A Review on the Role of AFAP1-AS1 in the Pathoetiology of Cancer

AFAP1-AS1 is a long non-coding RNA which partakes in the pathoetiology of several cancers. The sense protein coding gene from this locus partakes in the regulation of cytophagy, cell motility, invasive characteristics of cells and metastatic ability. In addition to acting in concert with AFAP1, AFAP1-AS1 can sequester a number of cancer-related miRNAs, thus affecting activity of signaling pathways involved in cancer progression. Most of animal studies have confirmed that AFAP1-AS1 silencing can reduce tumor volume and invasive behavior of tumor cells in the xenograft models. Moreover, statistical analyses in the human subjects have shown strong correlation between expression levels of this lncRNA and clinical outcomes. In the present work, we review the impact of AFAP1-AS1 in the carcinogenesis.


INTRODUCTION
Actin filament-associated protein 1 antisense RNA 1 (AFAP1-AS1, NC_000004.12) is a long non-coding RNA (lncRNA) which contributes in the pathoetiology of several cancers (1). It is transcribed from AFAP1 gene locus on 4p16.1. It has two alternatively spliced variants. Its second exon overlaps with exons 14-16 of AFAP1 gene. The motor fiber-associated protein encoded by AFAP1 has been shown to organize a platform for joining a number of tumor-related proteins such as SRC and protein kinase C (2). This platform can influence the organization and activity of actin filaments, therefore participating in cytophagy, cell motility, invasive characteristics of cells and metastatic ability (3). Both AFAP1 and FAP1-AS1 participate in the carcinogenesis through modulation of related signaling pathways. AFAP1 has acknowledged roles in the pathogenesis of a number of cancers, namely breast (4) and prostate cancer (5), yet its expression has been found to decreased in gastric cancer samples (6). AFAP1-AS1 is mainly regarded as an oncogenic lncRNA (1). However, the oncogenic effect of this lncRNA is not necessarily exerted through AFAP1-dependent routes. A number of deletion type copy-number variants (CNVs) have been identified in AFAP1-AS1 coding gene through application of whole genome sequencing (7). AFAP1-AS1 has been shown to affect several aspects of carcinogenesis through modulation of expression of cancer-related miRNAs. Since it has been shown to be dysregulated in diverse types of cancer, this lncRNA is a putative marker for a wide variety of cancers. Functional impacts of AFAP1-AS1 in the carcinogenesis have been appraised through knock-down and over-expression studies in cell lines and animal models. Moreover, the impact of AFAP1-AS1 deregulation has been assessed in human samples. In the present review, we discuss the role of AFAP1-AS1 in the carcinogenesis based on the evidence from these three types of studies.

CELL LINE STUDIES
Lung Cancer AFAP1-AS1 has been found to be over-expressed in non-small cell lung cancer (NSCLC) cells H1975, PC-9, A549, and SPCA-1 compared with the human non-tumorigenic lung epithelial cell line BEAS-2B. Functional studies in these cells have confirmed the ability of this lncRNA in binding with and sequestering miR-139-5p, a down-regulated miRNA in NSCLC samples. AFAP1-AS1 silencing and miR-139-5p up-regulation could similarly inhibit proliferation, colony forming ability and chemoresistance of NSCLC cells, while increasing their apoptosis. The sequestering impact of AFAP1-AS1 on miR-139-5p leads to up-regulation of RRM2, a protein which has been demonstrated to increase chemoresistance of NSCLC cells via activation of EGFR/AKT pathway (8). Another study in NSCLC has shown up-regulation of FAP1-AS1 parallel with down-regulation of IL-12 and upregulation of IL-10 and IFN-g. Functionally, AFAP1-AS1 has been shown to induce activity of IRF7, RIG-I-like receptor signals and Bcl-2. Cumulatively, AFAP1-AS1 enhances migration and invasive properties of NSCLC cells through activating IRF7 and the RIG-I-like receptor signaling pathway (9). Moreover, the interaction between AFAP1-AS1 and EZH2 and subsequent recruitment of EZH2 to the promoter of p21 has been shown to repress expression of p21 in this type of cancer (10). AFAP1-AS1 has also been shown to enhance expression of AFAP1 in lung cancer cells. Expression of AFAP1-AS1 in lung cancer cells is regulated through CpG methylation marks in its promoter, since the DNA methyltransferase inhibitor agent decitabine has been demonstrated to activate AFAP1-AS1 expression. AFAP1-AS1 has been reported to increase expression levels of pro-invasive genes PPP1R13L, VASP and SPTAN1, while decreasing expression levels of a number of anti-metastatic genes such as STAT1, NF1, and FBN2 (11). Figure 1 summarizes the mentioned routes of participation of AFAP1-As1 in the pathogenesis of lung cancer.
AFAP1-AS1 can also affect lung cancer through a variety of other mechanisms being summarized in Figure 2. For instance, AFAP1-AS1 has been shown to regulate expression of numerous members of the small GTPase proteins as well as those participating in the actin cytokeratin signaling. Thus, the promoting effect of AFAP1-AS1 on cancer metastasis is most probably exerted through modulation of actin filament integrity (12). GTPases harmonize several cellular processes, such as cell polarity, migration, and cell cycle transition, thus they can participate in the pathogenies of cancer (13). Moreover, cytokeratins as members of intermediate filament protein family have been shown to affect carcinogenesis. They can also been used as cancer biomarkers (14).

