Non-coding RNA Activated by DNA Damage: Review of Its Roles in the Carcinogenesis

Long intergenic non-coding RNA 00657 (LINC00657) or “non-coding RNA activated by DNA damage” (NORAD) is an extremely conserved and copious long non-coding RNA (lncRNA). This transcript has pivotal role in the preservation of genome integrity. Several researches have appraised the role of NORAD in the evolution of human cancers with most of them indicating an oncogenic role for this lncRNA. Several miRNAs such as miR-199a-3p, miR-608, miR−155−5p, miR-590-3p, miR-495-3p, miR-608, miR-202-5p, miR-125a-3p, miR-144-3p, miR−202−5p, and miR-30a-5p have been recognized as targets of NORAD in different cancer cell lines. In addition, NORAD has interactions with cancer-related pathways, particularly STAT, TGF-β, Akt/mTOR, and PI3K/AKT pathway. Over-expression of NORAD has been related with poor clinical outcome of patients with diverse types of neoplasms. Collectively, NORAD is a prospective marker and target for combating cancer.


INTRODUCTION
Long intergenic non-coding RNA 00657 (LINC00657) or alternatively named as "non-coding RNA activated by DNA damage" (NORAD) is an extremely conserved and copious long non-coding RNA (lncRNA; Lee et al., 2016). This transcript has a crucial role in the conservation of genome stability since its inactivation results in striking aneuploidy in formerly karyotypically normal cells (Lee et al., 2016). This function of NORAD is exerted through sequestering Pumilio RNA Binding Family Members (Lee et al., 2016). In addition, NORAD has functional interactions with an element of DNA-damage system namely RNA Binding Motif Protein X-Linked (RBMX). NORAD regulates the capacity of RBMX to construct a ribonucleoprotein complex which encompasses a number of proteins such as topoisomerase I (Munschauer et al., 2018). Depletion of NORAD results in high rate of chromosome segregation impairments, abridged replication-fork speed and changed cellcycle movement (Munschauer et al., 2018). Due to the critical role of NORAD in the maintenance of genome stability and cell cycle progression, it is not surprising that dysregulation of this lncRNA leads to cancer. Therefore, several studies have appraised the role of this NORAD in initiation or progression of diverse types of malignancies. In the current review, we describe the role of NORAD in the evolution of human cancers based on the conducted experiments in cell lines, animal models and human subjects.

CELL LINE STUDIES
Expression of NORAD has been down-regulated in endometrial cancer cells. Forced up-regulation of this lncRNA suppressed growth of endometrial cancer cells and enhanced their apoptosis. Such effects have been exerted through NORAD binding with the anti-apoptotic protein Far Upstream Element Binding Protein 1 (FUBP1). Interaction between NORAD and FUBP1 has been shown to decrease nuclear localization of this antiapoptotic protein, releasing the pro-apoptotic gene promoters from FUBP1 occupation and enhancing apoptosis in these cells . A single study in colorectal cancer cells showed down-regulation of NORAD. Forced over-expression of NORAD reduced cell viability and invasiveness of these cells while enhanced cell apoptosis. This lncRNA has increased expression of Calpain 7 (CAPN7) and suppressed activity of PI3K/AKT pathway (Lei et al., 2018). However, two other studies in colorectal cancer cells reported the role of NORAD in increasing cell viability, proliferation, migration and invasion while inhibiting apoptosis Zhang et al., 2018). Other studies in diverse cancer cell lines also supported the oncogenic role of this lncRNA. For instance, in ovarian cancer cells, over-expression of NORAD has been correlated with downregulation of miR-199a-3p. NORAD silencing could suppress proliferation, invasiveness, migratory potential, and epithelialmesenchymal transition (EMT) of these cells. Functional studies confirmed the direct interplay between NORAD and miR-199a-3p (Xu C. et al., 2020). Besides, NORAD up-regulation has enhanced migration and invasion of hepatocellular carcinoma cells through sponging miR-202-5p, which acts as a tumorsuppressor miRNA through the TGF-β pathway (Yang et al., 2019a). The functional effect of NORAD in activation of TGF-β signaling has also verified in breast cancer cells (Zhou et al., 2019). In lung cancer cells, NORAD promotes EMTlike characteristics through activation of TGF-β signaling. In this type of cancer, importin β1 has been found to be a binding partner of NORAD. NORAD silencing has inhibited the physical interaction between importin β1 with Smad3 to some extent, thus blocking amassment of Smad complexes in the nucleus following induction with TGF−β. Therefore, NORAD facilitates the interaction between importin β1 and Smad3 to enhance nuclear amassment of Smad complexes following exposure to TGF-β (Kawasaki et al., 2018). Lentivirusmediated silencing of NORAD in epithelial cancer cells has inhibited proliferation, reduced chemoresistance and attenuated cell cycle progression. These roles are exerted through acting as a molecular sponge for hsa-miR-155-5p (Tong et al., 2019). In cervical cancer cells, NORAD enhances expression of SIP1 to increase cell proliferation, invasiveness and EMT. These effects are due to sponging miR-590-3p (Huo et al., 2018). In neuroblastoma, in addition to enhancement of cell proliferation and invasion, NORAD increases doxorubicin resistance possibly through suppression of apoptosis and autophagy. These effects are exerted through miR-144-3p/HDAC8 axis . In osteosarcoma cell lines, NORAD regulates cancer cell features via acting as a molecular sponge for hsa-miR-199a-3p . Another study has shown that transcription of NORAD is suppressed by the YAP/TAZ-TEAD complex, a transducer of Hippo pathway. NuRD complex also facilitates transcriptional silencing of NORAD through this route. NORAD exerts effective suppressive impact on migration and invasion of neoplastic cell lines, and blockage of NORAD expression contributes in the pro-migratory and invasive impacts of the YAP pathway. Functionally, NORAD uses its numerous repeated sequences to act as a multifaceted scaffold for binding and isolating S100P, thus inhibiting S100P-associated pro-metastatic cascades (Tan et al., 2019).
Non-coding RNA activated by DNA damage has also been found to increase expression of the PI3K/AKT/mTOR pathwayrelated proteins. Expression of these proteins has not not considerably influenced by miR-520a-3p mimic. However, cotransfection of NORAD and miR-520 mimic has upturned the expression of these proteins. NORAD silencing has not affected expression of PI3K/AKT/mTOR pathway-associated proteins, while anti-miR-520 has enhanced expression of these proteins. Taken together, NORAD has been shown to induce the activity of PI3K/AKT/mTOR signaling through sponging miR-520 (Wan et al., 2020). Table 1 displays summary of studies which evaluated expression of NORAD in cancer cell lines. Figure 1 depicts the role of Hippo cascade transducer YAP/TAZ-TEAD complex in inhibiting the expression of lncRNA NORAD in lung and breast neoplasms, and consequent attenuation of the tumor suppressor roles of NORAD in tumor cells. Figure 2 demonstrates the modulation of TRIP13 expression through lncRNA NORAD indicating that TRIP13 upregulation could suppress the impacts of miR-495-3p up-regulation on the proliferation, apoptosis, migratory potential, and invasiveness of prostate cancer cells. Figure 3 represents the oncogenic role of NORAD in gastric cancer progression via modulating the expression levels of RhoA/ROCK1.

