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1. Introduction

Acute myeloid leukemia (AML) is characterized by uncontrolled proliferation of myeloid progenitors that exhibit a severe block in their ability to differentiate into mature granulocytes or macrophages (1). The transcription factor CCAAT/enhancer binding protein α (C/EBPα) is a lineage-specific transcription factor in the hematopoietic system and is required for the formation of committed myeloid progenitors from multipotent precursor cells by coupling the direct transcriptional activation of myeloid-specific genes with the arrest of cell proliferation (2).

C/EBPα is specifically expressed in granulocytes, monocytes and eosinophils (3), although it is also found in hepatocytes, adipocytes and type II pneumocytes (4). Previous studies have illustrated the function of C/EBPα in hematopoiesis by promoting granulocyte and monocyte differentiation (2,5,6). Studies have eported that C/EBPα expression is detectable at a low level in the hematopoietic stem cell (HSC) population, and its expression increases as these cells develop into the common myeloid progenitor (CMP) and subsequently the granulocyte-monocyte progenitor (GMP), while conditional C/EBPα deficiency in adult mice blocked the transition from CMP to GMP, resulting in reduced formation of both granulocytes and monocytes (7). Non-conditional targeted disruption of C/EBPα was found to result in a selective block in early granulocyte maturation, and these mice died at birth due to severe hypoglycemia (8). Moreover, knock-in mice with a targeted mutation in the C/EBPα basic region, which led to its dysfunction, predisposed the mice to a myeloproliferative disorder (9). However, when expressed in 32Dcl3 cells, representative of granulocytic progenitors, exogenous C/EBPα promoted granulopoiesis (10). These studies suggest that C/EBPα is a critical regulator of myeloid development. In fact, growing evidence suggests that the function of C/EBPα is critically altered in subsets of AML patients based on various factors.

In this review, we summarized the fundamental role of C/EBPα in myeloid differentiation and the recently identified mechanisms of its activity.

2. Function of C/EBPα in myeloid differentiation

C/EBPα is a member of the basic leucine zipper (bZIP) transcription factor family, of which several members are also expressed in the myeloid lineage (e.g., C/EBPβ and C/EBPε) (2). In mammals, C/EBPα is a lineage-specific transcription factor that is required for the formation of committed myeloid progenitors from multipotent precursor cells. The C/EBPα molecule contains transactivation domains (TADs) at its N-terminus and a DNA-binding and dimerization bZIP structure at its C-terminus. Furthermore, C/EBPα is an intronless gene whose mRNA can be translated from two different AUG codons giving rise to two distinct isoforms (p42 and p30). p30 lacks two N-terminal transactivation domains that are only present on p42 (11) (Fig. 1). Unless otherwise indicated, C/EBPα represents the p42 isoform in the present review.

Figure 1 Structure of the two isoforms of C/EBPα. C/EBPα, CCAAT/enhancer binding protein α.

To date, numerous studies have been reported regarding the function of C/EBPα in AML. Firstly, genomic mutations have been detected in the C/EBPα gene in ~5–14% of AML patients (12,13). Among these mutations, N-terminal frameshift mutations prematurely truncate the full-length p42 form while preserving the p30 form, with the latter inhibiting the remaining wild-type C/EBPα p42 protein in a dominant-negative manner (12). In addition, C-terminal in-frame insertions or deletions were found to disrupt the basic zipper region, thus critically affecting DNA binding (12,14), which was further illustrated by reports that the critical basic region residues of the C/EBPα protein-DNA interaction were identified by an X-ray structure assay (14). Although there is a predominant C/EBPα mutation pattern in AML patients, the majority of AML patients always have more than one C/EBPα mutation (13,15). In addition, reduced expression could disrupt the function of C/EBPα for normal hematopoiesis (16). A detailed study showed that deletion of the C/EBPα gene led to arrest at the CMP to GMP transition, thereby causing reduced formation of both granulocytes and monocytes (7). In addition, studies have reported that C/EBPα heterodimerizes with AP-1 proteins (C/EBPα:c-Jun or C/EBPα:-c-Fos) for preference in directing monopoiesis (17), whereas C/EBPα homodimers cooperate with NF-κB p50 to promote granulopoiesis (18). Regulation of macrophage and neutrophil cell fates may also be altered by the relative PU.1:C/EBPα ratio (19).

3. Regulation of the C/EBPα signaling pathway

As a critical factor involved in myeloid differentiation, the function of C/EBPα must be tightly regulated to maintain the differentiation homeostasis. Detailed study of the regulation of C/EBPα could also highlight the understanding of the pathological mechanisms of AML.

