CCAT1: an oncogenic long noncoding RNA in human cancers

Abstract

Purpose

Long noncoding RNAs (lncRNAs) are a new class of noncoding RNAs that participate in a variety of biological processes such as cell proliferation, cell cycle, differentiation and apoptosis, mainly by regulation of gene expression at various levels, including chromatin, splicing, transcriptional and post-transcriptional levels. CCAT1 is a recently identified oncogenic lncRNA, which has been reported to be consistently upregulated in multiple cancer tissues and closely correlated with initiation and progression of cancers. The aim of this paper is to provide an overview of various roles of CCAT1 in human cancers.

Methods and results

We searched studies in electronic databases. Studies have shown the high expression pattern and oncogenic role of CCAT1 in different types of cancer, and aberrant expression of CCAT1 is involved in several processes correlated with carcinogenesis such as cell proliferation, apoptosis, migration and invasion by regulating different target genes and pathways.

Conclusion

LncRNA CCAT1 promises to be a novel diagnostic biomarker, therapeutic target, as well as prognostic biomarker in human cancers.

Introduction

Cancer is a major health problem and one of the leading causes of death worldwide. It is estimated that 1685,210 new cases of cancer will be diagnosed in the USA alone in 2016 and approximately 595,690 patients will die from cancer this year (Siegel et al. 2016). The molecular basis of cancer has been widely investigated in the past few decades, while our knowledge of the molecular mechanisms underlying carcinogenesis and progression of cancer is still deficient and the development of new treatments for patients has been slow. Genetic alterations alone cannot explain how cancer occurs well; however, epigenetic abnormalities in process partially account for the cause of cancer. Accumulating evidence has indicated that epigenetic alterations play critical roles in development and progression of cancers (Taby and Issa 2010). There are several epigenetic machineries in cancer, including DNA methylation, histone modifications, gene imprinting and chromatin remodeling as well as noncoding RNAs (ncRNAs) regulation (Sharma et al. 2010).

With the rapid development of the genome and transcriptome sequencing technologies and implement of genomics consortiums such as ENCODE and FANTOM, the classic view of the transcriptome landscape and its mRNA-centric paradigm for transcript annotation has undergone a fundamental change (de Hoon et al. 2015; Pennisi 2012). Scientists have found that more than 90 % of the genome can be transcribed, but only less than 2 % is subsequently translated, which means that the vast majority of genome serves as the template for the transcription of noncoding RNAs (ncRNAs) (Mattick and Makunin 2006). Over the past 10+ years, Kapranov’s group made effort to discover and characterize the extent of transcription from noncoding regions and revealed several new RNA classes and explored possible function of the noncoding portion of the human genome (Kapranov et al. 2007, 2010; St Laurent et al. 2013, 2015). Small ncRNAs (<200 nt), long noncoding RNAs (lncRNAs, >200 nt) and very long intergenic RNAs (>50 kb, vlincRNAs) are widespread in the human genome (Lazorthes et al. 2015). In the past decades, small ncRNAs, especially microRNAs (miRNAs) and small interference RNAs (siRNAs), have been widely investigated and identified as key regulators of various biological processes, including cell proliferation, differentiation, apoptosis, autophagy, necroptosis, migration, invasion and angiogenesis (Li et al. 2014; Su et al. 2015; Yoshino et al. 2013). However, in recent years, accumulating evidence has shown the potential role of lncRNAs in numerous diseases, including cancers. Some lncRNAs are upregulated in multiple cancer tissues and cells and exhibit pro-cancerous capability, such as HOX antisense intergenic RNA (HOTAIR) and metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) (Ji et al. 2003; Kim et al. 2013), while others are reported low expression and play tumor-suppressive roles, for instance, maternally expressed gene 3 (MEG3) and growth arrest-specific transcript 5 (GAS5) (Ma et al. 2015a; Zhou et al. 2012). Furthermore, certain lncRNAs have shown the developmental and tissue-specific expression patterns. These characteristics appear critical for their functional analysis and make it possible for application of lncRNAs in diagnosis, prognostic evaluation and treatment of cancer patients.

