Epigenetic Regulation by lncRNAs: An Overview Focused on UCA1 in Colorectal Cancer

Colorectal cancers have become the second leading cause of cancer-related deaths. In particular, acquired chemoresistance and metastatic lesions occurring in colorectal cancer are a major challenge for chemotherapy treatment. Accumulating evidence shows that long non-coding (lncRNAs) are involved in the initiation, progression, and metastasis of cancer. We here discuss the epigenetic mechanisms through which lncRNAs regulate gene expression in cancer cells. In the second part of this review, we focus on the role of lncRNA Urothelial Cancer Associated 1 (UCA1) to integrate research in different types of cancer in order to decipher its putative function and mechanism of regulation in colorectal cancer cells. UCA1 is highly expressed in cancer cells and mediates transcriptional regulation on an epigenetic level through the interaction with chromatin modifiers, by direct regulation via chromatin looping and/or by sponging the action of a diversity of miRNAs. Furthermore, we discuss the role of UCA1 in the regulation of cell cycle progression and its relation to chemoresistance in colorectal cancer cells.


Colorectal Cancer
Colon and rectal cancers (together nominated colorectal cancer (CRC)) have become the second leading cause of cancer deaths both in the United States and in Europe ( [1,2], respectively). CRC occurrence has been correlated to an unhealthy lifestyle (tobacco, alcohol, red meat, sedentariness, obesity), whereas physical activity and dietary fibers protect against CRC [3]. In addition, early diagnosis by stool-based CRC screening has decreased disease mortality [4]. However, most patients are only diagnosed after they have symptoms and frequently present metastatic lesions (e.g., 14% in the German DACHS study [5], 19-24% in US SEER study [6]). An additional 20% of the CRC patients develop metastases during their disease evolution [5,7]. Distant metastases occur mainly in the liver, peritoneum and lung tissues. Non-metastatic colon cancer is generally treated by surgical colectomy combined with chemotherapy (e.g., inhibitors of DNA synthesis, FOLFOX (FOLinic acid, 5-Fluorouracil (5-FU) and OXaliplatin) or CAPEOX (CAPEcitabine and OXaliplatin), ±anti-VEGF antibodies such as bevacizumab [8]). Isolated and local metastases of colon cancer are also surgically resected. For unresectable metastatic CRC, a continuum of systemic chemotherapy is provided (e.g., combined therapy with reduced folate leucovorin, topoisomerase I inhibitor irinotecan, anti-EGFR antibodies cetuximab, or panitumumab; described in the National Comprehensive Cancer Network Clinical Practice Guidelines [8]).
Several tumor tissue alterations are important for the diagnosis and prognosis of CRC [8,9]. For example, CRC patients present 5-20% tumors with microsatellite instability (MSI) related to Figure 1. The function of lncRNAs in the cell. LncRNAs exert different functions in the nucleus, ranging from genomic DNA organization in speckle bodies, histone, and DNA methylation and direct transcriptional regulation (Section 2.1). Through their interaction with mRNA and function in miRNA regulation, they affect protein translation (Section 2.2). In addition, they interact with proteins, affecting stability, activity, and/or complex recruitment (Section 2.3). LncRNAs exert different functions in the nucleus, ranging from genomic DNA organization in speckle bodies, histone, and DNA methylation and direct transcriptional regulation (Section 2.1). Through their interaction with mRNA and function in miRNA regulation, they affect protein translation (Section 2.2). In addition, they interact with proteins, affecting stability, activity, and/or complex recruitment (Section 2.3). Many lncRNAs are antisense to coding mRNA transcripts. Our text-mining using the UCSC genome table browser shows that there are at least 991 genes with a known antisense transcript (curated RefSeq track, GRCh38/hg38). The transcription of antisense lncRNA can directly inhibit transcription of sense coding genes [69][70][71], which may be mediated by Polymerase II collisions [26]. Both SPINT-AS1 and UTX-AS1 expression are negatively correlated with sense gene expression, and their overexpression is correlated with poor prognosis for CRC [72,73]. Transcripts of lncRNA may also bind to splicing machinery factors and activate proximal promoter regions, as shown for U1 snRNP/linc1319 that activated the malignant brain tumor (MBT) domain-containing protein Sfmbt2 [74]. In addition, long-range chromatin looping through lncRNA-DNA interaction may regulate protein-coding gene transcription [75,76]. Amaral et al. reported the association with chromatin looping for several lncRNAs differentially expressed in CRC cells (GAS5, H19, HAGLR, NEAT1, PINT, and CRNDE (in Supplementary Table S6 of Amaral et al. [76])). The lncRNA CCAT1-L that is upregulated in human CRC also regulates such chromatin looping at the MYC locus [77]. UCA1/CUDR promotes chromatin looping at the promoter of lncRNA HULC in liver cancer cells [78]. First reported for tumor suppressor lncRNA MEG3, one of the mechanisms that guide lncRNAs to target DNA sequences is the formation of the triplex RNA-DNA helixes of GA-rich regions [76,79]

