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Zinc finger antisense 1: A long noncoding RNA with complex roles in human cancers
Accepted Manuscript Zinc finger antisense 1: A long noncoding RNA with complex roles in human cancers
Xiaodi Jiang, Zhi Yang, Zhiwei Li PII: DOI: Reference:
S0378-1119(18)31218-6 https://doi.org/10.1016/j.gene.2018.11.075 GENE 43420
To appear in:
Gene
Received date: Revised date: Accepted date:
17 September 2018 21 November 2018 25 November 2018
Please cite this article as: Xiaodi Jiang, Zhi Yang, Zhiwei Li , Zinc finger antisense 1: A long noncoding RNA with complex roles in human cancers. Gene (2018), https://doi.org/ 10.1016/j.gene.2018.11.075
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ACCEPTED MANUSCRIPT Zinc finger antisense 1: a long noncoding RNA with complex roles in human cancers
Xiaodi Jianga; Zhi Yangb; Zhiwei Lia*
Department of Infectious Diseases, Shengjing Hospital of China Medical University, Shenyang, China
b
Department of General Surgery, The Fourth Hospital of China Medical University, Shenyang, China
*
Corresponding author: Zhiwei Li, Department of Infectious Diseases, Shengjing Hospital of China
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a
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Medical University, No. 39 Huaxiang Road, Tiexi District, Shenyang 110022, China. E-mail:
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[email protected]
Abstract: Zinc finger antisense 1 (ZFAS1), a newly identified long non-coding RNA, is a transcript
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antisense to the 5' end of the protein-coding gene zinc finger NFX1-type containing 1 which hosts three C/D-box small nucleolar RNAs (SNORDs) within sequential introns: Snord12, Snord12b, and
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Snord12c. ZFAS1 is dysregulated and acts as either an oncogene or a tumor suppressor in different
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human malignancies. ZFAS1 has been implicated in many aspects of carcinogenesis, including proliferation, invasion, metastasis, apoptosis, cell cycle, and drug resistance. The mechanisms underlying the effects of ZFAS1 are complex and involve multiple signaling pathways. In this review,
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the multiple pathological functions of ZFAS1 in diverse malignancies are systematically reviewed to
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elucidate the molecular basis of its biological roles and to provide new directions for future research.
Keywords: Long non-coding RNA; ZFAS1; Cancer; Oncogene; Tumor suppressor
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ACCEPTED MANUSCRIPT 1. Introduction Cancer is a group of complex diseases threatening human health worldwide. The molecular basis of cancer has been extensively studied over the past few decades. Accumulating evidence indicates that epigenetic processes play crucial roles in cancer involving histone modifications, DNA methylation, chromatin remodeling, and gene imprinting, as well as noncoding RNA (ncRNA) regulation. 1,2
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NcRNAs are transcribed from noncoding regions. Due to the lack of an open reading frame, ncRNAs have no or limited protein-coding potential.3 The ncRNAs are divided into diverse types according to
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their size and include microRNAs (miRNAs), long non-coding RNAs (lncRNAs), small nucleolar RNAs (snoRNAs), and PIWI-interacting RNAs.
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LncRNAs are ncRNAs longer than 200 nucleotides in length. Emerging evidence suggests that lncRNAs play a pivotal role in the development and progression of cancer. 4 LncRNAs are involved in
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various aspects of gene regulation including epigenetic regulation, imprinting, cellular homeostasis, transcription, and mRNA splicing.5-7 Furthermore, certain lncRNAs show developmental and tissue
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specific expression patterns.8-11 These characteristics, which are critical for their functional analysis, highlight the potential of lncRNAs as diagnostic, prognostic, and therapeutic targets in cancer.
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Although multiple lncRNAs have been studied in the past few decades, many lncRNAs have not been
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fully examined. The role of potentially thousands of lncRNAs has attracted intense scientific interest. Zinc finger antisense 1 (ZFAS1, also known as ZNFX1-AS1) is a newly identified lncRNA which is dysregulated in numerous human cancers and implicated in many aspects of carcinogenesis. ZFAS1 is
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highly expressed in the mammary gland and is downregulated in human invasive ductal carcinoma compared with normal breast tissue.12 Knockdown of ZFAS1 in mammary epithelial cells increases
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their proliferation and differentiation, indicating that ZFAS1 may act as a tumor suppressor gene in breast cancer.12 However, recent studies have demonstrated that ZFAS1 is upregulated in multiple types of tumors, suggesting that ZFAS1 may also act as a proto-oncogene in cancer. In this review, we provide an overview of current evidence supporting the role of ZFAS1 in human cancer and describe potential molecular mechanisms and their clinical significance (Tables 1 and 2).
2. Discovery and characterization of ZFAS1 ZFAS1 was originally identified by Askarian-Amiri et al. in 2011. To identify novel regulators in breast biology, they identified differentially regulated lncRNAs during mouse mammary gland development. 2
ACCEPTED MANUSCRIPT Among the highest and most differentially expressed genes was Zfas1, which was expressed in most tissues but exhibited the greatest abundance in mouse developing mammary glands, where it plays an important developmental role.12 ZFAS1 is its syntenic human ortholog. While there is relatively low primary sequence conservation between ZFAS1 and Zfas1, their predicted secondary structures have similar features.12
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ZFAS1 is located on chromosome 20q13, which contains a transcript antisense to the 5' end of the protein-coding gene zinc finger NFX1-type containing 1 (ZNFX1). Many antisense lncRNAs are
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known to regulate the expression of their protein coding counterparts in cis. 13,14 However, the lack of correlation between ZFAS1 and ZNFX1 expression indicates that there is no apparent cis regulation
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between them.15
ZFAS1 is expressed as at least five different isoforms in both cytoplasmic and nuclear
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compartments.15 They vary in size from 516 to 1006 bases with exons two and five common to all isoforms. Many of these lncRNAs appear to be ribosome-associated.16,17 A study by Hansji et al.
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indicated that ZFAS1 co-localized with polysomes, where it was predominantly associated with the small ribosomal subunit.15 ZFAS1 expression is strongly correlated with that of a number of mRNAs
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encoding ribosomal proteins involved in ribosome biogenesis. ZFAS1 may thus be involved in the
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regulation of the ribosome through interactions with mature ribosomes in the cytoplasm as well as through interactions with immature ribosomes in the nucleus.15 ZFAS1 hosts three C/D-box snoRNAs (SNORDs) within sequential introns: Snord12, Snord12b, and
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Snord12c.12 SnoRNAs are a functionally diverse group of 60–150 nt trans-acting ncRNAs that guide modifications of selected nucleotides in rRNAs or spliceosomal RNAs.18-20 From a structural basis,
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snoRNAs are divided into SNORDs and H/ACA box snoRNAs. Both types of snoRNAs are dysregulated in various types of cancer and have been implicated in the development and progression of human malignancy.21-23 In addition to the evidence that snoRNAs are involved in cancer development, there are some preliminary data showing that the genes that host snoRNAs may also have important functions in regulating the biological behavior of cancer.24
3. ZFAS1 deregulation in human cancers 3.1 ZFAS1 in breast cancer Breast cancer is a malignant tumor originating from breast tissues, is the most common cancer with 3
ACCEPTED MANUSCRIPT gradually increased incidence in women all over the world.25 The first study focusing on ZFAS1 in breast cancer was published in 2011. The authors reported that ZFAS1 was downregulated in breast cancer compared with normal breast tissue.12 Knockdown of ZFAS1 in mammary epithelial cells increased proliferation and differentiation, suggesting that ZFAS1 may serve as a tumor suppressor gene in breast cancer. However, the number of tumor tissue samples evaluated in this study was limited
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(only five) and effects on proliferation were evaluated at only one time point (48 h) without biological replicates. Subsequently, another study further analyzed the genome-wide RNA transcript profile of
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ZFAS1 using the RNAseq data set from The Cancer Gene Atlas and found that ZFAS1 expression was not significantly different in breast cancer patients compared with healthy controls.15 However, the
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expression of ZFAS1 was significantly reduced in basal and human epidermal growth factor receptor 2-positive breast cancer subtypes compared to normal breast tissue. It was also more highly expressed
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in estrogen receptor (ER)+ compared to ER- subtypes.15 Similarly, another group investigated ZFAS1 levels in invasive breast carcinoma, ductal carcinoma in situ, and normal adjacent breast tissues by
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chromogenic in situ hybridization.26 They demonstrated that ZFAS1 was negative or only weakly expressed in all groups of specimens, indicating there was no significant difference in ZFAS1
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expression in breast cancer.
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Conversely, Fan et al. determined that ZFAS1 expression was downregulated in breast cancer cell lines, and additional experiments further demonstrated that overexpression of ZFAS1 inhibited breast cancer cell proliferation, migration, and invasion in vitro.27 They suggested that ZFAS1 acts as a tumor
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suppressor in breast cancer. Obviously, further large multicenter studies with more stringent standards are required to resolve the discrepancies in ZFAS1 expression and function in breast cancer between
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these studies.