Among all of the lncRNAs, colon cancer-associated transcript-1 (CCAT1), also known as cancer-associated region long noncoding RNA-5 (CARLo-5) or CCAT1-S with a length of 2628 nucleotides, which map to chromosome 8q24.21 containing single-nucleotide polymorphisms (SNPs) strongly associated with various cancers, has been a rising star of oncogenic lncRNAs (Nissan et al. 2012; Xiang et al. 2014). CCAT1 RNA contains three ORFs, but none of them can be finally translated to protein product. Recently, CCAT1 has been reported to be consistently upregulated in multiple cancer tissues and closely correlated with initiation and progression of cancers. In this review, we will focus on CCAT1, summarize the current researches regarding the expression, functions and regulation mechanisms of CCAT1 and discuss the potential value of CCAT1 on the diagnosis, prognostic evaluation and treatment of human cancers.

CCAT1 as an oncogenic lncRNA in human cancers

CCAT1 lncRNA was first reported to be upregulated in colon cancer by Nissan et al. (2012). They identified nine transcripts differentially expressed in colon cancer by representational difference analysis (RDA) with HT29 RNA as tester and normal colon tissue RNA as driver, including CCAT1. In this study, qRT-PCR analysis demonstrated that higher level of CCAT1 was expressed in colon cancer tissues, but low or undetectable level was found in normal mucosa tissues. The expression levels of CCAT1 in colon cancer are on average 235-fold higher than that of CCAT1 in normal colon mucosa. What is important is that CCAT1 is strongly expressed in all stage of adenoma-to-carcinoma sequence, even in early phase of carcinogenesis in adenomatous polyps and in tumor proximal colonic epithelium, suggesting its high specificity. Furthermore, CCAT1 was detected to be upregulated in all of H&E-positive colon cancer-associated lymph nodes, and 40 % of H&E and immunohistochemistry (IHC)-negative lymph nodes, indicating the high sensitivity. Soon after, the experiment conducted by Alaiyan and coworkers also obtained similar results by both qRT-PCR and in situ hybridization (ISH), demonstrating the vital role of CCAT1 in tumorigenesis and metastasis of colon cancer (Alaiyan et al. 2013). Ye et al. (2015) identified that the expression of CCAT1 was significantly higher in colon cancer samples than that in adjacent normal samples and its overexpression was closely associated with local infiltration, tumor staging and vascular invasion. He et al. (2014) also confirmed that the overexpression of CCAT1 in colon cancer tissues was corrected with clinical stage and lymph nodes metastasis of colon cancer patients and aberrant upregulation of CCAT1 could induce cell proliferation, migration and invasion. Except for colon cancer, aberrant overexpression of CCAT1 has been identified in many other human cancer tissues and cells, such as gastric cancer, lung cancer and hepatocellular cancer. Cabanski et al. (2015) conducted a pan-cancer transcriptome analysis of lncRNAs comparing tumors and matched adjacent normal samples using publicly available RNA-seq data and discovered 229 lncRNAs that differentially expressed in multiple cancers. Among them, CCAT1 was confirmed to be upregulated in colon and rectal adenocarcinoma (CRC), lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). Moreover, knockdown of CCAT1 in two lung cancer cell lines, NCI-H322 M and NCI-H522, resulted in inhibition of cell growth, indicating that CCAT1 could induce lung cancer cell proliferation. Luo et al. (2014) demonstrated that CCAT1 was significantly upregulated in nonsmall cell lung cancer (NSCLC) tissues compared with adjacent normal tissues by qRT-PCR analysis. They reported that overexpression of CCAT1 was closely associated with advanced clinical stage and lymph nodes metastasis, indicating the potential oncogenic capacity. Furthermore, by loss-of-function assay, the authors found that CCAT1 could induce the cell proliferation and promote migration and invasion of NSCLC cell lines. A study conducted by Feng Yang and his colleagues found that the expression of CCAT1 was upregulated in gastric cancer tissues in comparison with adjacent normal gastric mucosa tissues and its overexpression was closely associated with tumor growth, lymph node metastasis and distant metastasis (Yang et al. 2013). Moreover, by applying gain-of-function and loss-of-function assay, the authors demonstrated that CCAT1 could promote gastric cancer cell proliferation and migration, indicating that CCAT1 might function as an oncogenic lncRNA. Similar results were obtained by Zhang et al. (2014b) and Mizrahi et al. (2015). In addition, Mizrahi et al. compared the expression of CCAT1 between gastric cancer specimens and gastric mucosa samples obtained from subjects without cancer. Interestingly, the expression of CCAT1 is higher in adjacent normal tissues than that in healthy controls, suggesting a potential “field effect.” They also identified that the expression of CCAT1 in recurrent gastric cancer tissues was highest, indicating that it might be a biomarker for surveillance. Several experiments demonstrated that CCAT1 expression was significantly overexpressed in hepatocellular carcinoma (HCC) compared with adjacent noncancerous hepatic samples and its upregulation was associated with tumor sizes, vascular invasion, Edmondson–Steiner grade and AFP (Deng et al. 2015; Wang et al. 2015; Zhu et al. 2015a, b). In addition, by using gain- and loss-of-function assay in HCC cells, CCAT1 was verified to promote cell proliferation, migration and invasion, indicating that CCAT1 served as an oncogenic lncRNA in HCC.

