*Review* **Zinc Fingers and Homeobox Family in Cancer: A Double-Edged Sword**

**Yonghua Bao <sup>1</sup> , Haifeng Zhang <sup>1</sup> , Zhixue Han <sup>1</sup> , Yongchen Guo 2,\* and Wancai Yang 3,\***


**Abstract:** The zinc fingers and homeobox (ZHX) family includes ZHX1, ZHX2, and ZHX3, and their proteins have similar unique structures, containing two C2H2-type zinc finger motifs and four or five HOX-like homeodomains. The members of the ZHX family can form homodimers or heterodimers with each other or with a subunit of nuclear factor Y. Previous studies have suggested that ZHXs can function as positive or negative transcriptional regulators. Recent studies have further revealed their biological functions and underlying mechanisms in cancers. This review summarized the advances of ZHX-mediated functions, including tumor-suppressive and oncogenic functions in cancer formation and progression, the molecular mechanisms, and regulatory functions, such as cancer cell proliferation, migration, invasion, and metastasis. Moreover, the differential expression levels and their association with good or poor outcomes in patients with various malignancies and differential responses to chemotherapy exert opposite functions of oncogene or tumor suppressors. Therefore, the ZHXs act as a double-edged sword in cancers.

**Keywords:** zinc fingers; homeoboxes; ZHX1; ZHX2; ZHX3; cancer

**1. Introduction**

The zinc fingers and homeobox (ZHX) family have three members, including ZHX1– 3 [1–4]. They share similar gene and protein structures [5]. ZHX family members contain a unique protein structure, two C2-H2 (Cys-Xaa2-Cys-Xaa12-His-Xaa4-His) zinc finger motifs and four or five HOX-like homeodomains (HDs).

The humansZHX1 and ZHX2 are located on chromosome 8 [1,4], and ZHX3 is located on chromosome 20 [6]. ZHX1–3 can constitute homodimers or heterodimers and can interact with the alpha subunit of nuclear factor Y (NFYA) to repress transcription [1–3,6–8].

#### *1.1. ZHX1 Gene and Biological Functions*

ZHX1 was initially identified from a murine bone marrow stromal cell [9]. Human ZHX1 was identified by screening a human liver cDNA library for NFY-interacting protein isolation [4]. The ZHX1 gene of humans is located in Chr8q24.13, spanning approximately 27 kb. ZHX1 has eight transcripts, including two main ones, 4.5 kilobases (kb) and 5 kb, respectively. These two transcripts are expressed ubiquitously, although the latter is more abundant in most tissues [4].

Human ZHX1 contains 873 amino acid residues and is structurally composed of two C2-H2 motifs and five homeodomains [4]. This is why it is classified as a zinc finger of homeodomain transcription factor. ZHX1 forms homodimers or heterodimers through the homeodomain 1 region (amino acids 272–432). ZHX1 functions as a transcriptional repressor via an acidic region (amino acids 831–873). Achieving complete inhibitory activity requires dimerization [7].

ZHX1 proteins of humans and mice possess 91% amino acid similarity [8]. The ZHX1 gene of mice is located in chromosome 15, approximately 29 kb in length [2]. ZHX1 mRNA

**Citation:** Bao, Y.; Zhang, H.; Han, Z.; Guo, Y.; Yang, W. Zinc Fingers and Homeobox Family in Cancer: A Double-Edged Sword. *Int. J. Mol. Sci.* **2022**, *23*, 11167. https://doi.org/ 10.3390/ijms231911167

Academic Editor: Laura Paleari

Received: 8 August 2022 Accepted: 19 September 2022 Published: 22 September 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

is widely expressed in adult mice, with a high expression in the brain and lower levels in the liver and kidney [9]. It has been reported that the mouse ZHX1 gene expression could be induced by IL-2 in CTLL-2 cells [10].

Besides interacting with NFYA, ZHX1 binds DNA methyltransferase 3B [11] and the transcriptional corepressor BS69 to induce repression activity [12].

#### *1.2. ZHX2 and Biological Functions*

Human ZHX2 was cloned as a novel ubiquitous transcription factor and a ZHX1 interacting protein from a yeast two-hybrid screen on a size-fractionated brain cDNA library [1]. The ZHX2 gene is located on Chr8q24.13, spanning approximately 193 kb. This gene has two transcripts (splice variants) coding 837aa and 166aa. ZHX2 mRNA is expressed in various tissues, with the highest levels in the ovaries, and sequentially lower levels in the lung, heart, kidney, brain, and liver. The pancreas, spleen, testis, and skeletal muscle have intermediate expression levels. A genome-wide association study has demonstrated that individuals with the ZHX2 polymorphism G779A have a strong response to the smallpox vaccine [13].

