles in regulating the cellular mechanisms underlying colorectal neoplasia. These actions impact cell differentiation and proliferation, epithelial–mesenchymal transition, apoptosis, homeostasis, senescence, and maintenance of stemness. With the goal of highlighting promising points of therapeutic intervention, we review the oncogenic and tumor suppressor roles of zinc finger proteins with respect to colorectal cancer tumorigenesis and progression.


Zinc Finger Proteins Are Transcriptional Regulators in Colorectal Cancer (CRC)
With more than 1.9 million new cases of CRC worldwide in 2020 [1] and 150,000 new cases projected in the US in 2023 [2], CRC is the third most prevalent cancer and second leading cause of cancer-related death in the world [3]. Potential contributors to increased CRC risk include the increasing prevalence of aging populations, ingestion of so-called Western diets, obesity, and insufficient physical exercise-all clearly interrelated factors. For currently unknown reasons, there has also been a shift towards the development of colorectal cancer in younger people, i.e., those younger than age 50 years [2]. Prior colon adenomas, inflammatory bowel disease, and a family history of CRC, including hereditary disorders (e.g., familial adenomatous polyposis and Lynch syndrome), further increase CRC risk [4]. Various molecular mechanisms are implicated in the development of CRC, including altered cellular signaling pathways, epigenetic modifications, genomic instability, and metabolic dysfunction. The complexity of CRC pathogenesis is further confounded by extensive cross-talk between these and other mechanisms [5]. The observation that zinc finger proteins play a key role as transcriptional regulators in the development and progression of CRC prompted the current review.
For the purposes of this review, a zinc finger (ZNF) is defined as a small, functional, autonomously folded domain that requires the coordination of one or more zinc ions to stabilize its structure [6]. In 1985, xenopus transcription factor IIIA (TFIIIA) was the first protein shown to possess such a zinc finger domain [6]. ZNFs are DNA-binding domains that comprise the largest family of transcription factors. They can also bind RNA, facilitate interactions between proteins, have structural roles, and provide other, as-yet-undefined biological functions [6]. Hence, zinc finger proteins (ZFP) can act as transcriptional repressors and activators for a wide array of genes, thus modulating a multitude of biological processes, including cell differentiation, cell proliferation, migration, invasion, epithelial-mesenchymal transition (EMT), apoptosis, and stemness ( Figure 1). As fundamental regulators of cancer progression via both oncogenic and tumor suppressor functions, ZFPs may also serve as therapeutic targets [7]. In this review, we discuss the roles of ZFPs in the development and progression of CRC, as either tumor suppressors or oncogenes, and highlight potential points of therapeutic intervention.
biological functions [6]. Hence, zinc finger proteins (ZFP) can act as transcriptional repressors and activators for a wide array of genes, thus modulating a multitude of biological processes, including cell differentiation, cell proliferation, migration, invasion, epithelial-mesenchymal transition (EMT), apoptosis, and stemness ( Figure 1). As fundamental regulators of cancer progression via both oncogenic and tumor suppressor functions, ZFPs may also serve as therapeutic targets [7]. In this review, we discuss the roles of ZFPs in the development and progression of CRC, as either tumor suppressors or oncogenes, and highlight potential points of therapeutic intervention. Figure 1. The roles of zinc finger proteins (ZFPs) in maintaining cellular homeostasis. ZFPs contain a zinc finger motif, vital for binding DNA and executing transcriptional regulation of target genes involved with mechanisms that maintain normal cellular physiology (i.e., signaling, DNA damage response, etc.). This figure illustrates the mechanisms whereby ZFP regulation of target gene transcriptional activity alters key cellular phenotypes. Created with Biorender.com. (Table 1) Most ZFPs implicated in CRC progression are involved in manipulating the cellular phenotype to promote proliferative and invasive behavior and achieve metastatic potential through multiple mechanisms. Figure 1. The roles of zinc finger proteins (ZFPs) in maintaining cellular homeostasis. ZFPs contain a zinc finger motif, vital for binding DNA and executing transcriptional regulation of target genes involved with mechanisms that maintain normal cellular physiology (i.e., signaling, DNA damage response, etc.). This figure illustrates the mechanisms whereby ZFP regulation of target gene transcriptional activity alters key cellular phenotypes. Created with Biorender.com. (Table 1) Most ZFPs implicated in CRC progression are involved in manipulating the cellular phenotype to promote proliferative and invasive behavior and achieve metastatic potential through multiple mechanisms.
Non-canonical Wnt pathways encompassing cascades independent of the β-catenin/ TCF complex classically control cell migratory and polarity phenotypes; the two most common non-canonical pathways are the planar cell polarity and calcium-activated pathways [9,125].
Numerous ZFPs downregulate Wnt signaling by different mechanisms, thereby attenuating CRC cell motility and invasive behavior. This can occur by restricting the expression of EMT-promoting genes, e.g., blocking pro-EMT Wnt signaling via OVOL2-mediated recruitment of histone deacetylase to the TCF4-β-catenin complex [10]. FLYWCH1 stimulates reduced cell motility and enhances cell attachment through β-catenin interactions and transcriptional modulation of β-catenin/TCF4 to impede the expression of downstream genes including ZEB1, EPHA4, and E-cadherin [13]. ZFP36, negatively correlated with Wnt/β-catenin signaling activity, represses the expression of EMT-related transcription factors including MACC1, ZEB1, and SOX9 [14]. While R-spondin proteins usually enhance Wnt signaling through ZNRF3 turnover, RSPO2 promotes the stabilization of membraneassociated ZNRF3 through LGR5-dependent interactions [15]. These interactions reduce CRC cell motility and proliferation, likely by inhibiting the Wnt5a-activated non-canonical pathway [16]; this highlights the complexity and interactions between signaling pathways underlying CRC pathogenesis.
ZNF277, a senescence-regulating transcription factor upregulated in CRC, is also a transcriptional target of β-catenin. Previous work by our group demonstrated that ZNF277 mRNA and protein expression modulate key cancer pathways, including the HOXD family and p21 WAF1 , and play a key role in M 3 muscarinic receptor-dependent murine CRC progression [30,31].