3.2 ZFAS1 in colorectal cancer Colorectal cancer (CRC) is the fourth main cause of cancer-related mortality and the third most commonly diagnosed cancer worldwide.28 Recent studies have shown that ZFAS1 is remarkably upregulated in colorectal cancer tissues and cell lines. 29-32 Moreover, high ZFAS1 expression was significantly associated with Helicobacter pylori infection, lymph nodes metastasis, vascular invasion, and advanced Tumour Node Metastasis (TNM) stage.30-32 Patients with increased ZFAS1 expression showed shorter relapse-free survival and overall survival.30 Cox multivariate analyses confirmed ZFAS1 expression as an independent prognostic factor in CRC. Biologically, silencing ZFAS1 4
ACCEPTED MANUSCRIPT suppresses CRC cell proliferation, migration and invasion, and promotes cell apoptosis through many pathways.29-32 In addition, Fang et al. reported that ZFAS1 was also increased in the plasma of CRC patients.32 Thus, ZFAS1, a potential oncogenic lncRNA in CRC, may offer a hopeful diagnostic and therapeutic choice for the treatment of CRC. 3.3 ZFAS1 in gastric cancer
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Gastric cancer (GC) represents a major health burden worldwide, being the second main cause of cancer-related deaths.33 ZFAS1 expression is also upregulated in gastric cancer,34-37 and its increased
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level has been significantly correlated with lymphatic metastasis, TNM stage, and poor prognosis. 35,36 The silencing of ZFAS1 inhibited GC cell proliferation, cell cycle progress, migration, invasion, and
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the epithelial-mesenchymal transition (EMT), and enhanced cellular sensitivity to cis-platinum or paclitaxel.37
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Circulating tumor cells (CTCs) are considered useful as a “liquid biopsy” reflecting the progression of cancer.38 Circulating nucleic acids such as lncRNAs may also reflect CTC levels and act as CTC
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markers.39 Recent studies have shown that lncRNAs are relatively stable in plasma or serum, 40,41 although the precise mechanism is unclear. One possible explanation is that nucleic acid substances
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could be packaged and protected by several microparticles such as exosomes, microvesicles, and
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apoptotic bodies.42,43 ZFAS1 has been found to be upregulated in serum and serum exosomes of GC patients, and surgery can reduce its presence in plasma.34,35 This suggests that ZFAS1 in the plasma may act as a noninvasive diagnostic and prognostic biomarker for GC.
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3.4 ZFAS1 in hepatocellular carcinoma
Hepatocellular carcinoma (HCC) is one of the most common human malignancies worldwide and the
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third leading cause of cancer-related death.44 Portal vein tumor thrombus is the main route for intrahepatic metastasis of HCC cells and is strongly correlated with poor prognosis for patients with HCC.45,46 Li et al. demonstrated that the ZFAS1 transcripts are significantly upregulated in most HCC tissues compared with nontumor tissues from the same donor. 47 Significantly higher ZFAS1 expression was found in portal vein tumor thrombotic tissues than in primary HCC and healthy liver tissues. Meanwhile, upregulation of ZFAS1 was more frequently observed in HCC patients with microvascular invasion than in those without microvascular invasion. HCC patients with high ZFAS1 expression had higher recurrence rates and shorter overall survival than those with low expression of ZFAS1. 47 Furthermore, overexpression of ZFAS1 promoted HCC cell invasion and tumor metastasis in vitro and 5
ACCEPTED MANUSCRIPT in vivo. These results indicate that ZFAS1 is upregulated and correlated with tumor recurrence and metastasis in HCC. Luo et al. demonstrated that plasma levels of ZFAS1 are higher in HCC patients than in healthy controls or in patients with cirrhosis and hepatitis B. 48 In addition, the expression of ZFAS1 is correlated with serum alpha fetoprotein (AFP). These results suggest the potential of ZFAS1 as a
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diagnostic marker to distinguish HCC patients from healthy controls. 3.5 ZFAS1 in bladder cancer
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Bladder cancer is one of the most common urological malignancies worldwide.49 Despite considerable improvements in treatment strategies for bladder cancer, such as adjuvant chemo-radiotherapies and
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immunological therapy, one third of all patients experience recurrence.50-52 Therefore, further investigation of the mechanisms involved in the proliferation, metastasis, and invasion of bladder
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cancer is necessary to develop effective therapeutic approaches for the treatment of this disease. Yang et al. demonstrated that ZFAS1 expression was upregulated in bladder cancer tissues compared
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with paired adjacent normal tissues by analyzing The Cancer Genome Atlas database. 53 Furthermore, they confirmed that levels of ZFAS1 were elevated in bladder cancer tissues and cell lines compared
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with normal bladder tissues and normal uroepithelium cell lines. Further investigation revealed that the
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expression level of ZFAS1 was positively associated with clinical stage, muscularis invasion, lymph node metastasis, and distant metastasis in bladder cancer patients.53 Wang et al. also confirmed that ZFAS1 expression was significantly upregulated in bladder cancer tissues and cell lines. 54 Survival
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analysis showed that patients with elevated ZFAS1 expression had shorter progression-free survival and overall survival. Moreover, silencing of ZFAS1 markedly suppressed bladder cancer cell
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proliferation and colony formation, arrested the cell cycle, promoted cell apoptosis, and inhibited cell migration.54
3.6 ZFAS1 in ovarian cancer Ovarian cancer is the leading cause of mortality among gynaecological malignancies.49 Xia et al. reported that ZFAS1 was upregulated in epithelial ovarian cancer tissues and was negatively correlated to the overall survival rate of patients with epithelial ovarian cancer. 55 Moreover, depletion of ZFAS1 inhibited proliferation, migration, invasion, and chemoresistance to cisplatin and paclitaxel. Similarly, a significant correlation between ZFAS1 expression and chemosensitivity was confirmed in 233 patients with high grade serous ovarian cancer from the Gene Expression Omnibus datasets. 56 In vitro 6
ACCEPTED MANUSCRIPT experiments demonstrated that the ZFAS1 expression was upregulated by cisplatin in ovarian cancer cell lines.56 These findings suggested that ZFAS1 may participate in platinum resistance in ovarian cancer. 3.7 ZFAS1 in glioma Glioma is the most common brain cancer, accounting for >60% of primary brain tumors in adults.49 A
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relationship between ZFAS1 and human glioma progression was recently reported. ZFAS1 was upregulated in glioma tissues and cell lines, and high expression of ZFAS1 was significantly correlated
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with advanced tumor stage and poor overall survival. 57,58 Multivariate Cox regression analysis showed that the ZFAS1 level could serve as an independent prognostic factor for glioma. 57 Functionally,
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ZFAS1 acted as an oncogene in glioma, and knockdown of ZFAS1 promoted apoptosis and significantly inhibited cell proliferation, migration, and invasion. 57,58 Furthermore, silencing of ZFAS1
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resulted in cell cycle arrest at the G0/G1 phase and correspondingly decreased the percentage of S
potential therapeutic target for glioma. 3.8 ZFAS1 in other cancers
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phase cells in glioma cell lines.57 Thus, ZFAS1 may act as a valuable prognostic biomarker and
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Expression analysis of ZFAS1 in non-small cell lung cancer (NSCLC) tissues showed significant
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upregulation, and higher expression levels of ZFAS1 were found in more advanced tumor tissues. 59 ZFAS1 expression levels were significantly associated with tumor differentiation grade, lymph node metastasis, and TMN stage. Furthermore, patients with higher ZFAS1 expression levels had worse
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overall survival.59 Univariate and multivariate analyses indicated that high ZFAS1 expression was an independent prognostic factor for poor survival of NSCLC patients.
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Liu et al. revealed a functional role for ZFAS1 in osteosarcoma growth and metastasis. They found that expression of ZFAS1 was upregulated in osteosarcoma samples and cell lines, and overexpression of ZFAS1 was significantly associated with unfavorable prognosis of osteosarcoma patients. 60 Functional assays also demonstrated that ZFAS1 enhanced the growth and metastatic ability of osteosarcoma cells in vitro and in vivo.60 Chen et al. demonstrated that ZFAS1 was upregulated in tumors relative to normal prostate tissues, and high expression of ZFAS1 was correlated with worse outcomes in prostate cancer patients.61 The relationship between ZFAS1 and esophageal squamous cell carcinoma (ESCC) progression was recently reported. ZFAS1 expression was significantly higher in ESCC tissues compared with 7
ACCEPTED MANUSCRIPT corresponding adjacent normal tissues.62 Survival analysis showed that ESCC patients with high ZFAS1 expression had poor overall survival, and ZFAS1 expression was determined to be an independent prognostic factor.62
4. Regulatory mechanisms of ZFAS1
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As mentioned above, multiple studies have shown that ZFAS1 is implicated in many aspects of carcinogenesis, which has prompted a large number of researchers to explore its possible molecular
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mechanism. As shown in Figure 1, ZFAS1 mediates different functions such as proliferation, invasion, metastasis, apoptosis, cell cycle, and chemoresistance via interactions with different types of molecules.