The mouse ZHX2 gene encodes an 836aa protein, having 87% amino acid identify with the human ZHX2 protein [14]. ZHX2 contains two C2-H2 zinc finger motifs and five HDs. This region, called the P domain, is rich in proline and resides between HD1 and HD2. ZHX2 forms a homodimer with itself or a heterodimer with ZHX1 at the region containing HD1. In addition, ZHX2 can interact with NFYA at the region between HD1 and HD2. Further studies have revealed that nuclear ZHX2, not cytoplasmic ZHX2, acts as a transcriptional repressor. The repressor domain resides in a region between the HD1 and P domains [1,3].

ZHX2 was previously thought to suppress gene transcription. However, in vitro and in vivo studies showed that ZHX2 positively regulates major urinary proteins' (Mups) gene expression [15].

#### *1.3. ZHX3 and Biological Functions*

The human ZHX3 gene is located on Chr20q12, spanning approximately 139 kb, and has 21 transcripts (splice variants). ZHX3 has 956 amino acid residues and, like ZHX1, contains two C2-H2 motifs and five HDs [6]. ZHX3 forms a heterodimer with ZHX1 and interacts with NFYA. The pleiotropic HD1 region is responsible for the dimerization with ZHX1, interaction with NFYA, and repressor function. In addition, two nuclear localization signals were mapped to the N-terminus at zinc fingers 1 and the HD2 region. Moreover, protein–protein interaction assays showed that ZHX3 formed a heterodimer with ZHX2 via a region containing HD1. In mice, there are three ZHX3 transcripts of 9.5 kb, 6.5 kb, and 4.4 kb in length, which are ubiquitously and differentially expressed. Functional studies have revealed that nuclear ZHX3 is a ubiquitous transcriptional repressor and functions as a dimer [8,16].

Accumulating evidence has demonstrated that ZHX family members play important roles in cell development, differentiation, and various cancers [17–22]. The aberrant expression and dysfunction of ZHXs are associated with the occurrence and progression of various diseases. These include hematological, neurological, and glomerular diseases, as well as carcinogenesis and its progression [22]. The expression levels are also linked to the outcomes of several malignancies. Interestingly, the biological functions of ZHXs in cancers are not consistent. For example, ZHXs act as tumor suppressors or oncogenes in different types of cancers. The expression levels of ZHXs are also associated with poor or good outcomes in patients with various malignancies, and act as a "double-edged sword". This review focuses on the clinical significance of the oncogenic or tumor-suppressive functions of ZHX family members in cancers.

#### **2. Oncogenic or Tumor-Suppressive Functions of ZHXs in Cancers**

#### *2.1. ZHX1 and Cancer*

ZHX1 has recently been involved in the tumorigenesis of various tumors. The regulation of ZHX1 expression, its clinical significance, and role in cancer is controversial. ZHX1 acts as an oncogene or tumor suppressor in different types of cancer (Table 1). ZHX1 contributes to cancer cell proliferation, mobility, migration, and invasion (Figure 1). In glioblastoma [23], the messenger RNA and protein expression of ZHX1 are increased compared with normal brain tissue, and ZHX1 overexpression was associated with short overall survival. In vitro, the knockdown of ZHX1 expression suppressed cell proliferation, mobility, migration, and invasion in glioblastoma. However, increasing the expression of ZHX1 enhanced malignant features, which are linked to the regulation of TWIST and SNAIL, both of which are transcriptional factors for the epithelial–mesenchymal transition (EMT) and metastasis.

It has been reported that long non-coding RNA (lncRNA), microRNA (miRNA), and ZHX1 participate in tumor initiation and progression. In vitro and in vivo studies have shown that LncRNA MALAT1 facilitates glioblastoma cell proliferation and progression by reducing miR-199a to promote ZHX1 expression. The LncRNA MALAT1-elevated expression is associated with the poor prognosis of glioblastoma patients [24]. Another study on glioma also showed that ZHX1 is an oncogene, and it was identified as a target gene of miR-23b-3p. LncRNA SNHG17 can absorb miR-23b-3p like a sponge to elevate ZHX1expression; therefore, increased SNHG17 and ZHX1 expressions and the reduced miR-23b-3p expression were found in glioma tissues. Oncogenic SNHG17 improves cell proliferation, migration, and invasion in glioma [25]. In addition, lncRNA LINC01140 promote the development of glioma by downregulating miR-199a-3p expression and indirectly upregulating ZHX1 expression [26]. These findings strongly suggest that targeting the lncRNA/miRNA/ZHX1 pathway might provide a new therapeutic strategy for tumors of the nervous system.