ZFPs Modulate Other Signaling Pathways
While Wnt signaling is a key modulator of colon cancer progression, many other pathways are also exploited by ZFPs. Notably, the Ras/ERK pathway, mutated in many cancers, is inhibited by ZC3H13, an anti-oncogene downregulated in CRC, thus attenuating cell proliferation and invasion [33]. Conversely, ZDHHC9, which is upregulated in CRC, potentially modulates the N-Ras and H-Ras pathway via its palmitoyl transferase activity; the precise mechanism of how this promotes tumorgenicity remains obscure [34].
Activation of intracellular Janus kinases (JAKs) via ligand-mediated receptor activation leads to downstream phosphorylation and dimerization of STAT proteins; this STAT complex functions as a nuclear transcription factor that promotes the expression of genes beneficial for tumorigenesis [126]. For example, in addition to regulating the Ras/ERK pathway, ZNF143 controls the JAK/STAT pathway to alter the expression of IL-8, a proangiogenic cytokine, thereby curtailing tumor progression [35]. ZNF460 promotes CRC metastasis by stimulating JAK2/STAT3 signaling [37].
In the absence of an inhibitor, IκB, the NF-κB transcription factor, comprising p65 and p50 subunits, is translocated to the nucleus where it modulates the expression of target genes that promote cell proliferation, migration, and invasion [127]. Activation of NF-κB signaling can occur via transcriptional repression of ZCCHC10 by miR-410-3p [38]. Alternatively, ZFPs such as ZFP91 can interact with the p65 subunit of NF-κB to stimulate transcription of the alpha-subunit of the transcription factor hypoxia-inducible factor 1 (HIF-1α) to regulate processes that promote tumorigenesis, including cell proliferation, invasion, metastasis, angiogenesis, and cell cycle progression [39].
Phosphatidylinositide 3-kinase (PI3K) is activated downstream of many receptor types to phosphorylate cell membrane-adherent phosphoinositides. Following a series of subsequent recruitment and binding events, involving various enzyme complexes, the AKT kinase is ultimately turned on to induce mTOR, MDM2, and FOXO activation resulting in accelerated cell differentiation and proliferation [128]. miR-708-5p, an enhancer of PI3K/AKT signaling, negatively regulates ZNF549 to induce CRC cell proliferation and migration [40]. Interestingly, while ZNF549 expression in primary tumors is reduced compared to normal tissue, its expression is increased in advanced-stage tumors, suggesting additional roles for this ZFP in CRC progression [40]. Conversely, GLI1, a vital player in Hedgehog signaling, enhances PI3K/AKT signaling in a Foxm1-dependent manner to increase CRC metastasis [42], while ZNF692 promotes cell proliferation and invasion by altering the expression of proteins involved in cell cycle regulation and angiogenesis, including cyclin D1, CDK2, and p27 Kip1 , via the PI3K/AKT pathway [43]. ZBED6, a repressor of IGF2 expression, attenuates cell proliferation, likely via PI3K signaling, and has also been implicated as a modulator of the Wnt, Hippo, TGF, and EGFR pathways [44].
TGF-β ligand-receptor binding results in the phosphorylation of cytosolic SMAD proteins and the formation of SMAD multimer complexes that are translocated to the nucleus and bind specific DNA motifs, thus regulating target gene expression [129]. Interestingly, the enhancement of TGF-β signaling by ZNF37A-induced repression of the tumor microenvironment regulator THSD4 stimulates cytokine generation by cancer-associated fibroblasts to promote tumor spread [45]. TGF-β pathway activation to increase invasive potential can also occur via upregulation of ZNF326 target genes, e.g., LTBP4, a TGF-β1 receptor activator [46]. Both ZNF367 [47] and ZNF280A [48] inhibit the Hippo tumor suppressor pathway, augmenting Yes-associated protein (YAP) signaling to enhance CRC cell proliferation. ZMYND8, a multifunctional transcription factor, histone reader, and DNA repair protein, is also a target of YAP-mediated signaling that induces the cholesterol synthesis necessary for CRC cell proliferation [50].