Besides, overexpression of CCAT1 has also been demonstrated in breast cancer, gallbladder cancer and ovarian cancer cells (Liu et al. 2013; Ma et al. 2015b; Zhang et al. 2015). To sum up, we have found the high expression pattern and oncogenic role of CCAT1 in different types of cancer, as shown in Table 1. In fact, aberrant expression of CCAT1 is involved in several processes correlated with carcinogenesis such as cell proliferation, apoptosis, migration and invasion.

Table 1.

Functions and applications of upregulated CCAT1 in different cancers

Cancer type	Functions	Applications	References
Colorectal cancer	↑Proliferation/migration/invasion	Diagnosis biomarker; staging biomarker; prognosis biomarker	Alaiyan et al. (2013), Cabanski et al. (2015), He et al. (2014), Kim et al. (2014), Nissan et al. (2012), Ye et al. (2015)
Lung cancer	↑Proliferation/migration/invasion; ↑EMT	Prognosis biomarker	Cabanski et al. (2015), Luo et al. (2014)
Gastric cancer	↑Proliferation/migration; ↓Apoptosis	NA	Mizrahi et al. (2015), Yang et al. (2013), Zhang et al. (2014), Zhou et al. (2016)
Hepatocellular cancer	↑Proliferation/migration/invasion	Prognosis biomarker	Deng et al. (2015), Wang et al. (2015), Zhu et al. (2015a, b)
Breast cancer	NA	Prognosis biomarker	Zhang et al. (2015)
Gallbladder cancer	↑Proliferation/invasion	NA	Ma et al. (2015a, b)
Ovarian cancer cells	NA	NA	Liu et al. (2013)
Acute myeloid leukemia	↑Proliferation ↓Monocytic differentiation	NA	Chen et al. (2016)

Factors involved in overexpression of CCAT1

The expression patterns and functions of CCAT1 in human cancers have been widely studied, whereas less research is available on the mechanism of CCAT1 deregulation. Current studies have suggested that the aberrant expression of lncRNAs can be mediated by several mechanisms, especially transcriptional regulation. For instance, E2F1 can bind ERIC’s promoter and elevate the expression level of lncRNA ERIC, and P53 can induce the expression of TUG1, which is a direct transcriptional target of P53 (Feldstein et al. 2013; Zhang et al. 2014a). c-Myc is also a pivotal transcription factor, which is significantly amplified in various cancers, such as colon cancer, breast cancer and prostate cancer (Nesbit et al. 1999). It can dimerize with its partner protein Max (Myc-associated protein X) to bind to E-box (CACGTG) DNA recognition sequence and activate transcription of target genes, which participate in cell proliferation, differentiation, apoptosis and adhesion (Blackwood and Eisenman 1991; Cole and Henriksson 2006; Dang et al. 2006). In recent years, a number of studies have focused on the interplay between lncRNAs and Myc in cancer (Deng et al. 2014). There is one E-box element in the promoter region of CCAT1. Moreover, several experiments demonstrated that c-Myc could enhance both transcriptional activity and expression of CCAT1 through directly binding to E-box element, consequently promoting tumorigenesis and metastatic progress of multiple cancers (He et al. 2014; Yang et al. 2013; Zhu et al. 2015b). Once the E-box element was mutated, the effect of c-Myc on CCAT1 could not be observed. Interestingly, Kim et al. (Kim et al. 2014) found that CCAT1 expression was closely associated with rs6983267 allele corrected with increased cancer susceptibility and the Myc enhancer region including rs6983267 at 8q24.21 could modulate CCAT1 expression by long range interacting with the active regulatory region of CCAT1 promoter region. More interestingly, Xiang et al. (2014) identified a new lncRNA CCAT1-L, which overlaps with CCAT1, and knockdown of CCAT1-L resulted in disruption of CCAT1, suggesting that CCAT1 might be derived from CCAT1-L.