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4.1 ZFAS1 epigenetically represses KLF2 and NKD2 expression.
Nie et al. demonstrated that ZFAS1 could directly bind with the enhancer of zeste homolog 2 (EZH2),
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lysine-specific demethylase 1 (LSD1), and CoREST (histone demethyltransferase of REST complex) in gastric cancer cells.36 Furthermore, they found that the tumor suppressors naked cuticle homolog 2
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(NKD2) and Kruppel-like factor 2 (KLF2) were key downstream mediators of ZFAS1. KLF2 is a member of the Kruppel-like factor family that includes transcription factors with Cys2/His2 zinc-finger
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domains.63 NKD2, one of the naked cuticle family, has been shown to function as a tumor suppressor in a
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variety of cancers.64,65 Further investigations revealed that ZFAS1 simultaneously recruits EZH2 and LSD1 to the NDK2 and KLF2 promoter regions and represses their transcription via trimethylation of histone H3 at lysine 27 and demethylation of dimethylated histone H3 lysine 4 (Figure 2).36 Similarly,
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Yang et al. reported that knockdown of ZFAS1 repressed bladder cancer cell proliferation via upregulation of KLF2 and NKD2 expression.53 These results suggest that ZFAS1 exerts oncogenic
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affects at least partly by epigenetically repressing KLF2 and NKD2 expression.
4.2 ZFAS1 regulates EMT pathway EMT is a critical mechanism in cancer metastasis. A number of studies have shown that ZFAS1 facilitates tumor invasion and metastasis by promoting EMT. Lv et al. demonstrated that silencing ZFAS1 could impair migration and invasion by inhibiting EMT through reduced expression of matrix metallopeptidase 2 (MMP2), MMP9, N-cadherin, integrin β1, zinc finger E-box binding homeobox 1 (ZEB1), Twist, and Snail as well as by increasing E-cadherin levels in glioma.57 Similarly, Gao et al. showed that knockdown of ZFAS1 significantly decreased protein expression of N-cadherin and Snail 8
ACCEPTED MANUSCRIPT and increased E-cadherin in glioma.58 In addition, Fang et al. showed that ZFAS1 may function as an oncogene by modulating ZEB1 to induce EMT.46 Yang et al. suggested that knockdown of ZFAS1 repressed cell migration and invasion by downregulating ZEB1 and ZEB2 expression in bladder cancer.53 Liu et al. also demonstrated that ZFAS1 directly interacted with ZEB2 to regulate its stability.60 Furthermore, through domain mapping and RNA pull-down assay, they found that the 3’-end fragment of
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ZFAS1 was essential to bind ZEB2. These findings suggest that ZFAS1 may promote cancer
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progression via regulated EMT signaling pathways.
4.3 ZFAS1 serves as a miRNA sponge
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In spite of the controversy surrounding the competing endogenous RNA (ceRNA) hypothesis, its role as a plausible generic mechanism for regulating gene expression has triggered new fields of
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research.66,67 In recent years, accumulating evidence has suggested that certain lncRNAs can act as ceRNAs which function as sponges of common miRNAs to prevent their endogenous suppressive
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effect on key targets.68,69 Recently, ZFAS1 has also been identified in crosstalk with several miRNAs in human cancer. Xia et al. demonstrated that ZFAS1 promoted the expression of Specificity protein 1
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(Sp1) in ovarian cancer by competitive antagonism against miR-150-5p to promote cell proliferation
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and chemotherapy resistance in ovarian cancer cells.55 Interestingly, Sp1 could directly bind the ZFAS1 promoter and activate transcription.60 This finding suggested that a ZFAS1/miR-150-5p/Sp1 feedback loop participates in cancer progression. Another study found that ZFAS1 increases ZEB1, MMP14 and
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MMP16 expression and promotes HCC metastasis by sponging miR-150 and inhibiting its function.47 The miR-150 family is the main target of the ZFAS1 sponge, but other miRNAs can interact with it as
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well. Liu et al. reported that ZFAS1 could function as a ceRNA of B lymphoma Moloney murine leukemiavirus insertion region 1 (BMI1).60 ZFAS1 positively regulated malignant phenotypes by competitively binding miR-200b and miR-200c and upregulating BMI1. Similarly, Xie et al. found that ZFAS1 sponges miR-484 to promote cell proliferation and invasion in CRC. 31 Wang et al. demonstrated that ZFAS1 facilitates bladder cancer tumorigenesis by sponging miR-329.54
4.4 Other regulatory mechanisms In addition to the EMT pathway and acting as a ceRNA, ZFAS1 can also activate various signaling pathways. Thorenoor et al. reported that ZFAS1 functions as an oncogene in CRC via indirect 9
ACCEPTED MANUSCRIPT destabilization of p53, and through direct and indirect interactions with the cyclin-dependent kinase 1 (CDK1)/cyclin B1 complex leading to cell cycle progression and inhibition of apoptosis. 29 In addition, Gao et al. discovered that knockdown of ZFAS1 decreased the expression of Notch signal-related proteins hairy enhancer of split-1 and Notch intracellular domain in glioma cells.58
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5. Conclusion and future directions The data described in this review suggests that ZFAS1 is upregulated in a variety of human cancers and
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acts an oncogene; In contrast, ZFAS1 acts as a tumor suppressor in breast cancer. The contradictory functions of ZFAS1 in different cancers may be largely attributed to differences in tumor tissue origin,
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extracellular microenvironment, and upstream and downstream regulatory factors. ZFAS1 is dysregulated in diverse malignancies and implicated in many aspects of carcinogenesis,
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including proliferation, invasion, metastasis, apoptosis, cell cycle, and chemoresistance. The mechanism underlying the effects of ZFAS1 is complex and involves multiple signaling pathways. However, the
require further systematic investigation.
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precise molecular mechanisms upstream and downstream of ZFAS1 are not thoroughly understood and
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ZFAS1 dysregulation is significantly associated with multiple clinicopathological characteristics and
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survival rate, suggesting its potential clinical utility as a biomarker. ZFAS1 is extremely prospective to be a potential therapeutic target due to forceful tumor specificity and reduced systemic toxicity. Nevertheless, the expression and function of ZFAS1 in body fluids remains quite obscure, and the
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detection of ZFAS1 is still far from ready for clinical applications. Research of ZFAS1 function is still at a preliminary stage, and much more work is needed to gain a comprehensive understanding of the
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potential and limits of ZFAS1 for cancer diagnosis and treatment.
Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 81502073).
Compliance with Ethical Standards Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. 10
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Conflict of interest We declare that we have no conflict of interest.
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ACCEPTED MANUSCRIPT cancer diagnostics in liquid biopsies. Ann Oncol. 2016;27:557-558. 44. Au JS, Frenette CT. Management of Hepatocellular Carcinoma: Current Status and Future Directions. Gut Liver. 2015;9:437-448. 45. Shi J, Lai EC, Li N, et al. Surgical treatment of hepatocellular carcinoma with portal vein tumor thrombus. Ann Surg Oncol. 2010;17:2073-2080.
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46. Yang P, Li QJ, Feng Y, et al. TGF-β-miR-34a-CCL22 signaling-induced Treg cell recruitment promotes venous metastases of HBV-positive hepatocellular carcinoma. Cancer Cell. 2012;22:291-303.
hepatocellular carcinoma. Cancer Res. 2015;75:3181-3191.
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47. Li T, Xie J, Shen C, et al. Amplification of long noncoding RNA ZFAS1 promotes metastasis in
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48. Luo P, Liang C, Zhang X, et al. Identification of long non-coding RNA ZFAS1 as a novel biomarker for diagnosis of HCC. Biosci Rep. 2018. pii: BSR20171359.
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49. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics, 2012. CA Cancer J Clin. 2015;65:87-108.
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50. Chou R, Selph SS, Buckley DI, et al. Treatment of muscle-invasive bladder cancer: A systematic review. Cancer. 2016;122:842-851.
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51. Racioppi M, D'Agostino D, Totaro A, et al. Value of current chemotherapy and surgery in advanced
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and metastatic bladder cancer. Urol Int. 2012;88:249-258. 52. Sonpavde G, Goldman BH, Speights VO, et al. Quality of pathologic response and surgery correlate with survival for patients with completely resected bladder cancer after neoadjuvant chemotherapy.
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Cancer. 2009;115:4104-4109.