ZFPs Modulate Epigenetic Modifications
Epigenetic modifications are broadly characterized by altered gene expression in the absence of DNA sequence changes. The most well studied modifications involved in CRC pathogenesis include altered target gene promoter methylation status and/or recruitment of histone modifiers (i.e., acetylators and methylators) to alter gene expression levels [130]. Non-coding RNAs such as long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) have also emerged as epigenetic modulators [131]. Promoter methylation often inactivates or downregulates target tumor suppressor ZFPs, including ZBTB18 [51], GATA4/GATA5 [52], and ZNF677 [53], to promote CRC pathogenesis via CRC cell proliferation and invasion. This can also occur via promoter hypermethylation, as seen with ZFP82, which normally interacts with the corepressor KAP1, to attenuate rRNA transcription and ribosome biogenesis via histone deacetylation [54], and SPOP, which facilities polyubiquitination and subsequent degradation of histone deacetylase 6 (HDAC6) to reduce deacetylation of non-histone proteins and prevent EMT [56,57]. Interestingly, PRDM5, a tumor suppressor underscored by its ability to inhibit cell proliferation after its transfection into human CRC cells, is also inactivated by trimethylation of histone H3 lysine [58]. Conversely, UHRF1, an E3 ubiquitin ligase, can initiate and maintain DNA hypermethylation to silence tumor suppressor genes and promote metastasis [59].
Interactions with histone deacetylases by ZFPs can also promote CRC metastasis. MTA1, whose expression is increased in CRC liver metastases, was shown to interact with histone deacetylase and modulate chromatin structure and transcriptional repression [61]. Similarly, MORC2 interacts with the histone deacetylase Sirutin1 to suppress downstream Myc-regulated gene 1N (NDRG1) transcription, ultimately promoting EMT and CRC lung metastasis [62]. Alternatively, ZNF518B expression promotes CRC metastasis and recurrence by recruiting histone methyltransferases (e.g., EZH2 and G9A) to silence downstream tumor suppressor genes [63]. ZNF146 enhances CRC cell proliferation and motility [65] by telomere dysregulation through interactions with hRAP, a telomeric protein that modulates telomere length [64]. Heterochromatin silencing via heterochromatin protein 1 interactions is a potential mechanism whereby ZNF382 inhibits tumorigenesis by altering the expression of oncogenes and other factors involved in NF-κB signaling [66].
Epigenetic modifications of DNA or chromatin are not the only mechanisms utilized to induce CRC oncogenicity. KLF5, a downstream mediator of activated K-Ras and H-Ras, targets lncRNAs to modulate the expression of protein-coding genes via interactions with co-regulatory transcription factors, such as AR and HSF1 [67]. Similarly, Sp1 induces expression of the lncRNA ZFAS1, which binds miR-150-5p, to upregulate VEGFA and subsequently promote cell proliferation, migration, and invasion [68,69]. Primary tumors and metastases overexpress THAP11 which binds to host cell factor-1 (HCF-1), a transcriptional coregulator, to target promoters, ultimately modulating transcription and cell proliferation via histone modifications [70].