Interaction of lncRNAs with RNA
Evidence of direct physical lncRNA-mRNA interaction enhancing the stability of mRNA is sparse, but was shown for lncRNA MACC1-AS1 in gastric cancer cells and MAPT AS1 in breast cancer cells [80,81]. Similarly, the transforming growth factor-β (TGFβ)-induced antisense RNA Zeb2/Sip1 in bladder carcinoma cells binds to the 5'UTR-splicing site of the ZEB2 transcript and prevents its degradation [82,83]. Nonetheless, the stability of mRNA is modified via the binding of miRNAs frequently, which recruits a miRNA-induced silencing complex (miRISC) triggering mRNA deadenylation and decay [84]. On one hand, lncRNAs can be processed to generate miRNAs changing the miRNome of cells. Several tumor-related lncRNAs have been shown to be miRNA-precursors (Table 1). On the other hand, lncRNAs are frequently reported to function as competing endogenous RNAs (ceRNAs) to relieve the miRNA-mediated degradation of mRNAs [85]. Besides lncRNA, ceRNAs may also include mRNAs, circle RNAs, and pseudogenes [86]. Despite the fact that the stoichiometric relationship between miRNAs/regulated genes and the affinity of ceRNAs to miRNAs may question the feasibility of gene regulation through changes in ceRNA levels [87], several dedicated databases list lncRNA-miRNA interactions [86,88] and the number of original reports on their interactions still increases every year (at least 130 articles on lnc-ceRNAs January-June 2018). Recently, an lncRNA/miRNA/mRNA interactome was reported based on CRC expression data from the TCGA database. These analyses illustrated a ceRNA network of 25 principal miRNAs and 64 lncRNAs, including CRNDE, H19, HULC and UCA1 [17,89]. As discussed below, UCA1 was reported to interact with 29 miRNAs in several types of cancers. Five of these miRNAs were reported as differentially expressed and in the ceRNA network (miR- 143, -144, -145, -182, and -206; [89]). It remains unclear whether the binding of miRNAs to lncRNAs also triggers lncRNA decay, since this decay may be very slow.
Direct interaction of lncRNA-mRNA affects mRNA splicing, but may also interfere with protein translation. Antisense transcripts that overlap a gene translation start site and encode an inverted retrotransposon short interspersed nuclear element (SINE) may stimulate protein translation [90]. In addition, the interaction of lncRNA GAS5 in a complex with Eukaryotic Translation Initiation Factor 4E (eIF4E) inhibited the cMYC translation [91].