53. Yang H, Li G, Cheng B, Jiang R. ZFAS1 functions as an oncogenic long non-coding RNA in
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bladder cancer. Biosci Rep. 2018;38. pii: BSR20180475. 54. Wang JS, Liu QH, Cheng XH, Zhang WY, Jin YC. The long noncoding RNA ZFAS1 facilitates bladder cancer tumorigenesis by sponging miR-329. Biomed Pharmacother. 2018;103:174-181. 55. Xia B, Hou Y, Chen H, et al. Long non-coding RNA ZFAS1 interacts with miR-150-5p to regulate Sp1 expression and ovarian cancer cell malignancy. Oncotarget. 2017;8:19534-19546. 56. Liu R, Zeng Y, Zhou CF, et al. Long noncoding RNA expression signature to predict platinum-based chemotherapeutic sensitivity of ovarian cancer patients. Sci Rep. 2017;7:18. 57. Lv QL, Chen SH, Zhang X, et al. Upregulation of long noncoding RNA zinc finger antisense 1 enhances epithelial-mesenchymal transition in vitro and predicts poor prognosis in glioma. Tumour 14
ACCEPTED MANUSCRIPT Biol. 2017;39:1010428317695022. 58. Gao K, Ji Z, She K, Yang Q, Shao L. Long non-coding RNA ZFAS1 is an unfavourable prognostic factor and promotes glioma cell progression by activation of the Notch signaling pathway. Biomed Pharmacother. 2017;87:555-560. 59. Tian FM, Meng FQ, Wang XB. Overexpression of long-noncoding RNA ZFAS1 decreases survival
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in human NSCLC patients. Eur Rev Med Pharmacol Sci. 2016;20:5126-5131. 60. Liu G, Wang L, Han H, et al. LncRNA ZFAS1 promotes growth and metastasis by regulating BMI1
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61. Chen X, Yang C, Xie S, Cheung E. Long non-coding RNA GAS5 and ZFAS1 are prognostic
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markers involved in translation targeted by miR-940 in prostate cancer. Oncotarget. 2017;9:1048-1062. 62. Shi H, Liu Z, Pei D, Jiang Y, Zhu H, Chen B. Development and validation of nomogram based on
patients. Oncotarget. 2017;8:59048-59057.
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lncRNA ZFAS1 for predicting survival in lymph node-negative esophageal squamous cell carcinoma
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63. Kaczynski J, Cook T, Urrutia R. Sp1- and Krüppel-like transcription factors. Genome Biol. 2003;4:206.
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64. Wu J, Lingrel JB. KLF2 inhibits Jurkat T leukemia cell growth via upregulation of
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cyclin-dependent kinase inhibitor p21WAF1/CIP1. Oncogene. 2004;23:8088-8096. 65. Zhao S, Kurenbekova L, Gao Y, et al. NKD2, a negative regulator of Wnt signaling, suppresses tumor growth and metastasis in osteosarcoma. Oncogene. 2015 Sep 24;34:5069-79.
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66. Denzler R, Agarwal V, Stefano J, Bartel DP, Stoffel M. Assessing the ceRNA hypothesis with quantitative measurements of miRNA and target abundance. Mol Cell. 2014;54:766-776.
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67. Thomson DW, Dinger ME. Endogenous microRNA sponges: evidence and controversy. Nat Rev Genet. 2016;17:272-283. 68. Liu XH, Sun M, Nie FQ, et al. Lnc RNA HOTAIR functions as a competing endogenous RNA to regulate HER2 expression by sponging miR-331-3p in gastric cancer. Mol Cancer. 2014;13:92. 69. Lv M, Zhong Z, Huang M, Tian Q, Jiang R, Chen J. lncRNA H19 regulates epithelial-mesenchymal transition and metastasis of bladder cancer by miR-29b-3p as competing endogenous RNA. Biochim Biophys Acta. 2017;1864:1887-1899.
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ACCEPTED MANUSCRIPT Figure Legends Figure 1. Overview of the regulatory mechanisms of ZFAS1 involved in cancer progression. ZFAS1 exerts multiple biological effects on proliferation, invasion, metastasis, cell cycle, apoptosis, and chemoresistance by interacting with different types of molecules. ZFAS1 can simultaneously interact with EZH2 and LSD1 to repress expression of underlying targets KLF2 and NKD2. ZFAS1
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promotes migration and invasion by inducing the EMT through reduced expression of epithelial markers and increasing levels of mesenchymal markers. ZFAS1 also facilitates malignant phenotypes
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by acting as a ceRNA. ZFAS1 functions as an oncogene via indirect destabilization of p53 and through direct and indirect interactions with the CDK1/cyclin B1 complex leading to cell cycle progression and
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inhibition of apoptosis. In addition, a ZFAS1/miR-150-5p/Sp1 feedback loop plays a pivotal role in cancer progression.
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Abbreviations: ZFAS1, zinc finger antisense 1; EZH2, enhancer of zeste homolog 2; LSD1, lysine-specific demethylase 1; KLF2, Kruppel-like factor 2; NKD2, naked cuticle homolog 2; EMT,
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epithelial-mesenchymal transition; ceRNA, competing endogenous RNA; CDK1, cyclin-dependent kinase 1; Sp1, Specificity protein 1; ZEB1/2, zinc finger E-box binding homeobox 1/2; MMP14/16,
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matrix metallopeptidase 14/16; BMI1, B lymphoma Moloney murine leukemiavirus insertion region 1.
Figure 2. Model of regulation showing epigenetic repression of KLF2 and NKD2 expression by ZFAS1. ZFAS1 simultaneously recruits EZH2 and LSD1 to the NDK2 and KLF2 promoter region and represses
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their transcription mediated by H3K27me3 and demethylation of H3K4me2. Abbreviations: ZFAS1, zinc finger antisense 1; EZH2, enhancer of zeste homolog 2; LSD1,
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lysine-specific demethylase 1; KLF2, Kruppel-like factor 2; NKD2, naked cuticle homolog 2; H3K27me3, trimethylation of histone H3 at lysine 27; H3K4me2, histone H3 lysine 4 dimethylation.
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ACCEPTED MANUSCRIPT Abbreviations: CDK1 - cyclin-dependent kinase 1 CDH1/2 - cadherin 1/2 CeRNA - competing endogenous RNA CRC - colorectal cancer CTCs - circulating tumor cells EMT - epithelial-mesenchymal transition EpCAM - epithelial cell adhesion molecule ER - estrogen receptor
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EZH2 - enhancer of zeste homolog 2 GC - gastric cancer
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Hes-1 - hairy enhancer of split-1 HCC - hepatocellular carcinoma
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H3K27me3 - trimethylation of histone H3 at lysine 27 H3K4me2 - histone H3 lysine 4 dimethylation KLF2 - kruppel like factor 2
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LncRNA - long non-coding RNA LSD1 - lysine-specific demethylase 1
MMP2/9/14/16 - matrix metallopeptidase 2/9/14/16 ncRNA - noncoding RNA PCNA - proliferating cell nuclear antigen RPL28 - ribosomal protein L28 Sp1 - Specificity protein 1
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NAA10 - Nα-acetyltransferase 10
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SnoRNAs - small nucleolar RNAs
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NKD2 - naked cuticle homolog 2
NICD - Notch intracellular domain
NSCLC - non-small cell lung cancer
ZEB1/2 - zinc finger E-box binding homeobox 1/2
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BMI1 - B lymphoma Moloney murine leukemiavirus insertion region 1 ZFAS1 - zinc finger antisense 1 ZNFX1 - zinc finger NFX1-type containing 1
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ZO-1 - zonula occludens-1
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Cancer type
Expression
Role
Biological function
Related gene and protein
References
Breast cancer
No difference or
Tumor
Proliferation, differentiation, EMT,
E-cadherin, N-cadherin, vimentin
12,15,26,27
downregulation
suppressor
migration, invasion, apoptosis, cell
proliferation, cell cycle, apoptosis,
CDK1/cyclin B1, p53, miR-590-3p,
29-32
EMT, migration, invasive,
ZEB1, E-cadherin, ZO-1, N-cadherin,
metastasis
vimentin, miR-484
Proliferation, migration, invasive,
EpCAM, CDH1, CDH2, vimentin,
Colorectal cancer
Gastric cancer
Upregulation
Upregulation
Oncogene
Oncogene
EMT, apoptosis, cell cycle,
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cycle
34-37
ZEB1, Snail, MMP14, Twist, cyclin D1, Bcl2, N-cadherin, Slug, Bax,
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chemoresistance
E-cadherin, EZH2, LSD1, NKD2,
Hepatocellular
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KLF2, PCNA, Cyclin D1, Cyclin E,
Upregulation
Oncogene
Migration, invasive, metastasis
ZEB1, MMP14, MMP16, miR-150
47,48
Upregulation
Oncogene
Proliferation, migration, invasion,
E-cadherin, vimentin, KLF2, NKD2,
53,54
apoptosis, cell cycle
ZEB1, ZEB2
Proliferation, migration, invasion,
Sp1, miR-150-5p
55,56
Proliferation, migration, invasive,
MMP2, MMP9, N-cadherin, integrin
57,58
EMT, apoptosis, cell cycle,
β1, ZEB1, Twist, Snail, E-cadherin，
Upregulation
Oncogene
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Ovarian cancer
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carcinoma Bladder cancer
Cyclin B1, MMP2
chemoresistance
Upregulation
Non-small cell lung
Upregulation
Osteosarcoma
－
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cancer
Oncogene
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Glioma
Upregulation
Oncogene
Hes-1, NICD
－
－
59
Proliferation, migration, invasive,
BMI1, miR-200b, miR-200c, ZEB2,
60
apoptosis, cell cycle, metastasis
SP1
Upregulation
－
－
miR-940, NAA10, RPL28
61
Esophageal squamous
Upregulation
－
－
－
62
cell carcinoma
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Prostate cancer
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Table 1 Functional characterization of ZFAS1 in various cancers Abbreviations: ZFAS1, zinc finger antisense 1; EMT, epithelial-mesenchymal transition; CDK1, cyclin-dependent kinase 1; ZEB1/2, zinc finger E-box binding homeobox 1/2; ZO-1, zonula occludens-1; EpCAM, epithelial cell adhesion molecule; CDH1/2, cadherin 1/2; MMP2/9/14/16, matrix metallopeptidase 2/9/14/16; EZH2, enhancer of zeste homolog 2; LSD1, lysine-specific demethylase 1; NKD2, naked cuticle homolog 2; KLF2, kruppel like factor 2; PCNA, proliferating cell nuclear antigen; Hes-1, hairy enhancer of split-1; NICD, Notch intracellular domain; BMI1, B lymphoma Moloney murine leukemiavirus insertion region 1; SP1, Specificity protein 1; NAA10, Nα-acetyltransferase 10; RPL28, ribosomal protein L28.