ZFPs and the DNA Damage Response
Evasion of DNA damage response pathways during cell growth and proliferation is important for CRC progression. Non-homologous end joining (NHEJ) and homologous recombination (HR) are the major double-strand DNA break repair pathways, the latter requiring proto-oncogenes BRAC1 and BRCA2 for successful repair [132]. Other types of DNA damage, such as single-stranded DNA mishaps, utilize mismatch repair (MMR) and base excision repair (BER) to repair or remove mismatched nucleotides [133]. Defects in any of these pathways contribute to genomic instability and the accumulation of mutations that promote cancer cell phenotypes. ZEB1, a well-studied ZFP, promotes colitis-associated CRC by repressing transcriptional repair molecules, including N-methyl-purine glycosylase, involved in the BER pathway. ZEB1 upregulation in CRC cells also stimulates macrophages to generate reactive oxygen species and IL-1β, which results in a positive feedback loop of increased DNA damage and impaired DNA repair [78].
KLF4 counteracts the suppressive effects of p53 on NHEJ and HR mechanisms to modulate DNA repair [74], and in colitis-associated CRC models, it maintains genomic stability by directing p53 to centrosomes [72]. Meanwhile, the inactivation of the DNA helicase protein HLTF in CRC promotes genomic instability and subsequent malignant transformation [81].

ZFPs Modulate p53 Levels
To maintain cell homeostasis through regulation of the cell cycle, DNA repair response, and cell death pathways, p53, a vital tumor suppressor, modulates numerous cell processes via its role as a transcription factor. p53 levels are tightly regulated by numerous proteins, including MDM2, an E3 ubiquitin ligase responsible for p53 degradation, thus maintaining p53 levels appropriate for the cell state [134]. ZBTB7A, a tumor suppressor [84], can also function as an oncogene by downregulating p53, thereby enhancing ETS proto-oncogene 1 (ETS-1) function and stimulating CRC cell proliferation and invasion [82]. Both GLI3 [86] and the p52 isoform of ZFP398 [88] help stabilize the p53-MDM2 complex by binding to p53 and MDM2. The resulting ubiquitination and exosomal removal of p53 facilitate neoplasia and cell proliferation [88].

ZFPs Modulate E-Cadherin Expression
The transmembrane glycoprotein E-cadherin, a well-studied cell-adhesion molecule, is commonly expressed in epithelial cells to prevent metastatic behavior [135]. In CRC, SNAI1 upregulation induces EMT and metastasis by inhibiting transcription and subsequent expression of the vitamin D receptor and E-cadherin, and promoting β-catenin nuclear translocation to potentiate Wnt pathway signaling [89]. SPRY2, a receptor tyrosine kinase modulator, also downregulates vitamin D 3 -dependent E-cadherin expression to augment cellular invasion and de-differentiation [92]. NANOS1, a downstream target gene of Ecadherin, can also compromise the anti-migratory properties of E-cadherin. hNanos1, inversely correlated with E-cadherin expression, induces cytoplasmic translocation of p120 catenin (p120ctn), a regulatory protein that complexes with cadherin proteins at the cell membrane to maintain adherens junctions, subsequently disrupting cell-cell adhesion stability [93]. However, to attenuate cell migration and invasion, ZC3H12C overexpression enhances E-cadherin expression and suppresses vimentin expression in human CRC cells without altering cell survival [94].