HUMAN STUDIES
Based on the assessment of data available in The Cancer Genome Atlas (TCGA) as well as an independent cohort of patients with endometrial cancer, expression of NORAD has been decreased in endometrial cancer samples compared with normal tissue samples in association with cancer progression. Notably, has been identified as the underlying mechanism of NORAD downregulation in these samples . A single study in patients with colorectal cancer demonstrated down-regulation of NORAD in tumor tissues particularly in samples obtained from patients developed distant metastasis. Down-regulation of NORAD has been associated with poor patients' outcome, advanced tumor size and TNM stage (Lei et al., 2018). Apart from these two studies, other studies have reported up-regulation     Frontiers in Cell and Developmental Biology | www.frontiersin.org FIGURE 1 | A schematic representation of the crosstalk between Hippo signaling cascade and lncRNA NORAD in lung and breast neoplasms. YAP/TAZ is mainly modulated via the canonical Hippo cascade, MST1/2-SAV1, and LATS1/2-MOB1. LATS1/2 could in turn phosphorylate YAP/TAZ and suppress its function through either ubiquitination and proteasome-mediated degradation or 14-3-3-mediated cytoplasmic sequestration. Unphosphorylated YAP/TAZ is transferred to the nucleus, where it could interact with TEAD transcription factors and trigger the expression of various target genes. LATS1/2 could be upregulated via STK25, TAOK, NF2, and MAP4KS, while being inhibited through GPCR-RHOA-mediated F-actin function mechanical cues as well as NUAK2. In addition, expression of MST1/2 is regulated by TAOK and MARK4. Expression of YAP/TAZ is also modulated in an independent manner from LATS. Besides, PTPN14 and AMOT could interact with YAP/TAZ and sequester it in the cell membrane. Expression of YAP/TAZ is downregulated via the β-catenin demolition complex or TIAM1 through a direct interaction. Phosphorylation of YAP/TAZ is triggered by CDK1, AMPK, Aurora A, NLK, and various RTKs. In addition, p38 and VGLL4 could interact with TEAD and inhibit the function of YAP/TAZ (Yamaguchi and Taouk, 2020). Mounting evidence has collectively demonstrated that the Hippo pathway transducer YAP/TAZ-TEAD complex could play an effective role in suppressing the expression level of lncRNA NORAD in both lung and breast cancers. Its downregulation correlates with enhancement of migration, invasion as well as metastasis in tumor cells (Tan et al., 2019).
of NORAD in tumoral samples compared with non-tumoral samples from the same tissue. Such over-expression has also been verified in other cohorts of patients with colorectal cancer Zhang et al., 2018). Besides, expression of this lncRNA has been up-regulated in hepatocellular carcinoma (HCC) samples compared with adjacent tissues in correlation with poor overall survival (Yang et al., 2019a). Over-expression of NORAD in cervical cancer patients has been correlated with higher stage, lymph nodes and vascular involvement, and poor survival (Huo et al., 2018). Table 2 depicts the results of studies which evaluated expression of NORAD in clinical samples.