In addition, epigenetic modulation, such as DNA methylation and histone modification, is also a critical mechanism underlying lncRNAs deregulation. For example, lncRNA MEG3, encoded by an imprinted gene located at 14q32, is silenced by hypermethylation of differentially methylated regions (DMRs), and treatment of GC cells with 5-aza-dC can increase MEG3 expression level (Zhang et al. 2010). Polycomb group protein enhancer of zeste homolog 2 (EZH2) can directly repress the expression of lncRNA SPRY4 intronic transcript 1 (SPRY4-IT1) through epigenetic maintenance of H3K27me3 (Sun et al. 2014). Hence, epigenetic regulation may also be correlated with the aberrant expression of CCAT1 in human cancer, although there is currently not enough evidence to prove this in cancer.

The molecular mechanisms of how CCAT1 inducing human cancers

A growing number of reports have shown that CCAT1 has a pivotal role in malignancies and exhibits its oncogenic activity by inducing cancer cell proliferation, migration and invasion. Nevertheless, the molecular mechanism by which CCAT1 exerts its oncogenic capacity remains largely unknown (Fig. 1). Kim et al. (2014) found that knockdown of CCAT1 by siRNA led to inhibition of colon-derived cell proliferation and increase in cell population in the G1 phase, suggesting that CCAT1 might induce cell proliferation by regulating cell cycle. Moreover, the authors identified 14 upregulated and 24 downregulated cell cycle-related genes by profiling cells treated with CCAT1-siRNA and demonstrated that CCAT1 could inhibit the expression of CDKN1A mRNA, which is a crucial regulator of G1 arrest. Luo et al. (2014) also reported that knockdown of CCAT1 could induce a significant G0/G1 arrest in NSCLC and silencing of CCAT1 by siRNA significantly enhanced the expression of p16, p21 and p27, which are GO/G1 arrest markers, by Western blotting assay. Furthermore, they demonstrated that silencing of CCAT1 resulted in upregulation of E-cadherin expression while downregulation of fibronectin and vimentin, which are critical hallmarks of epithelial–mesenchymal transition (EMT). In addition, knockdown of CCAT1 inhibited the expression of a series of transcription factors mediating EMT, such as Snail and Twist. These data indicated that the effect of CCAT1 on cell migration and invasion might be mediated by modulation of EMT. Another study showed that CCAT1 silencing could inhibit gastric cancer cell proliferation by induction of GO/G1 arrest and apoptosis (Zhang et al. 2014b). By Western blotting analysis, the authors demonstrated that knockdown of CCAT1 led to upregulation of GO/G1 arrest markers, p16, p21 and p27, and pro-apoptotic factors, caspase-3 and Bax/Bcl-2 ratio. What’s more, they found that silencing of CCAT1 resulted in inactivation of ERK/MAPK pathway, which plays a pivotal role in modulation of diverse cellular processes, such as cell proliferation, apoptosis and differentiation.

Fig. 1.

