Multiple ankyrin repeats single KH domain (mask) protein was initially identified in
In recent years, many studies containing relevant informa-tion on ANKHD1 in cancer biology and its clinical relevance, as well as the increasing complexity of signaling networks in which this protein acts, have been reported. Thus, understanding ANKHD1-related cellular and molecular functions may pave the avenues for new insights into this protein in the field of oncology. In the present review, we analyze the evidence of ANKHD1 in the malignant phenotype of cancer cells (
The entire human
The transcript variant 1 (start: 139781399 and end: 139919441 bp; orientation: plus strand; 34 exons; mRNA: 8233 bp, tran-script ID: ENST00000360839.7) is the longest transcript variant and encodes the ANKHD1 isoform 1 (canonical; 2542 amino acid [aa] protein). The transcript variant 2 (start: 139781399 and end: 139852062 bp; orientation: plus strand; 11 exons; mRNA: 2161 bp, transcript ID: ENST00000394722.7) presents an alternate in-frame splice site in the 5` coding region, lacks several exons, uses an alternate 3` terminal exon and encodes the ANKHD1 isoform 2, a shorter isoform that presents a distinct C-terminus compared to isoform 1 (616 aa protein). The transcript variant 3 (start: 139781399 and end: 139852062 bp; orientation: plus strand; 11 exons; mRNA: 2194 bp, tran-script ID: ENST00000394723.7) lacks several exons, uses an alternate 3` terminal exon, and encodes the ANKHD1 isoform 3 that also has a distinct C-terminus compared to isoform 1 (627 aa protein). The transcript variant 4 (start: 139781399 and end: 139852062 bp; orientation: plus strand; 10 exons, mRNA: 2084 bp, transcript ID: ENST00000616482.4) also lacks several exons, uses an alternate 3` terminal exon, and encodes the shortest ANKHD1 isoform (isoform 4; 581 aa protein), which also presents a distinct C-terminus compared to isoform 1.
Finally, the transcript variant 5 (also known as
Although ANKHD1 presents several isoforms, the specific functions of each isoform, as well as the subcellular localiza-tion pattern and their expression in different tissues and cell types, have been little explored so far. Smaller isoforms of ANKHD1 lack the KH domain, which has been implicated in the binding to microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), modulating their availability and/or stability (10, 11). On the other hand, the repeats of ARD are quite preserved in all isoforms and appear to be necessary and sufficient for many of the protein interactions that we will discuss in the following lines. Larger ANKHD1 isoforms (1 and 5) present nuclear localization sequences (also known as NLS) that seem to play a key role in ANKHD1 nuclear translocation and in its function as a co-activator of YAP1 (12). Indeed, transfection assays using smaller ANKHD1 isoforms revealed a predominantly cytoplasmic distribution of these proteins (13).
The human protein ANKHD1 is orthologous to the mask protein of
In a screen for genetic modulators of the JAK/STAT signaling pathway in
In 2013, it was described that mask acts as a yorkie (yki) cofactor, regulating the activity of the Hippo signaling pathway (19, 20). This discovery had a major impact, since the Hippo pathway has been related to morphogenesis and tissue homeostasis in normal development and is largely deregulated in cancer (21, 22). The interaction of ANKHD1/YAP1 (ortho-logizes mask/yki) has also been validated in human cells. Indeed, ANKHD1 has been shown to be important for nuclear translocation of YAP1, which is essential for its role as a transcriptional coactivator, as well as for the activation of YAP1-mediated genes (19, 20). In addition, implications of ANKHD1/YAP1 axis were highlighted for cell cycle progres-sion, migration, and invasion
Despite strong indications that ANKHD1 regulates the JAK/STAT and Hippo pathway to exercise its role on cell prolife-ration, it has been shown that ANKHD1 may acts through other signaling repertoires to exert this cellular function. In leukemia cells that do not express significant levels of YAP1 and activation of the JAK/STAT pathway, the inhibition of ANKHD1 reduces proliferation and migration
Accordingly, other studies have expanded the repertoire of signaling pathways and cellular processes mediated by ANKHD1. It has been reported that ANKHD1 interacts and represses CDKN1A (also known as p21) expression, promoting cell cycle progression (29, 30), and that ANKHD1 silencing induced DNA damage markers, and reduced core histones, PCNA expression and S-phase progression (31). In another context, ANKHD1 interacts with SMYD3, a lysine methyltransferase, and mediates the pro-migratory and pro-invasive phenotype through the activation of SMYD3-modulated genes, especially
The aberrant expression of ANKHD1, as well as its potential prognostic value, has been reported in some types of cancer. In this setting, ANKHD1 is highly expressed in primary samples and cell lines derived from patients with acute leukemia and multiple myeloma (15, 29), which has been associated with cell proliferation, migration, and tumorigenesis (26, 30). Similarly, ANKHD1 expression is elevated in patients with renal cell carcinoma (10). In prostate cancer cells, ANKHD1 promotes cell cycle progression and increases tumor growth (23).
In breast cancer patients, high ANKHD1 expression was associated with reduced relapse-free survival in two independent data sets (20). In a similar way, high ANKHD1 was associated with reduced overall survival and recurrence-free survival in hepatocellular carcinoma patients (32). It was also demonstrated that ANKHD1 is widely expressed in both, normal and tumor colorectal tissues, but high ANKHD1 expression was associated with invasion of adjacent tissues of colorectal cancer patients (24). Functional assays confirmed that ANKHD1 promotes colorectal cancer cell migration and invasion
The complexity of the ANKHD1-mediated signaling network has grown in recent years. Due to the unique characteristics of this protein, current evidence indicates that ARD present in ANKHD1 serves as a signaling anchor providing stability for proteins and allowing or preventing interactions between different proteins (12, 26). Understanding the functions of the KH domain of ANKHD1 is still a topic to be explored and may considerably diversify the role of ANKHD1 in cellular processes, since the KH domain can bind, sequester or stabilize nucleic acids (
Another point to be discussed is that the functional studies that silence ANKHD1 do not specify which isoforms are depleted. In this sense, only the study using the ANKHD1 isoform 3 showed that its silencing induced caspase activation and apoptosis (13). Of note, the expression of the different ANKHD1 isoforms may be tissue- and context-specific (14). Taken together, these data suggest that ANKHD1 may perform different functions according to the predominant isoform and the repertoire of ligands available in each cell type.
In drosophila, mask is the only protein form, but in humans, there are two orthologous proteins. Despite recent data, indi-cating ANKHD1 or ANKRD17 acted similarly in the nuclear translocation of YAP1 (12), results from knockout mice suggest additional non-redundant roles for ANKHD1 and ANKRD17, since the knockout of one of the proteins results in develop-mental failure and embryo lethality (18, 35). More in-depth studies on the impact of ANKHD1 knockout in murine models are still needed.
In summary, the data generated so far indicate that ANKHD1 has an important repertoire of interacting protein already related to the development and progression of cancer, high-lighting the positive regulation of YAP1, JAK/STAT, and STMN1. The clinical impact of ANKHD1 in different neoplasms is still scarce, but the results so far suggest that the high expression of ANKHD1 is related to worse clinical outcomes. Functional studies are consistent, even though by different signaling path-ways, that ANKHD1 promotes cell proliferation, migration, invasion, and tumor growth.
This study was supported by the grants #2017/24993-0, #2019/23864-7, and #2019/25421-5, São Paulo Research Foundation (FAPESP), and grant #402587/2016-2, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). The authors thanks Fernanda T. Udinal, from the Hemocentro Foundation of Ribeirão Preto, São Paulo, Brazil, for the English language review.
The authors have no conflicting interests.