ZFPs Modulate CRC Cell Metabolism
As opposed to metabolic pathways such as oxidative phosphorylation, CRC cells, like many other cancer cell types, utilize aerobic glycolysis for energy generation. Glutaminolysis, the breakdown of glutamine, is upregulated in CRC to maintain anaplerosis, which sustains glycolytic pathways needed for tumor cell growth and proliferation [136]. ZBTB7C, downregulated in CRC, is thought to block Myc and tumor cell glutaminolysis, thus increasing immune cell proliferation due to the glutamine surplus in the tumor microenvironment and attenuating CRC cell proliferation [98]. Enhanced tumor cell aerobic glycolysis is also exploited by several oncogenes to promote cell proliferation and metastasis, e.g., YY1, via GLUT3 transcription upregulation [95], and ZFP1, via p53-dependent glycolysis [99]. ZNF568 can accomplish this by inhibiting p53-mediated mitochondrial metabolism [99].

ZFPs Modulate the Expression of Factors That Stimulate Angiogenesis
A major mechanism underlying CRC metastasis is angiogenesis, the formation of new blood vessels, via modulation of vascular endothelial growth factor (VEGF) signaling [137]. VEGF targets tyrosine kinase receptors to regulate downstream signaling cascades that alter pro-angiogenic behavior such as vascular permeability and cell survival [137]. The availability of VEGF can be upregulated by matrix metalloproteinase-2 (MMP2), a multifunctional protein in cancer [138]. ZNF384 [100], SNAI2 [103], and ZEB2 [107] are upregulated in CRC and enhance MMP2 expression and/or activity, promoting angiogenesis. SNAI2, which expedites p53/p21 degradation by upregulating MDM2 [101], is essential for mutant-KRAS cancer cell survival after EMT [104]. ZNF24 represses VEGF expression [109] to suppress angiogenesis, while ZKSCAN3, an inducer of VEGF to promote CRC development and invasion, is implicated in carcinoembryonic antigen (CEA)-producing tumor liver metastasis [110].

ZFPs Coordinate Cell Cycle Regulation and Apoptotic Mechanisms in CRC (Table 2)
Cell cycle regulation, essential to maintain cell integrity, is monitored at key checkpoints. This process is highly dependent on cyclins, cyclin-dependent kinases (CDKs), and p53, which regulate cell cycle progression and prevent inappropriate cell growth and genomic replication [139]. Apoptosis, induced by cell stress signals, can occur via either the intrinsic or extrinsic pathway; the former is mediated by mitochondrial and Bcl-2 proteins, and the latter is executed by activation of cell death receptors (e.g., TRAIL). Both pathways ultimately activate caspase signaling cascades that promote programmed cell death [140]. Evading cell cycle arrest and apoptosis is essential for CRC cell survival and proliferation and tumor expansion and spread.

ZFPs and Cell Cycle Checkpoint Regulation
The backbone of cell cycle regulation consists of CDKs activated by cyclins in the presence of mitogenic signals [176]. ZFP36L1 and ZFP36L2, both downregulated in CRC, are postulated to promote G1-phase cell cycle arrest without triggering cell death, likely by downregulating cyclin D expression [141]. Other regulatory components, such as checkpoint kinase 1 (Chk1), are vital to ensure cell cycle arrest until DNA damage is repaired [139]. To achieve G2-M cell cycle arrest, XAF1 activates Chk1 and then inactivates Cdc25C, a CDK activator, and the Cdc2-cyclin B complex [143].