Breast Cancer
In breast cancer cells, AFAP1-AS1 silencing could decrease proliferation and migratory potential, and increase cell apoptosis. miR-497-5p has been recognized as a target of AFAP1-AS1 in breast cancer cells. Since this miRNA targets SEPT2, AFAP1-AS1 up-regulation results in up-regulation of SEPT2 (17). miR-145 is another target of AFAP1-AS1 in triple negative breast cancer cells (TNBC) MDA-MB-231 breast cancer cells. According to the results of luciferase reporter assay, miR-145 can directly target MTH1. Thus, the effects of AFAP1-AS1 in enhancement of proliferation and invasiveness of TNBC are exerted through miR-145/MTH1 axis (18). Moreover, in this type of cancer, AFAP1-AS1 can sequester miR-2110 to enhance expression of Sp1 (19). AFAP1-AS1 has also been shown to enhance EMT of TNBC cells via influencing Wnt/b-catenin signaling (20). Finally, AFAP1-AS1 has been found to have significant over-expression in trastuzumab-resistant breast cancer cells versus responsive cells. Expression of this lncRNA has been enhanced by H3K27ac at its promoter. Most notably, trastuzumab resistant cells have been shown to secrete AFAP1-AS1 into exosomes, thus disseminating trastuzumab resistance in other cells. The impact of exosomal AFAP1-AS1 in induction of trastuzumab resistance is exerted via its interaction with AUF1 and subsequent induction of ERBB2 translation (21). Figure 3 depicts the impact of AFAP1-AS1 in carcinogenesis and therapy resistance of breast cancer cells.

Gastric Cancer
Similarly, AFAP1-AS1 has an oncogenic role in gastric cancer. AFAP1-AS1 silencing has significantly suppressed proliferation FIGURE 3 | The impact of AFAP1-AS1 in breast cancer progression and resistance to therapy. In addition to increasing cell proliferation and invasion, this lncRNA can increase expression of Her-2 protein, thus increasing resistance to trastuzumab. and cell cycle transition in this kind of cancer. Besides, reduction in the levels of this lncRNA can inhibit invasive capacity through affecting EMT (25). Down-regulation of KLF2 is another mechanism by which AFAP1-AS1 enhances proliferative and migratory aptitudes of gastric cancer cells (26). AFAP1-AS1 silencing in gastric cancer cells has led to a significant increase in the levels of Bax, cleaved PARP, Caspase 3, and Caspase 9, while decreasing Bcl-2 level. AFAP1-AS1 silencing has also reduced p-AKT levels and enhanced expression of PTEN in gastric cancer cells. Taken together, AFAP1-AS1 regulates proliferation and apoptotic processes in gastric cancer cell through PTEN/p-AKT cascade (27). AFAP1-AS1 can also promote proliferation and metastatic ability of gastric cancer cell through sequestering miR-155-5p and enhancing expression of FGF7 (28). Figure 5 shows the oncogenic role of AFAP1-AS1 in gastric cancer.

Esophageal Cancer
AFAP1-AS1 have also been shown to bind with miR-26a, therefore influencing expression of its target gene, i.e. ATF2. Exosomes originated from M2 macrophages have higher expression of AFAP1-AS1 and ATF2 and reduced expression of miR-26a, compared with M1 macrophages. These exosomes could transfer AFAP1-AS1 to esophageal cancer cells, thus downregulating miR-26a and enhancing ATF2 levels in the recipeint cells. These expression changes affect phenotype of esophageal cancer cells (29). The regulatory role of AFAP1-AS1 on miR-498/VEGFA axis is another mechanism of participation of this lncRNA in the pathetiology of esophageal cancer (30).

ANIMAL STUDIES
Investigations, particularly those conducted in BALB/c nude mice models have verified the oncogenic roles of AFAP1-AS1 in different types of cancers. AFAP1-AS1 knock-down has consistently led to significant reduction in tumor size/weight, attenuation of tumor growth rate and enhancement of response of cancer cells to therapeutic modalities ( Table 2). In NSCLC, AFAP1-As1 silencing not only reduces tumorigenicity, but also confers chemosensitivity (8). Moreover, its silencing can affect IRF7 and RIG-I-like receptor signals (9). In breast cancer, AFAP1-AS1 down-regulation can affect trastuzumab resistance (21).