Regulators targeting C/EBPα in myeloid differentiation and leukemogenesis

Many researches have focused on the factors regulating C/EBPα expression and its functions in leukemic diseases from different aspects, such as transcriptional repression by fusion genes, ubiquitination modification and epigenetic regulation.

Among the variant translocations in AML, the t(11:17) translocation is the most frequent, and renders resistance to all-trans retinoic acid (ATRA) treatment (20). After translocation, RARα is fused to PLZF to produce two fusion proteins, promyelocytic eukemia zinc finger-retinoic acid receptor α (PLZF-RARα) and RARα-PLZF, both of which participate in leukemia development (21). Among them, RARα-PLZF recruits HDAC1 and causes histone H3 deacetylation at C/EBPα target loci, thereby decreasing the expression of C/EBPα (Fig. 2). In line with this result, HDAC inhibitors were found to restore C/EBPα expression to a modest extent (22). In addition to RARα-PLZF, C/EBPα expression could also be downregulated through direct transcriptional repression by the fusion oncoprotein ML1-ETO (23). These findings provide molecular evidence for a mechanism through which fusion proteins act as modifier oncogenes that subvert differentiation in the granulocytic lineage by inhibiting the activity of C/EBPα.

Figure 2 PLZF-RARα negatively regulates C/EBPα expression. The fusion protein, PLZF-RARα, recruits HDAC1 and causes histone H3 deacetylation at the C/EBPα promoter, which further decreases the expression of C/EBPα. PLZF-RARα, promyelocytic eukemia zinc finger-retinoic acid receptor α; C/EBPα, CCAAT/enhancer binding protein α.

Epigenic modification is envisioned as an important epigenetic mechanism that regulates the expression of myeloid-specific genes in the hematopoietic system during leukemogenesis (24). Hypermethylation of the C/EBPα promoter was first reported preferentially in AML-M2 patients (25). The methylation status of the C/EBPα gene in chronic myeloid leukemia (CML) patients was also investigated, and the data suggested that aberrant methylation in the CpG island of the C/EBPα gene promoter could be a common event in CML (26). The role of EZH2 in hematopoietic development and leukemia is still controversial. Some groups suggest that EZH2 acts as a tumor suppressor in the myeloid lineage (27). However, it was also reported that ectopic expression of EZH2 causes a block in myeloid differentiation (28). A recent study found that EZH2 functions as an oncogene to block differentiation through suppressing C/EBPα expression. Notably, C/EBPα was found to be downregulated by EZH2 through methylation modification of its promoter in MA9-induced leukemia (29). Consistent with the above data, HDAC inhibitors restore C/EBPα target gene expression (22). A highly significant association was found between the frequency of C/EBPα gene epigenic modification and myeloid leukemia, while the role of C/EBPα methylation/deacetylation in the development, progression and prognosis in myeloid leukemia warrants further research.

Ubiquitination is an essential posttranslational modification for the modulation of C/EBPα activity. E3 ligases specifically targeting lysine 48 (K48)-linked ubiquitination of C/EBPα could promote the degradation of C/EBPα through the proteasome and thus terminate the downstream signaling transduction. Trib1 and Trib2 are two members of the Tribbles family that function as adapters to recruit E3 ubiquitin ligases and enhance ubiquitylation of its target protein. Consequently, Trib1 and Trib2 induce C/EBPα degradation and inhibit its function (30). E3 ubiquitin ligases, constitutively photomorphogenic 1 (COP1) (31) and E6-associated protein (E6AP) (32), were found to promote the degradation of C/EBPα by promoting its K48-linked polyubiquitination, thereby blocking myeloid differentiation of hematopoietic cells for tumorigenesis. Notably, during this process, COP1, which contains a COP1-binding motif, is recruited by Trib1 and is essential for downregulation of C/EBPα expression (31) (Fig. 3). However, whether any deubiquitinating enzymes exist to specifically remove K48-linked ubiquitination of C/EBPα and stabilize its expression or whether E3 ligases exist to induce K63-linked polyubiquitination to active C/EBPα function warrant further investigation. The answer to this question may help to elucidate the complexities of modulation of C/EBPα activity through ubiquitination/deubiquitination.

Figure 3 Regulation of C/EBPα by ubiquitin. E3 ubiquitin ligases, such as COP1 and E6AP, mediate C/EBPα proteasomal degradation, which further blocks myeloid differentiation. During this process, E3 ubiquitin ligases are recuited by Trib1 and Tirb2. C/EBPα, CCAAT/enhancer binding protein α; COP1, constitutively photomorphogenic 1; E6AP, E6-associated protein.

Downstream regulators targeting C/EBPα in myeloid differentiation and leukemogenesis

In addition to its important functions as a key target gene, several studies have demonstrated that C/EBPα may participate in leukemogenesis through regulation of a number of genes directly or indirectly.