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Cancer type
Overexpression of ZFAS1 and clinical features
References
Breast cancer
－
－
Colorectal cancer
Poorer relapse-free survival and overall survival, positive lymphatic invasion, advanced TNM
30-32
stage, positive vascular invasion, positive Helicobacter pylori infection Gastric cancer
Advanced TNM stage, positive lymphatic metastasis, poorer overall survival and progression-free
35,36
survival Hepatocellular carcinoma
Positive portal vein tumor thrombus, positive microvascular invasion, higher recurrence rate,
Bladder cancer
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poorer overall survival
Advanced clinical stage, positive muscularis invasion, positive lymph node metastasis, positive
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53,54
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distant metastasis, poorer progression-free survival and overall survival Poorer overall survival
Glioma
Advanced tumor stage, poorer overall survival
Non-small cell lung cancer
Advanced TMN stage, poorer tumor differentiation grade, positive lymph node metastasis, poorer
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Ovarian cancer
overall survival
55 57,58 59
Poorer overall survival
Prostate cancer
Poorer disease free survival
61
Esophageal squamous cell carcinoma
Poorer overall survival
62
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Osteosarcoma
Table 2 Clinical significance of ZFAS1 in various cancers
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Abbreviations: ZFAS1, zinc finger antisense 1
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Highlights
1. ZFAS1 is dysregulated in numerous human cancers. 2. ZFAS1 is involved in several processes correlated with carcinogenesis.
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3. Molecular basis of the biological role of ZFAS1 is complex
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Figure 1
Figure 2
Xiaodi Jiang, Zhi Yang, Zhiwei Li PII: DOI: Reference:
S0378-1119(18)31218-6 https://doi.org/10.1016/j.gene.2018.11.075 GENE 43420
To appear in:
Gene
Received date: Revised date: Accepted date:
17 September 2018 21 November 2018 25 November 2018
Please cite this article as: Xiaodi Jiang, Zhi Yang, Zhiwei Li , Zinc finger antisense 1: A long noncoding RNA with complex roles in human cancers. Gene (2018), https://doi.org/ 10.1016/j.gene.2018.11.075
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Zinc finger antisense 1: a long noncoding RNA with complex roles in human cancers
Xiaodi Jianga; Zhi Yangb; Zhiwei Lia*
Department of Infectious Diseases, Shengjing Hospital of China Medical University, Shenyang, China
b
Department of General Surgery, The Fourth Hospital of China Medical University, Shenyang, China
*
Corresponding author: Zhiwei Li, Department of Infectious Diseases, Shengjing Hospital of China
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a
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Medical University, No. 39 Huaxiang Road, Tiexi District, Shenyang 110022, China. E-mail:
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[email protected]
Abstract: Zinc finger antisense 1 (ZFAS1), a newly identified long non-coding RNA, is a transcript
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antisense to the 5' end of the protein-coding gene zinc finger NFX1-type containing 1 which hosts three C/D-box small nucleolar RNAs (SNORDs) within sequential introns: Snord12, Snord12b, and
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Snord12c. ZFAS1 is dysregulated and acts as either an oncogene or a tumor suppressor in different
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human malignancies. ZFAS1 has been implicated in many aspects of carcinogenesis, including proliferation, invasion, metastasis, apoptosis, cell cycle, and drug resistance. The mechanisms underlying the effects of ZFAS1 are complex and involve multiple signaling pathways. In this review,
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the multiple pathological functions of ZFAS1 in diverse malignancies are systematically reviewed to
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elucidate the molecular basis of its biological roles and to provide new directions for future research.
Keywords: Long non-coding RNA; ZFAS1; Cancer; Oncogene; Tumor suppressor
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ACCEPTED MANUSCRIPT 1. Introduction Cancer is a group of complex diseases threatening human health worldwide. The molecular basis of cancer has been extensively studied over the past few decades. Accumulating evidence indicates that epigenetic processes play crucial roles in cancer involving histone modifications, DNA methylation, chromatin remodeling, and gene imprinting, as well as noncoding RNA (ncRNA) regulation. 1,2
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NcRNAs are transcribed from noncoding regions. Due to the lack of an open reading frame, ncRNAs have no or limited protein-coding potential.3 The ncRNAs are divided into diverse types according to
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their size and include microRNAs (miRNAs), long non-coding RNAs (lncRNAs), small nucleolar RNAs (snoRNAs), and PIWI-interacting RNAs.
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LncRNAs are ncRNAs longer than 200 nucleotides in length. Emerging evidence suggests that lncRNAs play a pivotal role in the development and progression of cancer. 4 LncRNAs are involved in
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various aspects of gene regulation including epigenetic regulation, imprinting, cellular homeostasis, transcription, and mRNA splicing.5-7 Furthermore, certain lncRNAs show developmental and tissue
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specific expression patterns.8-11 These characteristics, which are critical for their functional analysis, highlight the potential of lncRNAs as diagnostic, prognostic, and therapeutic targets in cancer.
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Although multiple lncRNAs have been studied in the past few decades, many lncRNAs have not been
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fully examined. The role of potentially thousands of lncRNAs has attracted intense scientific interest. Zinc finger antisense 1 (ZFAS1, also known as ZNFX1-AS1) is a newly identified lncRNA which is dysregulated in numerous human cancers and implicated in many aspects of carcinogenesis. ZFAS1 is
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highly expressed in the mammary gland and is downregulated in human invasive ductal carcinoma compared with normal breast tissue.12 Knockdown of ZFAS1 in mammary epithelial cells increases
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their proliferation and differentiation, indicating that ZFAS1 may act as a tumor suppressor gene in breast cancer.12 However, recent studies have demonstrated that ZFAS1 is upregulated in multiple types of tumors, suggesting that ZFAS1 may also act as a proto-oncogene in cancer. In this review, we provide an overview of current evidence supporting the role of ZFAS1 in human cancer and describe potential molecular mechanisms and their clinical significance (Tables 1 and 2).
2. Discovery and characterization of ZFAS1 ZFAS1 was originally identified by Askarian-Amiri et al. in 2011. To identify novel regulators in breast biology, they identified differentially regulated lncRNAs during mouse mammary gland development. 2
ACCEPTED MANUSCRIPT Among the highest and most differentially expressed genes was Zfas1, which was expressed in most tissues but exhibited the greatest abundance in mouse developing mammary glands, where it plays an important developmental role.12 ZFAS1 is its syntenic human ortholog. While there is relatively low primary sequence conservation between ZFAS1 and Zfas1, their predicted secondary structures have similar features.12
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ZFAS1 is located on chromosome 20q13, which contains a transcript antisense to the 5' end of the protein-coding gene zinc finger NFX1-type containing 1 (ZNFX1). Many antisense lncRNAs are
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known to regulate the expression of their protein coding counterparts in cis. 13,14 However, the lack of correlation between ZFAS1 and ZNFX1 expression indicates that there is no apparent cis regulation
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between them.15
ZFAS1 is expressed as at least five different isoforms in both cytoplasmic and nuclear
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compartments.15 They vary in size from 516 to 1006 bases with exons two and five common to all isoforms. Many of these lncRNAs appear to be ribosome-associated.16,17 A study by Hansji et al.