Interaction of lncRNAs with Proteins
The interaction of lncRNAs with proteins can evoke the recruitment of effector complexes: as discussed above, the interaction with protein complexes affect epigenetic modifications and the interaction with heterogeneous nuclear ribonucleoproteins (hnRNPs) can regulate gene transcription in the nucleus (respectively reviewed by References [29,92]). The binding of lncRNAs to proteins can also function as a scaffold to increase protein stability (Table 1), e.g., the binding of the lncRNA Small Nucleolar RNA Host Gene (SNHG) 15 to SLUG blocks its ubiquitin-mediated degradation in colon cancer cells [68]. LncRNA binding to proteins can also decoy the protein function such as LINC01133 that titrates the splicing factor SRSF6 away from its targets, thereby preventing epithelial to mesenchymal transition in CRC cells [93]. Other lncRNAs also bind proteins of the splicing machinery (reviewed by Reference [94]), for example, lncRNA FAS-AS1 (SAF) interacts with SP45 resulting in alternatively spliced and anti-apoptotic FAS in cancer cells [95] and MALAT1 was shown to interact with serine/arginine (SR) splicing factors in nuclear speckle bodies [96]. Furthermore, interaction with signaling proteins may alter the activation of signaling pathways. For example, the binding of lncRNA NKILA to the NF-κB/IκB complex in breast cancer cells prevents IκB phosphorylation, thereby preventing NF-κB activation [97]. The cytoplasmic lncRNA LINK-A binding activates a protein tyrosine kinase complex (BRK/LRRK2), which results in the increased HIF1α signaling in breast cancer cells [98].
Although defined as long "non-coding" RNA, recent findings suggest that the presence of ribosomes on lncRNAs may indicate that the short open reading frames are a source of small peptide synthesis [99,100]. The detection of microproteins is challenging, but some evidence was reported; the lncRNA LINC00961 encodes a polypeptide (SPAR) that negatively regulates mTORC1 activation [101]; the LINC01420-derived microprotein was identified as a novel component of the mRNA decapping complex regulating mRNA decay [102] and a HOXB-AS3-derived peptide was shown to regulate pyruvate kinase M splicing and to affect the metabolic reprogramming of CRC cells [103]. The number of lncRNAs that are actually a source of small regulatory peptides remains to be investigated. Alternatively, the interaction of lncRNAs with ribosome complexes may trigger lncRNA degradation [104].
Accumulating evidence shows that lncRNAs are involved in the initiation, progression, and metastasis of cancer [105,106]. We have discussed how lncRNAs may regulate cancer-associated expression profiles on diverse levels. We have briefly cited several well-known lncRNAs that are implicated in CRC (Table 1), such as GAS5, H19, HOTAIR, CCAT1-L, CRNDE, and MALAT1. It has been shown that the gene desert of Chr8q24, which is a CRC risk locus, harbors 7 lncRNAs including CCAT1 (CARLo-5), and CASC19 (CARLo-6). In addition, numerous colorectal cancer associated lncRNAs, such as CCAT6 (MYCLo-2), CASC8 (CARLo-1), CASC21 (CARLo-2), PRNCR1 (CARLo-3), PCAT2 (CARLo-4), and CASC11 (CARLo-7), have been identified [107]. Recently, the analysis of lncRNA expression in different CRC molecular phenotypes highlighted the decreased expression of UCA1 in tumors with Mismatch Repair (MMR) defaults compared to tumors without such defaults [19]. The following sections focus on the role of UCA1 to integrate research on different cancer cells in order to decipher its putative functions and mechanisms of regulation in CRC cells.

Association of UCA1 Transcript Expression with Colorectal Cancer
Urothelial Cancer Associated 1 (UCA1) was first discovered in bladder cancer [108] and its long transcript is also the nominated Cancer Upregulated Drug-Resistant transcript (CUDR) [109]. Actually, three UCA1 transcript isoforms of 1.4 kb, 2.2 kb, and 2.7 kb have been described, but, in general, the most abundant 1.4 kb isoform is studied [110]. Analysis of UCA1 expression and patient survival data from the TCGA dataset shows that its expression was correlated with increased hazard ratio in different types of cancers, in particular with pancreatic adenocarcinoma ( Table 2, [111][112][113]). Although several patient studies reported that a high expression of UCA1 is correlated with bad disease prognosis in CRC (the number of patient of these studies N = 80 [114], N = 54 [115], N = 90 [116] and the N = 530 for the Asian meta-analysis [117]), no such evidence was observed in the TCGA COAD-READ study ( Table 2). Since separated analyses of colon (COAD) and rectal (READ) adenocarcinoma patients showed different Kaplan Meier survival curves, this discrepancy may originate from mixed colon and rectal adenocarcinoma patients in several studies (e.g., known mixed COAD:READ patient groups in [114,116]). Recently, a genome-wide analysis of lncRNA expression in different CRC phenotypes was realized, highlighting the decreased expression of UCA1 in tumors with Mismatch Repair (MMR) defaults compared to tumors without such defaults [19]. These differences are in line with the notion that different primary tumor locations (COAD vs. READ) and, therefore, carcinogenesis pathways define the molecular characteristics and epigenetic signature of the tumor [118].