Another study has demonstrated the oncogenic roles of lncRNA DLG1-AS1/miR-107/ZHX1 axis in cervical cancer [27]. lncRNA DLG1-AS1 was remarkably overexpressed in cervical cancer tissues, and the cervical cancer patients with a high DLG1-AS1 expression have worse outcomes. Manipulating gene expression in vitro showed that the knockdown of the DLG1-AS1 gene inhibited the proliferation of cervical cancer cells. Mechanical studies showed that the inhibition of miR-107 abrogated DLG1-AS1-mediated ZHX1 expression via competitive binding between ZHX1 and miR-107. Vice versa, increasing the expression of miR-107 rescued the role of DLG1-AS1 in cervical cancer cell proliferation, indicating that the oncogenic effects of DLG-AS in cervical cancer are miR-107/ZHX1-dependent. ZHX1 also acts as an oncogene in cholangiocarcinoma [28]. Immunohistochemical staining analysis revealed that ZHX1 is amplified and overexpressed in cholangiocarcinoma tissues. In vitro studies showed that overexpressing ZHX1 facilitated cholangiocarcinoma cell proliferation, migration, and invasion. Conversely, ZHX1 knockdown using specific small interfering RNAs decreased malignant characteristics. Mechanistic studies showed that ZHX1-mediated tumor promotion might be partially associated with early growth response 1 (EGR1).



**Table 1.** Studies on ZHX1 expression, function, clinical significance, and molecular mechanism.

**Figure 1.** ZHX1 and cancers: biological functions and potential signaling networking. **Figure 1.** ZHX1 and cancers: biological functions and potential signaling networking.

In contrast, ZHX1 exerts tumor‐suppressive functions in gastric cancer [29], in which ZHX1 expression was reduced. Interestingly, the reduced nuclear expression of ZHX1 was closely related to a larger tumor size, poorer differentiation, advanced TNM stages, and a deeper invasion. However, these features were not correlated with lymph node me‐ tastasis. In cultured gastric cancer cells, stable transfection with plasmids overexpressing ZHX1 significantly promoted apoptosis, and inhibited cell proliferation and migration. In nude mice, the ZHX1 overexpression inhibited the tumor growth that was associated with cell cycle arrest and a repressed expression of cyclin D1. In contrast, ZHX1 exerts tumor-suppressive functions in gastric cancer [29], in which ZHX1 expression was reduced. Interestingly, the reduced nuclear expression of ZHX1 was closely related to a larger tumor size, poorer differentiation, advanced TNM stages, and a deeper invasion. However, these features were not correlated with lymph node metastasis. In cultured gastric cancer cells, stable transfection with plasmids overexpressing ZHX1 significantly promoted apoptosis, and inhibited cell proliferation and migration. In nude mice, the ZHX1 overexpression inhibited the tumor growth that was associated with cell cycle arrest and a repressed expression of cyclin D1.

Another study on gastric cancers reported by Wang et al. also showed that ZHX1 was a tumor suppressor and a downstream target of tumor promoter miRNA‐199a‐3p [30]. MiR‐199a‐3p expression was increased in gastric cancer tissues and gastric cancer cell lines. The study also demonstrated that miR‐199a‐3p could negatively regulate ZHX1 gene expression in gastric cancer cells. Therefore, increasing the expression of miR‐199a‐ 3p inhibits ZHX1 expression, but reducing miR‐199a‐3p expression promotes ZHX1 ex‐ Another study on gastric cancers reported by Wang et al. also showed that ZHX1 was a tumor suppressor and a downstream target of tumor promoter miRNA-199a-3p [30]. MiR-199a-3p expression was increased in gastric cancer tissues and gastric cancer cell lines. The study also demonstrated that miR-199a-3p could negatively regulate ZHX1 gene expression in gastric cancer cells. Therefore, increasing the expression of miR-199a-3p inhibits ZHX1 expression, but reducing miR-199a-3p expression promotes ZHX1 expression. Furthermore, restoring ZHX1 expression inhibits cancer cell proliferation in vitro.