Homeostasis Governed by the Bcl-2 Protein Family Can Be Modulated by ZFPs
Bcl2 family proteins, comprising pro-and anti-apoptotic proteins, are differentially regulated to maintain cellular homeostasis. When apoptosis is induced by cell stress, anti-apoptotic proteins (e.g., Bax, Bcl-x L ) are upregulated by p53-induced BH3-only proteins, which target mitochondrial proteins to initiate caspase activation and programmed cell death [140]. ZIC1, downregulated by promoter hypermethylation in CRC, induces apoptosis by triggering the Bcl-x L /Bad/Caspase 3 cascade [149]. MECOM upregulation in CRC inhibits apoptosis by inducing Bcl-x L protein transcription and augmenting cells in the G0/G1 phase [151]. CPEB4 promotes CRC development by downregulating Bax and increasing Bcl-x L expression, thus suppressing apoptosis [154].

Epigenetic Modifications Modulated by ZFPs
As discussed above, epigenetic modifications provide a common mechanism to regulate ZFP function and can be manipulated by ZFPs to suppress CRC progression. Both ZBTB33 [157] and ZNF304 [158] promote tumor suppressor gene silencing by directly binding to CpG island methylator phenotype gene promoters and recruiting corepressor complexes to methylate target genes, thereby preventing CRC cell cycle arrest. In contrast, to induce apoptosis in an exosome-dependent manner, ZC3HAV1 binds directly to and degrades the mRNA transcript of anti-apoptotic TRAIL decoy receptor 4 (TRAILR4) [159]. Interestingly, the tumor-suppressive effects of ZC3HAV1 are dictated by the tumor microenvironment. In the presence of TRAIL signals, ZC3HAV1 stimulates apoptosis, but in their absence, ZC3HAV1 suppresses cell growth. PRDM2, frequently inactivated by DNA methylation, has been shown to promote cell apoptosis and G2/M arrest in colon cancer cells [160].

Other Mechanisms Whereby ZFPs Modulate CRC Progression
ZFPs can modulate CRC pathogenesis by modulating cell signaling and dependency on the tumor microenvironment. PLAGL1, an anti-proliferative gene, modulates apoptosis and cell cycle arrest in a PPARγ-dependent manner by upregulating the expression of target genes involved in cell growth [162]. KLF9 inhibits ISG15, an apoptosis-inhibiting cytokine, thereby suppressing tumorigenesis [164], while ZFX downregulates DUSP5, permitting constitutive MAPK signaling, to promote apoptotic resistance and cell cycle progression [166].
ZNF746 upregulation in CRC likely inhibits GSK3β and FWB7 to modulate sitespecific phosphorylation and dephosphorylation of oncogenic c-Myc, an action that blocks c-Myc ubiquitination and degradation to ultimately evade G1 cell cycle arrest [168]. RBBP6 upregulation in CRC facilitates MDM2-mediated p53 ubiquitination and degradation to promote tumorigenesis [169].
GLI2 activation by HIF-1α and cancer-associated fibroblast-secreted TGF-β2 recruits anti-apoptotic molecules, allowing evasion of chemotherapy-induced apoptosis in a hypoxic tumor microenvironment [170]. To augment cell migration and retard apoptosis, GLIS2, a GLI2-related KLF protein, inhibits transcription of the apoptotic gene PUMA and focal adhesion genes (e.g., cadherins), likely by modulating acetylation levels of gene enhancers [171]. (Table 3) Maintenance of stemness allows the self-renewal, differentiation, and propagation of a specific cell lineage. Cancer cells adopt this phenotype to enhance cell survival, cloning, and proliferation [177]. In CRC, many ZFPs contribute to this stem-like maintenance by manipulating Wnt signaling and epigenetic pathways. O Migration and invasion [188] O = oncogene; the NCBI Gene database was utilized to determine common alias(es).

ZFPs Modulate Wnt Signaling
To maintain stemness, upregulation of JADE3 expression in CRC potentiates canonical Wnt signaling and subsequent transcriptional upregulation of the stem cell regulator and marker LGR5 by recruiting histone acetylases at the LGR5 promoter [178]. Conversely, PRDM1 contributes to cancer cell stemness by preferential regulation of the non-canonical Wnt pathway, specifically the planar cell polarity pathway, detected by the presence of upregulated downstream targets such as Wnt5A and Fzd4 [179]. SALL4 was shown to promote metastasis [180] and stem cell self-renewal via its ability to modulate GLI1 expression [181] and potentiate the canonical Wnt/β-catenin pathway by direct interactions with β-catenin [182].