Regulation mechanism of CCATI in human cancers

Despite the controversy surrounding the competing endogenous RNAs (ceRNA) hypothesis, as a plausible generic mechanism for regulating gene expression, it has sparked new areas of research (Denzler et al. 2014; Thomson and Dinger 2016). In recent years, accumulating evidence suggests that certain lncRNAs can serve as ceRNAs, which function as sponges for common miRNAs and abolish their endogenous suppressive effect on key targets. An example of this type of regulation is exemplified by HOTAIR, which can act as an endogenous “sponge” to compete for miR-331-3p binding in gastric cancer and indirectly leads to altered expression of multiple target genes of miR-331-3p, such as oncogene human epithelial growth factor receptor 2 (HER2) (Liu et al. 2014). Similar report showed that PTENP1, a PTEN pseudogene-expressed noncoding RNA, could act as a tumor suppressor in prostate cancer cells and indirectly affect the activity of PTEN by competing for binding of miRNAs (Poliseno et al. 2010). Recently, CCAT1 has also been identified to cross talk with several miRNAs in human cancer. Deng et al. (2015) reported that CCAT1 could serve as a sponger for let-7 in HCC and resulted in de-suppression of its targets HMGA2 and c-Myc, antagonizing its function. It is well known that let-7 family exerts pivotal role in tumor inhibition through suppression of proliferation, migration and invasion, as well as induction of apoptosis (Boyerinas et al. 2010; Tang et al. 2016). Moreover, other reports have shown that c-Myc could directly bind to the promoter region of CCAT1 and activate its expression in several cancers, including HCC. Hence, CCAT1 and c-Myc maybe form a double-positive feedback loop to enhance the expression of each other. Similarly, Chen et al. (2016) found that CCAT1 inhibited myeloid cell differentiation and promoted cell proliferation by serving as a ceRNA for miR-155, thus altering the expression of c-Myc, a target of miR-151. Recent studies have demonstrated that there is reciprocal repression between lncRNAs and miRNAs. For example, lncRNA-GAS5 and miRNA-21 could modulate each other in a way similar to the microRNA-mediated silencing of target mRNAs (Zhang et al. 2013). Similarly, Ma et al. (2015b) demonstrated that CCAT1 promoted the proliferation and migration of GBC cells via negative regulation of miR-218-5p and then regulating its target Bmi1. Moreover, they found that CCAT1 and miR-218-5p bound to the same RISC complex and CCAT1 might promote the degradation of miR-218-5p by binding to the RISC complex, in which case CCAT1 served as a “miRNA” while miR-218-5p acted as the “miRNA targeting mRNA.” However, this study did not investigate the regulatory role of miR-218-5p in CCAT1. Zhou et al. (2016) identified that miR-490 could target CCAT1 and CCAT1 repressed miR-490, thus forming a reciprocal repression regulatory loop. What’s more, they found that CCAT1 modulated the expression of hnRNAP1 protein by negative regulation of miR-490 and CCAT1/miR-490/hnRNPA1 axis promoted GC migration. In all, these results enhance our understanding on the ceRNA regulatory network.

Clinical application of CCAT1 in human cancers

It is generally known that early diagnosis and early treatment can greatly increase the survival rate of cancer patients. Hence, exploration of useful biomarkers for early detection and diagnosis of cancer are required. Among the various methods currently being used, body fluid-based testing may be the ideal detection strategy for biomarkers because of the lower invasiveness and ease. However, many cancers lack serums markers with high sensitivity and specificity for general population screening; therefore, it is essential to explore novel valuable blood biomarkers. In recent years, growing evidence has shown that circulating lncRNA in plasma, serum or urine has the potential to be noninvasive diagnostic biomarkers for certain cancers (Tong et al. 2015; Wang et al. 2014; Zhou et al. 2015). For instance, plasma H19 could serve as a potential biomarker for diagnosis of GC, in particular for early tumor screening (Zhou et al. 2015). Recently, Nissan et al. (2012) found that CCAT1 expression levels in peripheral blood mononuclear cells (PBMCs) obtained from 7 of 16 (45 %) patients with CRC were significantly higher than that obtained from healthy individuals. Zhao et al. (2015) also demonstrated that among 13 lncRNAs aberrant expressed in CRC, plasma CCAT1 provided the highest diagnostic performance for detection of CRC (the area under the ROC curve (AUC), 0.842; P < 0.001; sensitivity, 75.7 %; specificity, 85.3 %). Intriguingly, CCAT1 combining with HOTAIR could provide a more powerful differential diagnosis between CRC patients and healthy controls (AUC, 0.954, P < 0.001, sensitivity, 84.3 %; specificity, 80.2 %) than use of CCAT1 alone. Most importantly, this combination was effective to detect CRC at an early stage (85 %). Furthermore, Kam et al. (2014) identified a CCAT1-specific peptide nucleic acid (PNA)-based molecular beacons (TO-PNA-MB) to serve as a diagnostic probe for in vitro, ex vivo and in situ detection of CRC. However, these study groups consisted of a relatively small number of patients. Future large-scale and multi-center randomized controlled studies are needed to evaluate the diagnostic value of CCAT1 in CRC or other human cancers.