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indicated that ZFAS1 co-localized with polysomes, where it was predominantly associated with the small ribosomal subunit.15 ZFAS1 expression is strongly correlated with that of a number of mRNAs
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encoding ribosomal proteins involved in ribosome biogenesis. ZFAS1 may thus be involved in the
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regulation of the ribosome through interactions with mature ribosomes in the cytoplasm as well as through interactions with immature ribosomes in the nucleus.15 ZFAS1 hosts three C/D-box snoRNAs (SNORDs) within sequential introns: Snord12, Snord12b, and
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Snord12c.12 SnoRNAs are a functionally diverse group of 60–150 nt trans-acting ncRNAs that guide modifications of selected nucleotides in rRNAs or spliceosomal RNAs.18-20 From a structural basis,
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snoRNAs are divided into SNORDs and H/ACA box snoRNAs. Both types of snoRNAs are dysregulated in various types of cancer and have been implicated in the development and progression of human malignancy.21-23 In addition to the evidence that snoRNAs are involved in cancer development, there are some preliminary data showing that the genes that host snoRNAs may also have important functions in regulating the biological behavior of cancer.24
3. ZFAS1 deregulation in human cancers 3.1 ZFAS1 in breast cancer Breast cancer is a malignant tumor originating from breast tissues, is the most common cancer with 3
ACCEPTED MANUSCRIPT gradually increased incidence in women all over the world.25 The first study focusing on ZFAS1 in breast cancer was published in 2011. The authors reported that ZFAS1 was downregulated in breast cancer compared with normal breast tissue.12 Knockdown of ZFAS1 in mammary epithelial cells increased proliferation and differentiation, suggesting that ZFAS1 may serve as a tumor suppressor gene in breast cancer. However, the number of tumor tissue samples evaluated in this study was limited
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(only five) and effects on proliferation were evaluated at only one time point (48 h) without biological replicates. Subsequently, another study further analyzed the genome-wide RNA transcript profile of
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ZFAS1 using the RNAseq data set from The Cancer Gene Atlas and found that ZFAS1 expression was not significantly different in breast cancer patients compared with healthy controls.15 However, the
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expression of ZFAS1 was significantly reduced in basal and human epidermal growth factor receptor 2-positive breast cancer subtypes compared to normal breast tissue. It was also more highly expressed
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in estrogen receptor (ER)+ compared to ER- subtypes.15 Similarly, another group investigated ZFAS1 levels in invasive breast carcinoma, ductal carcinoma in situ, and normal adjacent breast tissues by
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chromogenic in situ hybridization.26 They demonstrated that ZFAS1 was negative or only weakly expressed in all groups of specimens, indicating there was no significant difference in ZFAS1
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expression in breast cancer.
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Conversely, Fan et al. determined that ZFAS1 expression was downregulated in breast cancer cell lines, and additional experiments further demonstrated that overexpression of ZFAS1 inhibited breast cancer cell proliferation, migration, and invasion in vitro.27 They suggested that ZFAS1 acts as a tumor
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suppressor in breast cancer. Obviously, further large multicenter studies with more stringent standards are required to resolve the discrepancies in ZFAS1 expression and function in breast cancer between
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these studies.
3.2 ZFAS1 in colorectal cancer Colorectal cancer (CRC) is the fourth main cause of cancer-related mortality and the third most commonly diagnosed cancer worldwide.28 Recent studies have shown that ZFAS1 is remarkably upregulated in colorectal cancer tissues and cell lines. 29-32 Moreover, high ZFAS1 expression was significantly associated with Helicobacter pylori infection, lymph nodes metastasis, vascular invasion, and advanced Tumour Node Metastasis (TNM) stage.30-32 Patients with increased ZFAS1 expression showed shorter relapse-free survival and overall survival.30 Cox multivariate analyses confirmed ZFAS1 expression as an independent prognostic factor in CRC. Biologically, silencing ZFAS1 4
ACCEPTED MANUSCRIPT suppresses CRC cell proliferation, migration and invasion, and promotes cell apoptosis through many pathways.29-32 In addition, Fang et al. reported that ZFAS1 was also increased in the plasma of CRC patients.32 Thus, ZFAS1, a potential oncogenic lncRNA in CRC, may offer a hopeful diagnostic and therapeutic choice for the treatment of CRC. 3.3 ZFAS1 in gastric cancer
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Gastric cancer (GC) represents a major health burden worldwide, being the second main cause of cancer-related deaths.33 ZFAS1 expression is also upregulated in gastric cancer,34-37 and its increased
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level has been significantly correlated with lymphatic metastasis, TNM stage, and poor prognosis. 35,36 The silencing of ZFAS1 inhibited GC cell proliferation, cell cycle progress, migration, invasion, and
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the epithelial-mesenchymal transition (EMT), and enhanced cellular sensitivity to cis-platinum or paclitaxel.37
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Circulating tumor cells (CTCs) are considered useful as a “liquid biopsy” reflecting the progression of cancer.38 Circulating nucleic acids such as lncRNAs may also reflect CTC levels and act as CTC
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markers.39 Recent studies have shown that lncRNAs are relatively stable in plasma or serum, 40,41 although the precise mechanism is unclear. One possible explanation is that nucleic acid substances
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could be packaged and protected by several microparticles such as exosomes, microvesicles, and
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apoptotic bodies.42,43 ZFAS1 has been found to be upregulated in serum and serum exosomes of GC patients, and surgery can reduce its presence in plasma.34,35 This suggests that ZFAS1 in the plasma may act as a noninvasive diagnostic and prognostic biomarker for GC.
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3.4 ZFAS1 in hepatocellular carcinoma
Hepatocellular carcinoma (HCC) is one of the most common human malignancies worldwide and the
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third leading cause of cancer-related death.44 Portal vein tumor thrombus is the main route for intrahepatic metastasis of HCC cells and is strongly correlated with poor prognosis for patients with HCC.45,46 Li et al. demonstrated that the ZFAS1 transcripts are significantly upregulated in most HCC tissues compared with nontumor tissues from the same donor. 47 Significantly higher ZFAS1 expression was found in portal vein tumor thrombotic tissues than in primary HCC and healthy liver tissues. Meanwhile, upregulation of ZFAS1 was more frequently observed in HCC patients with microvascular invasion than in those without microvascular invasion. HCC patients with high ZFAS1 expression had higher recurrence rates and shorter overall survival than those with low expression of ZFAS1. 47 Furthermore, overexpression of ZFAS1 promoted HCC cell invasion and tumor metastasis in vitro and 5
ACCEPTED MANUSCRIPT in vivo. These results indicate that ZFAS1 is upregulated and correlated with tumor recurrence and metastasis in HCC. Luo et al. demonstrated that plasma levels of ZFAS1 are higher in HCC patients than in healthy controls or in patients with cirrhosis and hepatitis B. 48 In addition, the expression of ZFAS1 is correlated with serum alpha fetoprotein (AFP). These results suggest the potential of ZFAS1 as a
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diagnostic marker to distinguish HCC patients from healthy controls. 3.5 ZFAS1 in bladder cancer
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Bladder cancer is one of the most common urological malignancies worldwide.49 Despite considerable improvements in treatment strategies for bladder cancer, such as adjuvant chemo-radiotherapies and
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immunological therapy, one third of all patients experience recurrence.50-52 Therefore, further investigation of the mechanisms involved in the proliferation, metastasis, and invasion of bladder
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cancer is necessary to develop effective therapeutic approaches for the treatment of this disease. Yang et al. demonstrated that ZFAS1 expression was upregulated in bladder cancer tissues compared
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with paired adjacent normal tissues by analyzing The Cancer Genome Atlas database. 53 Furthermore, they confirmed that levels of ZFAS1 were elevated in bladder cancer tissues and cell lines compared
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with normal bladder tissues and normal uroepithelium cell lines. Further investigation revealed that the
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expression level of ZFAS1 was positively associated with clinical stage, muscularis invasion, lymph node metastasis, and distant metastasis in bladder cancer patients.53 Wang et al. also confirmed that ZFAS1 expression was significantly upregulated in bladder cancer tissues and cell lines. 54 Survival
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analysis showed that patients with elevated ZFAS1 expression had shorter progression-free survival and overall survival. Moreover, silencing of ZFAS1 markedly suppressed bladder cancer cell
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proliferation and colony formation, arrested the cell cycle, promoted cell apoptosis, and inhibited cell migration.54
3.6 ZFAS1 in ovarian cancer Ovarian cancer is the leading cause of mortality among gynaecological malignancies.49 Xia et al. reported that ZFAS1 was upregulated in epithelial ovarian cancer tissues and was negatively correlated to the overall survival rate of patients with epithelial ovarian cancer. 55 Moreover, depletion of ZFAS1 inhibited proliferation, migration, invasion, and chemoresistance to cisplatin and paclitaxel. Similarly, a significant correlation between ZFAS1 expression and chemosensitivity was confirmed in 233 patients with high grade serous ovarian cancer from the Gene Expression Omnibus datasets. 56 In vitro 6
ACCEPTED MANUSCRIPT experiments demonstrated that the ZFAS1 expression was upregulated by cisplatin in ovarian cancer cell lines.56 These findings suggested that ZFAS1 may participate in platinum resistance in ovarian cancer. 3.7 ZFAS1 in glioma Glioma is the most common brain cancer, accounting for >60% of primary brain tumors in adults.49 A
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relationship between ZFAS1 and human glioma progression was recently reported. ZFAS1 was upregulated in glioma tissues and cell lines, and high expression of ZFAS1 was significantly correlated
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with advanced tumor stage and poor overall survival. 57,58 Multivariate Cox regression analysis showed that the ZFAS1 level could serve as an independent prognostic factor for glioma. 57 Functionally,
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ZFAS1 acted as an oncogene in glioma, and knockdown of ZFAS1 promoted apoptosis and significantly inhibited cell proliferation, migration, and invasion. 57,58 Furthermore, silencing of ZFAS1
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resulted in cell cycle arrest at the G0/G1 phase and correspondingly decreased the percentage of S
potential therapeutic target for glioma. 3.8 ZFAS1 in other cancers
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phase cells in glioma cell lines.57 Thus, ZFAS1 may act as a valuable prognostic biomarker and
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Expression analysis of ZFAS1 in non-small cell lung cancer (NSCLC) tissues showed significant
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upregulation, and higher expression levels of ZFAS1 were found in more advanced tumor tissues. 59 ZFAS1 expression levels were significantly associated with tumor differentiation grade, lymph node metastasis, and TMN stage. Furthermore, patients with higher ZFAS1 expression levels had worse
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overall survival.59 Univariate and multivariate analyses indicated that high ZFAS1 expression was an independent prognostic factor for poor survival of NSCLC patients.