ZNF Structure and Function in CRC
In addition to highlighting the vital functions of ZFPs in CRC pathogenesis, it is important to recognize the role ZNF motifs play as core domains enabling ZFPs to execute specific tasks. ZNF motifs house consensus sequences commonly comprising cysteine (Cys) and histidine (His) residues coordinated by one or more zinc ions. The configuration of the ZNF structural fold varies depending on the combination and location of coordinated residues within the consensus sequence. Thirty types of ZNFs are classified based on structural composition, each type likely contributing to specific ZFP functions [189], particularly in CRC progression.
The majority of ZNFs belong to the Cys2His2 (C2H2)-type family. This ZNF family structurally comprises a single α-helix and anti-parallel β-sheet containing pairs of Cys and His residues coordinated by one zinc ion, which is necessary for target DNA sequence recognition and binding [190]. ZNF281, KLF4, and SNAI2 are among several ZFPs that harbor C2H2-type domains and modulate Wnt signaling, DNA damage response mechanisms, and angiogenesis, respectively, to modify CRC cell behavior [189].
E3-ubiquitin ligases, an abundant type of ZFP, can possess RING finger domains, characterized by a consensus sequence containing multiple Cys residues and a single His residue coordinated by two zinc ions. This domain is vital for ZNRF3, which regulates cytoplasmic β-catenin levels to negatively modulate Wnt signaling and cell proliferation [8]. UHRF1 possesses both RING finger and PHD domains responsible for silencing CRC metastatic tumor suppressor genes via epigenetic modifications [189].

Conserved Mutations in C2H2-Type ZFPs in CRC
The structural importance of ZNF motifs in ZFP function in CRC is evidenced by the functional consequences of ZNF-localized mutations. Computational and functional genomic analyses of CRC tumor samples reveal a high somatic mutation frequency at specific, conserved amino acid residues within C2H2-containing ZFP transcription factors, including ZNF382, ZNF281, and ZEB1 [191]. The most common mutation, an arginine-toisoleucine missense mutation at position 9, is predicted to alter the necessary orientation of the ZNF with respect to neighboring domains and impair the specificity of target DNA recognition and binding [191]. This, in concert with the finding that adjacent residues (i.e., position 8) are not frequently mutated in CRC [191], highlights the integral role of highly conserved residues in maintaining crucial ZNF structure and ZFP functions. It is possible that there is a threshold for the types of ZNF mutations tolerable for cells, particularly for ZFPs necessary for essential cellular functions. Additional studies are necessary to elucidate the molecular underpinnings by which such mutations modify ZFP-specific functions and downstream target gene expression; this will help lay the groundwork for personalized, targeted therapeutic development.

Evaluating the Potential of ZFPs as CRC Therapeutic Targets
As these multidimensional proteins play diverse roles in promoting CRC, ZFPs are poised to serve as targets for therapeutic development. A potential approach is the design and use of metallo-compounds to alter zinc coordination properties within the crucial ZNF motif. Several metal-based compounds are approved to treat various conditions, including rheumatoid arthritis, cancer (e.g., cisplatin), and parasitic infestation [192]. In vitro studies targeting SP1 [193] and ZFP36 [194] with copper complexes have demonstrated therapeutic potential by diminishing chemoresistance and modulating inflammation, respectively.
Future studies must evaluate the potential side effects of metal-complex treatments and monitor for metal toxicity. Another potential approach to modulate ZFP activity in CRC is to repurpose thalidomide analogs, already approved to treat select hematologic cancers. These small-molecule drugs degrade C2H2-type ZFPs by activating the E3 ubiquitin ligase CRL4 CRBN in vitro [195]. The use of thalidomide analogs to target oncogenic C2H2containing ZFPs involved in promoting CRC may be a promising avenue of exploration.