To date, several genes have been identified as being directly regulated by C/EBPα in myeloid leukemia; for example, Sox4, which is critical for normal differentiation and expansion of the lymphoid and myeloid lineages (33,34). In normal hematopoiesis, C/EBPα expression was found to be increased over the course of lineage commitment and then suppressed SOX4 expression through binding to its promoter (35). Importantly, leukemic transformation by C/EBPα mutation was partially reversed by Sox4 knockdown (36).

FMS-like tyrosine kinase-3 (FLT3) is a membrane-bound tyrosine kinase receptor. The interaction between the receptor FLT3 and its ligand FL led to crucial signaling during the early stages of the commitment of hematopoietic stem cells (37). Mutation or overexpression of the FLT3 gene enhanced the survival and expansion in a variety of leukemias and was associated with an unfavorable clinical outcome for AML patients (38). Kindler et al demonstrated the binding of C/EBPα in human AML on the FLT3 locus, and defined FLT3 as a direct downstream effector of C/EBPα. Furthermore, they demonstrated that bi-allelic C/EBPα mutations may reduce FLT3-mediated leukemogenic signals (37), which suggests that regulation of Flt3 expression could depend on strict C/EBPα activity thresholds in AML.

In addition, glycolytic enzyme hexokinase 3 (HK3) has been defined as a glycolytic enzyme most frequently expressed in myeloid cells and represents the dominant hexokinase in granulocytes accounting for most hexokinase activity (39). By comparing gene profiles in two cohorts of C/EBPα wild-type and mutant AML patients, C/EBPα was found to directly regulate HK3 by binding to its promoter (40). Furthermore, activation of HK3 transcription was found to be dependent on C/EBPα during all-trans retinoic acid (ATRA)-mediated neutrophil differentiation of APL cells (41).

As a leukocyte-specific gene, CORO1A has been linked to the inhibition of neutrophil apoptosis, with significantly lower CORO1A mRNA expression in C/EBPα-mutated AML (42,43). Recently, C/EBPα was also demonstrated as a direct transcriptional regulator of CORO1A in APL and C/EBPα-mutated AML patients (44).

In addition to the above-mentioned downstream effectors of C/EBPα-p42, there are still several potential genes regulated by C/EBPα-p30, such as PIN1 and Trib2. PIN1 appears to be important in tumorigenesis since it was found to be overexpressed in many types of cancers (45,46). Evidence for the role of PIN1 in leukemia includes the fact that it inhibits the ubiquitination of c-Jun, which further blocks granulocyte differentiation (46,47). PIN1 was detected as a target of C/EBPα-p30 in AML, as C/EBPα-p30 recruited the transcription factor E2F1 in the PIN1 promoter to elevate its expression (48). In addition to the role of Trib2 acting as an upstream effector of C/EBPα by mediating its proteasomal degradation, recent research also revealed that Trib2 can also form a feedback regulatory loop with C/EBPα. In normal myeloid progenitor cells, C/EBPα-p42 was found to bind to the Trib2 promoter and inhibit Trib2 activation. Conversely, C/EBPα-p30 activated the Trib2 promoter in preleukemic cells resulting in elevated Trib2 expression, ultimately contributing to the degradation of C/EBPα-p42 and uncontrolled proliferation in AML (49). That is, the exact role of Trib2 depends on the activity of C/EBPα-p30 or C/EBPα-p42 in its specific context. However, the mechanism of the switch between these two isoforms (C/EBPα-p30 or C/EBPα-p42) is still unknown.

In addition to binding to the promoter of several genes to participate directly in leukemogenesis, C/EBPα also indirectly regulates certain genes over the course. For example, transcription factor krüppel-like factor 5 (KLF5), an essential factor for granulocytic differentiation, was found to have a low level in AML (41,50). A study reported that KLF5 is indirectly regulated by C/EBPα, with its activation dependent on C/EBPα during ATRA-mediated neutrophil differentiation in APL cells (41).

DAPK2 is a proapoptotic protein that is mainly expressed in hematopoietic tissue. In addition to participating in different cell death pathways (51,52), Rizzi et al (53) and Fang et al (54) found a specific function for DAPK2 as an enhancer of neutrophil and erythroid differentiation. Other studies further confirmed that DAPK2 in myeloid cells is dependent on C/EBPα during granulocytic differentiation and this process seems to be indirect (55). Moreover, C/EBPα interferes with E2F1 transactivation of the c-Myc promoter in AML (56), which may influence proliferation and differentiation in HL-60 cells through VEGF (57) (Fig. 4).