PROGNOSTIC ROLE OF NORAD IN MALIGNANCIES
Apart from endometrial cancer in which up-regulation of NORAD determined good prognosis , in other Frontiers in Cell and Developmental Biology | www.frontiersin.org Heterogeneous expression patterns of Fubp1 could be seen among several cancer tissues, illustrating its multiple and sophisticated functions in cancer development . Accumulating evidence elucidates that epigenetic inactivation of NORAD could promote cell growth and reduce apoptosis in endometrial cancer cells. NORAD/FUBP1 interaction could inhibit FUBP1 nuclear localization, and thereby downregulating the recruitment of FUBP1 on promoters of target pro-apoptotic genes, triggering apoptosis in tumor cells .

ANIMAL STUDIES
Endometrial cancer is among few cancer types in which NORAD exerts anti-oncogenic effects. Such effects have been verified in animal models since NORAD silencing has enhanced tumor growth in the xenograft model. On the other hand, over-expression of FUBP1-binding fragment of NORAD has attenuated tumor growth in this model . Figure 4 illustrates the effect of lncRNA NORAD binding with FUBP1 in endometrial cancer cells.
Apart from this study, other in vivo studies have shown the role of NORAD in enhancement of tumor progression in animal models. For instance, NORAD increases the growth of neuroblastoma tumors in animal models via miR-144-3p/HDAC8 axis . Moreover, growth of osteosarcoma tumors in animals has been attenuated by NORAD silencing in the implanted cells . Further studies in malignant melanoma, cervical cancer, breast cancer and lung cancer supported oncogenic effects of NORAD in xenograft models. Table 3 recapitulates the results of studies which evaluated the role of NORAD in the development of cancer in animal models.

DISCUSSION
Numerous studies have evaluated the role of NORAD in the development of cancer. With the exception of two studies in endometrial and colorectal cancer, other studies indicate the oncogenic role of this lncRNA in diverse cancer types. Several miRNAs such as miR-199a-3p, miR-608, miR−155−5p, miR-590-3p, miR-495-3p, miR-608, miR-202-5p, miR-125a-3p, miR-144-3p, miR−202−5p, and miR-30a-5p have been recognized as targets of NORAD in different cancer cell lines. In addition, NORAD has interactions with cancer-related pathways such as STAT, TGF-β, Akt/mTOR, and PI3K/AKT pathway. The role of NORAD in activation of TGF-β has been verified in different cancers, namely hepatocellular carcinoma, breast cancer and lung cancer. This function is implicated in the enhancement of EMT features and invasive properties of cancer cells. Therefore, in addition its role in the initiation of cancer possibly through influencing genomic stability, NORAD partakes in the progression of cancer through enhancement of EMT. In addition, NORAD has a role in the modulation of response of cancer cells to a number of chemotherapeutic drugs such as doxorubicin and cisplatin (Huang et al., 2020;Wang et al., 2020).
In vivo studies in xenograft models of ovarian, cervical, breast, gastric, colorectal, liver and lung cancers as well as neuroblastoma and osteosarcoma have shown the efficacy of NORAD-targeting therapeutic options in reducing tumor burden. Therefore, this lncRNA is a putative target for treatment of cancer.
The prognostic value of NORAD has been verified in diverse cancer types such as lung, liver, pancreatic, colorectal, breast and cervical cancers where over-expression of this lncRNA was correlated with poor survival. Based on the significant difference in expression of this lncRNA between cancerous and noncancerous tissues, assessment of its expression might provide a diagnostic tool for cancer. However, the sensitivity and specificity of this marker should be assessed in diverse cancer types. Moreover, assessment of its expression in body fluid such as blood, serum and urine might help in the development of noninvasive diagnostic methods. The latter possible application of NORAD has not been assessed yet.
The data presented above indicate up-regulation of NORAD in almost all types of neoplasm. Moreover, functional studies have shown the pro-proliferative, pro-migratory, and pro-metastatic abilities of NORAD. Collectively, NORAD in an oncogenic lncRNA in most tissues and a possible target for inventions against cancer. Future investigations are required to support its application as diagnostic marker in the clinical settings.

AUTHOR CONTRIBUTIONS
MT and SG-F wrote the draft and revised it. ND, TA, BH, and AA collected the data and designed the tables and figures. All authors read and approved the submitted version.