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Liu et al. revealed a functional role for ZFAS1 in osteosarcoma growth and metastasis. They found that expression of ZFAS1 was upregulated in osteosarcoma samples and cell lines, and overexpression of ZFAS1 was significantly associated with unfavorable prognosis of osteosarcoma patients. 60 Functional assays also demonstrated that ZFAS1 enhanced the growth and metastatic ability of osteosarcoma cells in vitro and in vivo.60 Chen et al. demonstrated that ZFAS1 was upregulated in tumors relative to normal prostate tissues, and high expression of ZFAS1 was correlated with worse outcomes in prostate cancer patients.61 The relationship between ZFAS1 and esophageal squamous cell carcinoma (ESCC) progression was recently reported. ZFAS1 expression was significantly higher in ESCC tissues compared with 7
ACCEPTED MANUSCRIPT corresponding adjacent normal tissues.62 Survival analysis showed that ESCC patients with high ZFAS1 expression had poor overall survival, and ZFAS1 expression was determined to be an independent prognostic factor.62
4. Regulatory mechanisms of ZFAS1
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As mentioned above, multiple studies have shown that ZFAS1 is implicated in many aspects of carcinogenesis, which has prompted a large number of researchers to explore its possible molecular
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mechanism. As shown in Figure 1, ZFAS1 mediates different functions such as proliferation, invasion, metastasis, apoptosis, cell cycle, and chemoresistance via interactions with different types of molecules.
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4.1 ZFAS1 epigenetically represses KLF2 and NKD2 expression.
Nie et al. demonstrated that ZFAS1 could directly bind with the enhancer of zeste homolog 2 (EZH2),
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lysine-specific demethylase 1 (LSD1), and CoREST (histone demethyltransferase of REST complex) in gastric cancer cells.36 Furthermore, they found that the tumor suppressors naked cuticle homolog 2
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(NKD2) and Kruppel-like factor 2 (KLF2) were key downstream mediators of ZFAS1. KLF2 is a member of the Kruppel-like factor family that includes transcription factors with Cys2/His2 zinc-finger
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domains.63 NKD2, one of the naked cuticle family, has been shown to function as a tumor suppressor in a
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variety of cancers.64,65 Further investigations revealed that ZFAS1 simultaneously recruits EZH2 and LSD1 to the NDK2 and KLF2 promoter regions and represses their transcription via trimethylation of histone H3 at lysine 27 and demethylation of dimethylated histone H3 lysine 4 (Figure 2).36 Similarly,
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Yang et al. reported that knockdown of ZFAS1 repressed bladder cancer cell proliferation via upregulation of KLF2 and NKD2 expression.53 These results suggest that ZFAS1 exerts oncogenic
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affects at least partly by epigenetically repressing KLF2 and NKD2 expression.
4.2 ZFAS1 regulates EMT pathway EMT is a critical mechanism in cancer metastasis. A number of studies have shown that ZFAS1 facilitates tumor invasion and metastasis by promoting EMT. Lv et al. demonstrated that silencing ZFAS1 could impair migration and invasion by inhibiting EMT through reduced expression of matrix metallopeptidase 2 (MMP2), MMP9, N-cadherin, integrin β1, zinc finger E-box binding homeobox 1 (ZEB1), Twist, and Snail as well as by increasing E-cadherin levels in glioma.57 Similarly, Gao et al. showed that knockdown of ZFAS1 significantly decreased protein expression of N-cadherin and Snail 8
ACCEPTED MANUSCRIPT and increased E-cadherin in glioma.58 In addition, Fang et al. showed that ZFAS1 may function as an oncogene by modulating ZEB1 to induce EMT.46 Yang et al. suggested that knockdown of ZFAS1 repressed cell migration and invasion by downregulating ZEB1 and ZEB2 expression in bladder cancer.53 Liu et al. also demonstrated that ZFAS1 directly interacted with ZEB2 to regulate its stability.60 Furthermore, through domain mapping and RNA pull-down assay, they found that the 3’-end fragment of
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ZFAS1 was essential to bind ZEB2. These findings suggest that ZFAS1 may promote cancer
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progression via regulated EMT signaling pathways.
4.3 ZFAS1 serves as a miRNA sponge
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In spite of the controversy surrounding the competing endogenous RNA (ceRNA) hypothesis, its role as a plausible generic mechanism for regulating gene expression has triggered new fields of
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research.66,67 In recent years, accumulating evidence has suggested that certain lncRNAs can act as ceRNAs which function as sponges of common miRNAs to prevent their endogenous suppressive
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effect on key targets.68,69 Recently, ZFAS1 has also been identified in crosstalk with several miRNAs in human cancer. Xia et al. demonstrated that ZFAS1 promoted the expression of Specificity protein 1
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(Sp1) in ovarian cancer by competitive antagonism against miR-150-5p to promote cell proliferation
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and chemotherapy resistance in ovarian cancer cells.55 Interestingly, Sp1 could directly bind the ZFAS1 promoter and activate transcription.60 This finding suggested that a ZFAS1/miR-150-5p/Sp1 feedback loop participates in cancer progression. Another study found that ZFAS1 increases ZEB1, MMP14 and
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MMP16 expression and promotes HCC metastasis by sponging miR-150 and inhibiting its function.47 The miR-150 family is the main target of the ZFAS1 sponge, but other miRNAs can interact with it as
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well. Liu et al. reported that ZFAS1 could function as a ceRNA of B lymphoma Moloney murine leukemiavirus insertion region 1 (BMI1).60 ZFAS1 positively regulated malignant phenotypes by competitively binding miR-200b and miR-200c and upregulating BMI1. Similarly, Xie et al. found that ZFAS1 sponges miR-484 to promote cell proliferation and invasion in CRC. 31 Wang et al. demonstrated that ZFAS1 facilitates bladder cancer tumorigenesis by sponging miR-329.54
4.4 Other regulatory mechanisms In addition to the EMT pathway and acting as a ceRNA, ZFAS1 can also activate various signaling pathways. Thorenoor et al. reported that ZFAS1 functions as an oncogene in CRC via indirect 9
ACCEPTED MANUSCRIPT destabilization of p53, and through direct and indirect interactions with the cyclin-dependent kinase 1 (CDK1)/cyclin B1 complex leading to cell cycle progression and inhibition of apoptosis. 29 In addition, Gao et al. discovered that knockdown of ZFAS1 decreased the expression of Notch signal-related proteins hairy enhancer of split-1 and Notch intracellular domain in glioma cells.58
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5. Conclusion and future directions The data described in this review suggests that ZFAS1 is upregulated in a variety of human cancers and
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acts an oncogene; In contrast, ZFAS1 acts as a tumor suppressor in breast cancer. The contradictory functions of ZFAS1 in different cancers may be largely attributed to differences in tumor tissue origin,
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extracellular microenvironment, and upstream and downstream regulatory factors. ZFAS1 is dysregulated in diverse malignancies and implicated in many aspects of carcinogenesis,
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including proliferation, invasion, metastasis, apoptosis, cell cycle, and chemoresistance. The mechanism underlying the effects of ZFAS1 is complex and involves multiple signaling pathways. However, the
require further systematic investigation.
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precise molecular mechanisms upstream and downstream of ZFAS1 are not thoroughly understood and
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ZFAS1 dysregulation is significantly associated with multiple clinicopathological characteristics and
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survival rate, suggesting its potential clinical utility as a biomarker. ZFAS1 is extremely prospective to be a potential therapeutic target due to forceful tumor specificity and reduced systemic toxicity. Nevertheless, the expression and function of ZFAS1 in body fluids remains quite obscure, and the
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detection of ZFAS1 is still far from ready for clinical applications. Research of ZFAS1 function is still at a preliminary stage, and much more work is needed to gain a comprehensive understanding of the
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potential and limits of ZFAS1 for cancer diagnosis and treatment.
Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 81502073).
Compliance with Ethical Standards Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. 10
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Conflict of interest We declare that we have no conflict of interest.
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ACCEPTED MANUSCRIPT Figure Legends Figure 1. Overview of the regulatory mechanisms of ZFAS1 involved in cancer progression. ZFAS1 exerts multiple biological effects on proliferation, invasion, metastasis, cell cycle, apoptosis, and chemoresistance by interacting with different types of molecules. ZFAS1 can simultaneously interact with EZH2 and LSD1 to repress expression of underlying targets KLF2 and NKD2. ZFAS1
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promotes migration and invasion by inducing the EMT through reduced expression of epithelial markers and increasing levels of mesenchymal markers. ZFAS1 also facilitates malignant phenotypes
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by acting as a ceRNA. ZFAS1 functions as an oncogene via indirect destabilization of p53 and through direct and indirect interactions with the CDK1/cyclin B1 complex leading to cell cycle progression and
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inhibition of apoptosis. In addition, a ZFAS1/miR-150-5p/Sp1 feedback loop plays a pivotal role in cancer progression.
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Abbreviations: ZFAS1, zinc finger antisense 1; EZH2, enhancer of zeste homolog 2; LSD1, lysine-specific demethylase 1; KLF2, Kruppel-like factor 2; NKD2, naked cuticle homolog 2; EMT,
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epithelial-mesenchymal transition; ceRNA, competing endogenous RNA; CDK1, cyclin-dependent kinase 1; Sp1, Specificity protein 1; ZEB1/2, zinc finger E-box binding homeobox 1/2; MMP14/16,
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matrix metallopeptidase 14/16; BMI1, B lymphoma Moloney murine leukemiavirus insertion region 1.
Figure 2. Model of regulation showing epigenetic repression of KLF2 and NKD2 expression by ZFAS1. ZFAS1 simultaneously recruits EZH2 and LSD1 to the NDK2 and KLF2 promoter region and represses
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their transcription mediated by H3K27me3 and demethylation of H3K4me2. Abbreviations: ZFAS1, zinc finger antisense 1; EZH2, enhancer of zeste homolog 2; LSD1,
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lysine-specific demethylase 1; KLF2, Kruppel-like factor 2; NKD2, naked cuticle homolog 2; H3K27me3, trimethylation of histone H3 at lysine 27; H3K4me2, histone H3 lysine 4 dimethylation.
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ACCEPTED MANUSCRIPT Abbreviations: CDK1 - cyclin-dependent kinase 1 CDH1/2 - cadherin 1/2 CeRNA - competing endogenous RNA CRC - colorectal cancer CTCs - circulating tumor cells EMT - epithelial-mesenchymal transition EpCAM - epithelial cell adhesion molecule ER - estrogen receptor
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EZH2 - enhancer of zeste homolog 2 GC - gastric cancer
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Hes-1 - hairy enhancer of split-1 HCC - hepatocellular carcinoma
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H3K27me3 - trimethylation of histone H3 at lysine 27 H3K4me2 - histone H3 lysine 4 dimethylation KLF2 - kruppel like factor 2
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LncRNA - long non-coding RNA LSD1 - lysine-specific demethylase 1
MMP2/9/14/16 - matrix metallopeptidase 2/9/14/16 ncRNA - noncoding RNA PCNA - proliferating cell nuclear antigen RPL28 - ribosomal protein L28 Sp1 - Specificity protein 1
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NAA10 - Nα-acetyltransferase 10
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SnoRNAs - small nucleolar RNAs
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NKD2 - naked cuticle homolog 2
NICD - Notch intracellular domain
NSCLC - non-small cell lung cancer
ZEB1/2 - zinc finger E-box binding homeobox 1/2
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BMI1 - B lymphoma Moloney murine leukemiavirus insertion region 1 ZFAS1 - zinc finger antisense 1 ZNFX1 - zinc finger NFX1-type containing 1
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ZO-1 - zonula occludens-1
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Cancer type
Expression
Role
Biological function
Related gene and protein
References
Breast cancer
No difference or
Tumor
Proliferation, differentiation, EMT,
E-cadherin, N-cadherin, vimentin
12,15,26,27
downregulation
suppressor
migration, invasion, apoptosis, cell
proliferation, cell cycle, apoptosis,
CDK1/cyclin B1, p53, miR-590-3p,
29-32
EMT, migration, invasive,
ZEB1, E-cadherin, ZO-1, N-cadherin,
metastasis
vimentin, miR-484
Proliferation, migration, invasive,
EpCAM, CDH1, CDH2, vimentin,
Colorectal cancer
Gastric cancer
Upregulation
Upregulation
Oncogene
Oncogene
EMT, apoptosis, cell cycle,
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cycle
34-37
ZEB1, Snail, MMP14, Twist, cyclin D1, Bcl2, N-cadherin, Slug, Bax,
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chemoresistance
E-cadherin, EZH2, LSD1, NKD2,
Hepatocellular
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KLF2, PCNA, Cyclin D1, Cyclin E,
Upregulation
Oncogene
Migration, invasive, metastasis
ZEB1, MMP14, MMP16, miR-150
47,48
Upregulation
Oncogene
Proliferation, migration, invasion,
E-cadherin, vimentin, KLF2, NKD2,
53,54
apoptosis, cell cycle
ZEB1, ZEB2
Proliferation, migration, invasion,
Sp1, miR-150-5p
55,56
Proliferation, migration, invasive,
MMP2, MMP9, N-cadherin, integrin
57,58
EMT, apoptosis, cell cycle,
β1, ZEB1, Twist, Snail, E-cadherin，
Upregulation
Oncogene
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Ovarian cancer
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carcinoma Bladder cancer
Cyclin B1, MMP2
chemoresistance
Upregulation
Non-small cell lung
Upregulation
Osteosarcoma
－
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cancer
Oncogene
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Glioma
Upregulation
Oncogene
Hes-1, NICD
－
－
59
Proliferation, migration, invasive,
BMI1, miR-200b, miR-200c, ZEB2,
60
apoptosis, cell cycle, metastasis
SP1
Upregulation
－
－
miR-940, NAA10, RPL28
61
Esophageal squamous
Upregulation
－
－
－
62
cell carcinoma
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Prostate cancer
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Table 1 Functional characterization of ZFAS1 in various cancers Abbreviations: ZFAS1, zinc finger antisense 1; EMT, epithelial-mesenchymal transition; CDK1, cyclin-dependent kinase 1; ZEB1/2, zinc finger E-box binding homeobox 1/2; ZO-1, zonula occludens-1; EpCAM, epithelial cell adhesion molecule; CDH1/2, cadherin 1/2; MMP2/9/14/16, matrix metallopeptidase 2/9/14/16; EZH2, enhancer of zeste homolog 2; LSD1, lysine-specific demethylase 1; NKD2, naked cuticle homolog 2; KLF2, kruppel like factor 2; PCNA, proliferating cell nuclear antigen; Hes-1, hairy enhancer of split-1; NICD, Notch intracellular domain; BMI1, B lymphoma Moloney murine leukemiavirus insertion region 1; SP1, Specificity protein 1; NAA10, Nα-acetyltransferase 10; RPL28, ribosomal protein L28.
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Cancer type
Overexpression of ZFAS1 and clinical features
References
Breast cancer
－
－
Colorectal cancer
Poorer relapse-free survival and overall survival, positive lymphatic invasion, advanced TNM
30-32
stage, positive vascular invasion, positive Helicobacter pylori infection Gastric cancer
Advanced TNM stage, positive lymphatic metastasis, poorer overall survival and progression-free
35,36
survival Hepatocellular carcinoma
Positive portal vein tumor thrombus, positive microvascular invasion, higher recurrence rate,
Bladder cancer
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poorer overall survival
Advanced clinical stage, positive muscularis invasion, positive lymph node metastasis, positive
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53,54
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distant metastasis, poorer progression-free survival and overall survival Poorer overall survival
Glioma
Advanced tumor stage, poorer overall survival
Non-small cell lung cancer
Advanced TMN stage, poorer tumor differentiation grade, positive lymph node metastasis, poorer
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Ovarian cancer
overall survival
55 57,58 59
Poorer overall survival
Prostate cancer
Poorer disease free survival
61
Esophageal squamous cell carcinoma
Poorer overall survival
62
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Osteosarcoma
Table 2 Clinical significance of ZFAS1 in various cancers
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Abbreviations: ZFAS1, zinc finger antisense 1
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Highlights
1. ZFAS1 is dysregulated in numerous human cancers. 2. ZFAS1 is involved in several processes correlated with carcinogenesis.
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3. Molecular basis of the biological role of ZFAS1 is complex
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Figure 1
Figure 2