The ZBP1 family has the ability to regulate the expression of a plethora of RNA targets in order to link the processes of subcellular RNA localization, translational control, and RNA stability with cell polarity, migration, and proliferation (3
). ZBP1, which is actively expressed during embryonic development, becomes undetectable after birth and is silenced in most adult tissues. However, activation or elevated expression of the gene has been detected in the vast majority of mammalian tumors of different origins, and in some tumor types, ZBP1 activation has become a characteristic marker of the samples studied (10
). A number of studies have revealed the association of ZBP1 expression with tumorigenesis and progression, including induction of mammary tumors (41
), repression of cancer cell proliferation (26
), and inhibition of metastasis (45
). While much work has implicated the roles of ZBP1 in development and differentiation of human cancers (16
), the mechanism responsible for gene activation has not been extensively studied. Understanding the mechanism could eventually reveal the underlying basis leading to the characteristic expression of the ZBP1 gene in human cancers and may even provide therapeutic implications for inhibiting carcinogenesis and metastasis.
In 293T cells, CRD-BP, a mouse ortholog of chicken ZBP1, could be activated in response to β-catenin signaling (31
). In the present study, we confirmed this regulation and provided detailed evidence of molecular interaction between β-catenin and the ZBP1 promoter, which resulted in transactivation of the gene. We demonstrated that ZBP1 expression in mammalian breast cancer cells was correlated with nuclear translocation of endogenous β-catenin. We functionally characterized the ZBP1/CRD-BP promoter and identified an element (CTTTG-TC) located between nt −590 and −584 that was necessary for regulating gene expression. We further determined that β-catenin physically associated with the ZBP1 promoter through this putative binding element in vivo and specifically bound to the element in vitro. Noubissi et al. (31
) identified that β-catenin expressed from a transfected plasmid bound to the region −1333 to −1607 of the human ZBP1/CRD-BP promoter (corresponding to the rat promoter of −1107 to −1381 upstream of the translation site). However, it is worthwhile to mention that this region is not necessary for promoter activity because reporters under the control of the ZBP1/CRD-BP promoter with or without it did not change their transcriptional activity. In comparison, the promoter region containing the CTTTG-TC element in the region from −590 to −584 we characterized for β-catenin binding is essential for the ZBP1/CRD-BP promoter activity and is highly conserved in rat, mouse, and human genes (as is its flanking region), suggesting that mammalian breast cancers use this conserved element to control ZBP1 transcription.
β-Catenin plays a critical role in cell morphogenesis and human carcinogenesis through its dual function as a component of the adhesive complex at cell-cell junctions and as a transcriptional coregulator in the Wnt
signaling pathway (13
). Normal cells contain low levels of β-catenin, mostly associated with cell membranes, due to the presence of the adenomatous polyposis coli (APC) protein, which mediates the degradation of free cytoplasmic β-catenin (36
). Almost all colon cancers have either mutated β-catenin or deleted APC that allows free β-catenin to escape from cytoplasmic degradation and be imported into cell nuclei, where it activates the downstream targets of Wnt/
β-catenin signaling (28
). In breast cancers, aberrant activation of the β-catenin pathway has also been identified to induce a number of genes, including the cyclin D1, c-myc
, Twist, vimentin, and E-cadherin genes. Most of these targets were involved in various physiological and pathological processes of the disease (9
). We demonstrated that the ZBP1 gene is a target of the β-catenin pathway in breast cancers based on the in vivo and in vitro molecular interaction of β-catenin with the conserved element of the ZBP1 promoter, which had not been identified previously. Moreover, we identified β-catenin mRNA as a novel ZBP1 target. Gain of ZBP1 function increased stability of cytoplasmic β-catenin mRNA; apparently, this upregulation was contributed by the binding of ZBP1 to β-catenin mRNA. Thus, ZBP1 and β-catenin can regulate each other's expression, and this coregulation may open up new insights relating ZBP1 activation to breast cancers.
Both ZBP1 and β-catenin are well known to be highly expressed during normal embryonic development, as well as activated in a variety of human tumors (18
). The oncofetal pattern of ZBP1 expression could be the result of β-catenin activation during embryogenesis and tumorigenesis. However, the feedback regulation of ZBP1 to β-catenin mRNA could potentially amplify the role of β-catenin in the Wnt
signaling pathway to activate the genes associated with the pathway or to facilitate the function of β-catenin in cell-cell contacts. Targeted expression of ZBP1 in mammary tissues has been reported to induce mammary tumors, but this reduction was not by stabilizing c-myc
). Therefore, tumor induction in the animal could be partially contributed by ZBP1 regulation to other mRNA targets, including β-catenin mRNA. In addition to the ability of ZBP1 to induce tumorigenesis, overexpression of ZBP1 in a K562 line inhibited cell proliferation (26
); breast cancer cells without ZBP1 expression increased their metastatic potential (45
). Given the roles of ZBP1 as a localizing factor and a translational regulator, it is likely that the contradictory effects of ZBP1 expression on breast cancer cells could result from the diversity of the mRNA targets of ZBP1—some of which inhibit growth and others of which stimulate motility. For instance, depending on the cell type from which the tumor is derived, ZBP1 could either promote cancer progression by controlling c-myc
or β-catenin expression or repress metastasis by regulating localized translation of β-actin and other mRNAs.
In summary, we report an interconnected regulatory mechanism that leads to the characteristic expression of ZBP1 and β-catenin in breast cancer cells. β-Catenin binds to the conserved element of the mammalian ZBP1 promoter and transactivates gene expression, while ZBP1 associates with β-catenin mRNA and controls its cytoplasmic expression. This is the first demonstration that these two gene products are coregulated. The loss of function in either gene will affect the other's expression. The strong correlation between β-catenin signaling and ZBP1 activation is found in both breast cancer cell lines and breast patient tumors. Future studies will define the physiological significance of this interrelationship.