Although the clinical value of prognostic biomarkers is less than that of early diagnostic biomarkers, prognostic biomarkers can predict therapeutic effects to propose adequate treatments. Studies have verified the association between CCAT1 and prognosis of several human cancers, including HCC, breast cancer, CRC and NSCLC (Deng et al. 2015; He et al. 2014; Luo et al. 2014; Wang et al. 2015; Zhang et al. 2015; Zhu et al. 2015b). High expression of CCAT1 in tumor tissues may indicate poor disease-free survival and overall survival of tumor patients.

It is well known that a portion of conventionally staged N0 patients will develop recurrence of cancer, based on current histopathological nodal staging (positive or negative). Current histopathological nodal staging techniques may overlook occult lymph node metastasis, consequently resulting in clinical under-staging and under-treatment. Hence, it is important to improve the accuracy of lymph node staging in patients with cancer. Alaiyan and his coworkers found that CCAT1 expressions were significantly upregulated (over 100-fold) in all ten metastatic lymph nodes verified by histopathological examination obtained from CRC patients (Alaiyan et al. 2013). Furthermore, Nissan et al. (2012) demonstrated that CCAT1 was highly expressed even in a few pathologically staged node-negative (N0) patients with CRC, suggesting that these patients might have node micrometastases that could not be detected by nodal staging techniques utilized in clinic. Hence, CCAT1 may be used clinically for detection of occult lymph node metastases, which is hoped to improve staging accuracy and individualized therapeutic regimens. Further studies should be conducted to validate the positive quantitative threshold of CCAT1 for node staging and investigate the potential value of CCAT1 as a biomarker for clinical staging in other human cancers.

Several studies have shown that the expression of CCAT1 could be dramatically reduced by siRNA administration, leading to inhibition of oncogenic properties. Moreover, CCAT1 is significantly overexpressed in a variety of cancer tissues but seldom expressed or even not expressed in a panel of normal tissues (Nissan et al. 2012). The advantage makes CCAT1 a novel candidate antitumor therapeutic target. McCleland et al. (2016) reported that the bromodomain and extraterminal (BET) protein BRD4 inhibition, JQ1, led to growth arrest and differentiation in the epigenetically dysregulated CpG island methylator phenotype (CIMP) + class of tumors and CCAT1 expression predicted JQ1 sensitivity and BET-mediated c-Myc regulation. All these suggests that CCAT1 can be an ideal biomarker for identifying patients who are likely to benefit from BET inhibitors, which have been entered into clinical trials for several hematological malignancies (Shi and Vakoc 2014). However, the targeting of CCAT1 as a novel therapeutic approach requires a wider and deeper understanding of its biological functions and molecular mechanisms of action.

Conclusion

lncRNAs are emerging as a novel class of ncRNAs that are widely transcribed in the genome. Recently, high-throughput sequencing technologies such as microarray and RNA-seq have revealed thousands of lncRNAs that are crucial regulators of human gene expression. Studies have shown the high expression pattern and oncogenic role of CCAT1 in different types of cancer, and aberrant expression of CCAT1 is involved in several processes correlated with carcinogenesis such as cell proliferation, apoptosis, migration and invasion. However, our understanding of the role for CCAT1 in cancer biology is still in an early stage, and these discoveries have not been applied for tumor patients in clinical practice. Hence, further more large-scale researches are needed to elucidate the functions and exact molecular mechanisms of CCAT1 involved in carcinogenesis. What is important is that CCAT1 has the potential to serve as biomarkers for screening, diagnosis, staging and prognostication of cancer, as well as a novel target for molecular therapies. However, large case–control studies are needed to prove the sensi tivity and specificity of CCAT1 as a serum marker to monitor disease behavior and patient response to treatment.

Compliance with ethical standards

Conflict of interest

None.