Current Limitations to Targeting ZFPs for CRC Therapy
While we highlighted specific roles of ZFPs throughout this review, it is important to note that these proteins play multiple roles in the promotion of tumorigenic cell phenotypes (Tables 1-3). The ability of ZFPs to cross-regulate multiple different mechanisms, including signaling pathways, cell cycle checkpoints, and angiogenic markers, demonstrates the complexity of investigating the role of distinct ZFPs in CRC pathogenesis and identifying ZFPs as targets with therapeutic potential. While altered expression of certain ZFPs is linked to CRC survival (Figure 2), the precise underlying mechanisms remain elusive for most proteins. Additionally, there are conflicting data regarding whether some ZFPs, e.g., SALL1 [185,196], ZIC5 [197,198], ZNF148 [199][200][201], and ZNF750 [202,203], are CRC tumor suppressors or oncogenes; this raises the possibility they have dual functions depending on tumor stage-additional studies are needed to explore this possibility.

Current Limitations to Targeting ZFPs for CRC Therapy
While we highlighted specific roles of ZFPs throughout this review, it is important to note that these proteins play multiple roles in the promotion of tumorigenic cell phenotypes (Tables 1-3). The ability of ZFPs to cross-regulate multiple different mechanisms, including signaling pathways, cell cycle checkpoints, and angiogenic markers, demonstrates the complexity of investigating the role of distinct ZFPs in CRC pathogenesis and identifying ZFPs as targets with therapeutic potential. While altered expression of certain ZFPs is linked to CRC survival (Figure 2), the precise underlying mechanisms remain elusive for most proteins. Additionally, there are conflicting data regarding whether some ZFPs, e.g., SALL1 [185,196], ZIC5 [197,198], ZNF148 [199][200][201], and ZNF750 [202,203], are CRC tumor suppressors or oncogenes; this raises the possibility they have dual functions depending on tumor stage-additional studies are needed to explore this possibility. Many studies performed to understand the pathways and signaling mechanisms by which ZFPs suppress or promote CRC behavior stem are limited by the reliance on cell lines or murine models to draw conclusions regarding human disease. Additional work using human tissue, perhaps organoids, will help confirm their putative roles and mechanisms of action. While ZFPs play a complex role in CRC, ZFP lncRNA antisense (AS) transcripts are also emerging as players. ZFAS1 [204], ZEB2-AS1 [205], and ZEB1-AS1 [206] are among the lncRNA transcripts upregulated in CRC. Future studies investigating ZFPs, and their AS transcripts, will expand our understanding of their role in CRC development and progression and the potential to develop novel therapeutics based on this information.

Conclusions
ZFPs play key roles in CRC development and progression, primarily acting as oncogenes or tumor suppressors, by regulating cell proliferation, senescence, apoptosis, cell cycle, EMT, cell migration, invasion, stemness, and Wnt signaling. Future research can be directed at understanding the interactions between various ZFPs and identifying those with the most important effects on CRC metastasis and survival, thus identifying novel therapeutic opportunities.  Many studies performed to understand the pathways and signaling mechanisms by which ZFPs suppress or promote CRC behavior stem are limited by the reliance on cell lines or murine models to draw conclusions regarding human disease. Additional work using human tissue, perhaps organoids, will help confirm their putative roles and mechanisms of action. While ZFPs play a complex role in CRC, ZFP lncRNA antisense (AS) transcripts are also emerging as players. ZFAS1 [204], ZEB2-AS1 [205], and ZEB1-AS1 [206] are among the lncRNA transcripts upregulated in CRC. Future studies investigating ZFPs, and their AS transcripts, will expand our understanding of their role in CRC development and progression and the potential to develop novel therapeutics based on this information.

Conclusions
ZFPs play key roles in CRC development and progression, primarily acting as oncogenes or tumor suppressors, by regulating cell proliferation, senescence, apoptosis, cell cycle, EMT, cell migration, invasion, stemness, and Wnt signaling. Future research can be directed at understanding the interactions between various ZFPs and identifying those with the most important effects on CRC metastasis and survival, thus identifying novel therapeutic opportunities.

Conflicts of Interest:
The authors declare no conflict of interest.