Figure 4 C/EBPα functions though regulation of its target genes. After its translocation into the nucleus, C/EBPα-p42 promotes cell differentiation by regulating the gene expression directly (such as Sox4, FLT3, HK3 and CORO1A) or indirectly (such as KLF5, DAPK2 and C-myc). In addition, C/EBPα-p30 isoform blocks cell differentiation by targeting PIN1 and Trib2. Notably, Trib2 can be regulated by both C/EBPα-p42 and C/EBPα-p30 isoforms, and the switch mechanisms between them need further studies. C/EBPα, CCAAT/enhancer binding protein α; FLT3, FMS-like tyrosine kinase-3; HK3, hexokinase 3; KLF5, krüppel-like factor 5.

A number of new molecular genetic abnormalities have been identified in AML in the last few decade. Further studies are needed to analyze the interrelation between them involving C/EBPα, and a pivotal co-target gene would bear clinical significance.

Collaborating factors with C/EBPα

As mentioned earlier, C/EBPα-p30 directly activates Trib2 (49) and PIN1 (48) expression, by cooperating with E2F1 in AML. In addition, C/EBPα cooperates with several other proteins in the myeloid lineage, such as Hoxa9/Meis1. Homeobox A9 (HOXA9) is a homeodomain-containing transcription factor that plays a key role in HSC expansion and is commonly deregulated in human acute leukemias (58). Overexpression of HOXA9 always exists along with its cofactor meis homeobox 1 (MEIS1) in the pattern of Hoxa9/Meis1 in AML. Recent studies suggest that C/EBPα acts as a pioneer transcription factor in Hoxa9/Meis1-mediated leukemogenesis through regulating its target genes, Cdkn2a/b (59) and Sox4 (60).

Previous studies have also demonstrated that C/EBPα interacts with its different binding partners, including TBP and TFIIB (basal transcription initiation factors) (61), the SWI/SNF complexes (chromatin remodeling complexes) (62), Rb (tumor-suppressor protein) (63), Cdk2 and Cdk4 (cyclin-dependent kinases), p21 (cyclin-dependent kinase inhibitor) (64), GABPα (cell cycle regulator and transcription factor) (65). All of these may play a crucial role during the process of leukemogenesis.

Small molecules targeting C/EBPα

In search for small molecules that are able to reverse the low expression of the C/EBPα signature, a connectivity map was applied. This analysis predicted positive connectivity between the C/EBPα activation signature and histone deacetylase inhibitors. The results showed that histone deacetylase inhibitors reactivated the expression of the C/EBPα signature and promoted granulocytic differentiation of primary samples from the C/EBPα dysfunctional subset harboring biallelic C/EBPα mutations (66), which indicated that HDAC inhibitors could represent a promising therapeutic approach in this particular subtype of AML (67,68). Cytarabine (or Ara-C) is a pyrimidine antagonist, which interferes with DNA synthesis and is used in upfront and salvage regimens for AML (69). To improve the cytotoxic activity of Ara-C treatment, various novel drug combinations have been explored (70,71). Recently, it was demonstrated that miR-181a could sensitize a chemotherapy-resistant HL60 cell line to Ara-C treatment (72). Zhao et al found that the C/EBPα-p30 isoform could bind to the miR-181a-1 promoter to upregulate its expression. Furthermore, lenalidomide, a drug approved for myelodysplastic syndromes and multiple myeloma, sensitized leukemic cells to cytarabine (Ara-C) chemotherapy by enhancing translation of the C/EBPα-p30 isoform as well as miR-181a levels (73). Additionally, Ko et al found that the methylation status of let-7a-3 was inversely correlated with the methylation status in the distal promoter region of C/EBPα in AML (74).

Therefore, it may be promising to design various small molecules targeting the C/EBPα signaling pathway for the treatment of AML.

4. Conclusion

In this review, we summarized the regulatory mechanisms and the functional targets of C/EBPα in AML (Fig. 5). A more detailed molecular analysis of C/EBPα will ultimately highlight a number of new oncogenes that may supplement the prognostic information obtained by conventional karyotyping. Furthermore, targeted therapies should interfere with C/EBPα initiating or cooperating proteins, to improve the treatment of AML. Moreover, as growing evidence implicates aberrant C/EBPα activity in a variety of diseases, including solid tumors and rheumatoid arthritis, small molecular compounds specific for C/EBPα may provide potential strategies for the therapeutic intervention of a variety of diseases.

Figure 5 Schematic representation of the mechanism of C/EBPα functions during myeloid differentiation. C/EBPα, CCAAT/enhancer binding protein α.

Acknowledgments

This study was supported by grants from the National Natural Science Foundation of China (nos. 81300426, 30771103 and 81172792), the Project for Shandong Medical and Health science and Technology Plan Project (2013Ws0365), and the ‘Twelfth Five-Year’ National Science and Technology Support Program (2013BaI07B02).

References