CLINICAL STUDIES
Except from a single low-sample size study in gastric cancer which reported down-regulation of AFAP1-AS1 in tumoral tissues versus nearby samples (6), other studies consistently reported over-expression of AFAP1-AS1 in different neoplastic tissues compared with non-neoplastic tissues of the same origin ( Table 3). Even in the mentioned study, levels of AFAP1-AS1 were higher in patients who showed lymphatic or vascular invasion in comparison with those without these properties (6). Moreover, different statistical methods have been applied to assess correlations between expression level of AFAP1-AS1 and clinical outcomes, all of them reporting significant impact of upregulation of this lncRNA on increasing malignant behaviors of tumors and decreasing patients' survival. In pancreatic cancer, up-regulation of AFAP1-AS1 has been associated with lymph node involvement, perineural invasion, and poor clinical outcome. An in silico analysis of TCGA data of breast cancer patients has revealed AFAP1-AS1, as a differentially expressed lncRNA in basal tumors whose expression levels are associated with poor survival. Expression of this lncRNA has also been associated with hormone receptors status, HER2 expression, and PAM50 classification (81).
Tissue levels of AFAP1-AS1 could be used as a prognostic biomarker with the areas under ROC curves values of 0.86 and 0.93 for forecasting cancer progression in the periods of 6 and 12 months, respectively (66).
The ability of tissue levels of AFAP1-AS1 or its circulatory levels in differentiation of patients' samples from control samples has been appraised in different types of cancers ( Table 4). For instance, Li et al. have shown that over-expression of AFAP1-AS1 in serum samples of patients with NSCLC compared with normal controls can be used to distinguish these two sets of samples with an area under the curve (AUC) of 0.759. Combination of expression levels of this lncRNA with those of cyfra21-1 has increased AUC value to 0.860. Moreover, AFAP1-AS1 over-expression has been more prominent in patients with distant or lymph node metastasis, advanced clinical stage, and greater tumor burden (75). Serum levels of AFAP1-AS1 have also been shown to separate gastric cancer patients from controls with higher AUC value compared with conventional markers, i.e. CEA and CA19-9. Notably, serum levels of AFAP1-AS1 have been shown to be reduced following surgical treatment of patients (45).

DISCUSSION
AFAP1-AS1 has been found to be up-regulated in almost all kinds of malignant tissues. This lncRNA has multiple effects in the carcinogenesis process, most of them being exerted through AFAP1-independent manners. Most notably, AFAP1-AS1 can sequester a number of tumor suppressor miRNAs, thus releasing the targets of these miRNAs from inhibitory effects of miRNAs.
High-expression of AFAP1-AS1 was found to be an independent unfavorable prognostic factor.
Upregulation of AFAP1-AS1 was positively associated with tumor bulk as well as choroidal or optic nerve invasion. osteosarcoma. Moreover, similar interaction has been verified between this lncRNA and miR-145 in breast cancer and oral squamous cell carcinoma.
In fact, AFAP1-AS1 has multiple binding sites for miRNAs, thus regulating expression of a wide array of miRNAs. It is not clear whether binding of this lncRNA with a certain miRNA affects its interactions with other miRNAs. The crosstalk between AFAP1-AS1 and miRNAs can regulate activity of signaling pathways, angiogenic processes as well as EMT.
Lung cancer, nasopharyngeal carcinoma, colorectal cancer and cholangiocarcinoma are among cancers in which the interaction between AFAP1-AS1 and AFAP1 has been verified. However, the results of these studies are conflicting. For instance, AFAP1-AS1 silencing has been shown to increase expression of AFAP1 in a single study in lung cancer cells (12), while another study in this type of cancer has shown its effect on enhancement of expression of AFAP1 (11). Moreover, in a single study in MCF-7 breast cancer cells, AFAP1-AS1 silencing has not affected AFAP1 levels or actin filament integrity (40). Therefore, future studies are needed to elaborate the mechanistical impacts of AFAP1/AFAP1-AS1 interactions.
AFAP1-AS1 can affect response of cancer cells to a variety of anti-cancer modalities ranging from conventional chemotherapeutics to targeted therapeutics such as trastuzumab. Therefore, measurement of expression levels of this lncRNA can guide clinical oncologists to find the most appropriate therapeutic option for each patient. AFAP1-AS1 can also affect EMT and stemness of cancer cells, thus promoting their metastatic ability and increasing the propensity to tumor recurrence.
From a prognostic point of view, AFAP1-AS1 levels have been associated with tumor depth, tumor differentiation, TNM stage and other determinants of patients' survival, thus could be used as markers for prediction of clinical outcomes of patients with a variety of malignant conditions. Diagnostic application of AFAP1-AS1 has been appraised in several types of cancers, with the best results being obtained from studies in gastric and esophageal cancers.
Cumulatively, AFAP1-AS1 is a prototype of cancer-related lncRNAs that regulates carcinogenesis not only through modification of expression of its sense transcript, but also through a variety of other methods such as miRNA sequestering and epigenetically affecting expression of tumor suppressor genes.

AUTHOR CONTRIBUTIONS
SG-F and BH wrote the draft and revised it. MT designed and supervised the study. TK and MM collected the data and designed the figures and tables. All authors contributed to the article and approved the submitted version.