pression. Furthermore, restoring ZHX1 expression inhibits cancer cell proliferation in vitro. A more recent study indicated that an increased ZHX1 expression was associated with a better or worse prognosis for patients with gastric cancer in the clinical stages, in‐ cluding lymph node metastasis or distant metastasis, and surgical therapy [31]. For in‐ stance, a lower ZHX1 expression was related to a better prognosis of patients without lymph node metastasis, whereas a higher ZHX1 expression was related to a better A more recent study indicated that an increased ZHX1 expression was associated with a better or worse prognosis for patients with gastric cancer in the clinical stages, including lymph node metastasis or distant metastasis, and surgical therapy [31]. For instance, a lower ZHX1 expression was related to a better prognosis of patients without lymph node metastasis, whereas a higher ZHX1 expression was related to a better prognosis of patients without distant metastasis. Furthermore, a higher ZHX1 expression was associated with a better prognosis of the patients who received surgery therapy and in the patients whose cancer showed poor differentiation.

A study on clear cell renal cancers (ccRCC) [18] based on an online database showed that both ZHX1 and ZHX3 expression levels were downregulated, while ZHX2 was upregulated. In particular, the ZHX1 and ZHX3 expression levels were remarkably lower, but ZHX2 expression was higher in the advanced stages compared with the early stages. In addition, ZHX1 and ZHX3 expression levels were associated with the T stage, while ZHX1 expression was associated with the M stage. These results suggest that a lower expression of ZHX1 and ZHX3 is related to the progression of clear cell renal cancers. Furthermore, Kaplan–Meier and multivariate regression analysis showed that a reduction in ZHX1 mRNA expression is associated with worse survival [18]. Altogether, these findings indicate that ZHX1 is a tumor suppressor in renal cancer.

In hepatocellular carcinoma (HCC), ZHX1 has also been reported as a tumor suppressor. One study [32] detected a decreased ZHX1 expression from a HCC tumor and cell sample. In vitro studies have indicated that an increased expression of ZHX1 suppresses HCC SMMC-7721 cell proliferation. Another study also showed the tumor-suppressive roles of ZHX1 in HCC, in which miR-199a-3p targeting the ZHX1/PUMA signal was reported to inhibit tumorigenesis. In this case, miR-199a-3p inhibited HCC HepG2 cell growth in vitro and induced apoptosis by upregulating the expression of ZHX1 and PUMA [33]. In contrast, the knockdown of ZHX1 or PUMA reversed miR-199a-3p-mediated tumor inhibition roles, as determined by a mechanistic investigation, suggesting that the miR-199a-3p/ZHX1/PUMA signaling could be linked to BCL2, BCL2-associated X, and cleaved caspase 3.

The prognostic impact of ZHX1 and ZHX2 in chronic lymphocytic leukemia (CLL) has recently been reported [34]. CLL patients with reduced ZHX1 and ZHX2 expression have a worse prognosis. In addition, the accumulation of chromosomal abnormalities was negatively associated with ZHX1 and ZHX2 expression in CLL patients.

More studies support the tumor-suppressive roles of ZHX1 in T-cell acute lymphoblastic leukemia (T-ALL), showing that ZHX1 was significantly reduced in T-ALL cells; moreover, other tumor suppressors, such as FOXN2 and FOXN3, were also concurrently downregulated in the T-ALL cell lines, in which both FOXN2 and FOXN3 formed a regulator network and directly activated the transcription of ZHX1 [35]. Furthermore, the physiological expression profile of normal hematopoietic cells revealed that ZHX1 was highly expressed in T-cells and was lower in B-cells, suggesting biased functions of ZHX1 in lymphopoiesis [36]. Lastly, the Hodgkin lymphoma cell lines that showed no expression of orthodenticle homeobox 1 and 2 (OTX1/2) (e.g., OTX 1 and 2 negative) overexpressed ZHX1, correlating with the genomic amplification of the 8q24 locus, supporting the oncogenic potential of ZHX1 in Hodgkin lymphoma [36].

#### *2.2. ZHX2 and Cancer*

Several studies have demonstrated the tumor-suppressor activities of ZHX2 in hepatocellular carcinoma (HCC) and other malignancies (Table 2), but accumulating evidence has also shown that ZHX2 plays oncogenic roles in carcinogenesis and progression in various cancers (Table 2).

*Int. J. Mol. Sci.* **2022**, *23*, 11167