Regulation of UCA1 Transcript Expression
The UCA1 gene encodes 3 exons located on chromosome 19 and it is highly expressed in cancer cells. Indeed, its transcription is up-regulated by diverse oncogenic pathways. The Ras-responsive transcription factor Ets-2 was shown to regulate UCA1 transcription in both bladder and colorectal cells [115,120], UCA1 is upregulated by the major inducer of epithelial-mesenchymal transition (EMT) TGFβ in gastric and breast cancer cells [121,122] and by mediators of chemoresistance like Hippo (TAZ/YAP/TEAD) signaling in bladder and breast cancer cells [123,124]. BMP9 has an ambiguous role in tumor progression, but it was recently shown that BMP9 stimulated UCA1 expression in bladder cancer cells [124]. Interestingly, in these cells, UCA1 expression was also stimulated during hypoxia via Hypoxia-Inducible Factor-1α (HIF1α) and the secretion of UCA1-enriched exosomes was increased under those conditions [125,126].
Several chromatin remodeling factors inhibit UCA1 transcription. Although the transcription factor CCAAT/enhancer binding protein α (C/EBPα) upregulated the UCA1 expression [127], this activation was inhibited by the tumor repressor and part of an SWI/SNF chromatin remodeling complex, ARID1A [128]. Epigenetic inhibition of UCA1 in breast cancer cells was mediated by the Special AT-rich sequence Binding-protein 1 (SATB1) [129]. The Coactivator of AP1 and Estrogen Receptor (CAPERα)/ T-box3 (TBX3) repressor complex that mediates an arrest of cell growth also downregulated UCA1 in embryonic kidney cells [130].
Levels of UCA1 transcripts are also regulated post-transcriptionally; the RNA stability of UCA1 was downregulated by the interaction with insulin-like growth factor 2 messenger RNA binding protein (IMP1) [131] and by the interaction with miR-1 [132], whereas binding of UCA1 to heterogeneous nuclear ribonucleoprotein I (hnRNPI) increased its stability [133]. It remains to be explored if the described regulation of transcript levels in diverse cancer cells also regulates UCA1 in colorectal cells.

UCA1-Regulated Transcription
In common with a lot of other lncRNAs, UCA1 can regulate the transcription of genes via epigenetic modifications (Table 3). Recent studies showed that UCA1 can physically associate with EZH2 and suppress transcription via histone methylation (H3K27me3) on the promoter of cell cycle genes p21cip and p27Kip1 [45-47] and stimulate cyclin D1 expression [46]. The UCA1 interaction with SET1A in liver cancer cells enhanced the histone methylation (H3K4me3) loading onto the telomeric repeat binding factor 2 (TRF2) promoter region, increasing TRF2 expression and telomere length [134]. The binding of UCA1 to transcription regulating complexes can also function as a decoy. In gallbladder cancer cells, UCA1 interacted with Brahma related gene 1 (BRG1) of the chromatin SWI/SNF remodeling complex and prevented its binding to the p21 promoter locus [57]. Binding of UCA1 to heterogeneous nuclear ribonucleoprotein I (hnRNPI) in breast cancer cells resulted in the decreased stimulation of the p27 promoter by hnRNPI [133]. Another mechanism of transcriptional regulation by UCA1 occurs in hepatocytes where the UCA1/CUDR-induced chromatin loop recruits the transcription insulator CTCF and β-catenin enhancer resulting in the upregulation of β-catenin transcription [78]. Zhang et al. performed RNA immuno-precipitation assays showing a direct interaction of UCA1 with the mediators MOB1, Lats1, and YAP of the Hippo pathway, and demonstrated a major role of UCA1 for nuclear translocation of YAP and pancreatic cancer cell migration and invasion [113]. This pathway is also important in 5FU-chemoresistance CRC [135].

UCA1 and miRNA-Mediated Decay
With the exploring of the miRNA pathways, it has become clear that lncRNAs play an important role to fine-tune miRNA-mediated decay. In the last lustrum, over 40 articles have described the indirect regulation by UCA1 through sequestering of miRNAs and interfering with the degradation of downstream gene transcripts. At least 29 miRNAs were shown to interact with UCA1 (Table 4) and overall 32 different genes were reported with an altered expression mediated by the UCA1/miRNA interaction. In addition, our analysis of putative miRNA binding with miRCore [136], showed that another 9 miRNAs not previously described, may bind to the UCA1 transcript (highlighted in Table 4). These miRNAs also play a role in CRC (Table 4; references in "CRC" column). Focusing on CRC cells, the interaction of UCA1 with miR-143, first reported in bladder cancer cells [137], was confirmed and implicated UCA1 as an upstream effector of mTOR activation and as a regulator of K-Ras expression ( [138], Figure 2). Furthermore, UCA1 could sponge endogenous miR-204-5p and inhibit the degradation of its targets CREB1, BCL-2 and RAB22A indicating UCA1 promotes proliferation, inhibits apoptosis and plays a role in the acquired chemoresistance of these CRC cells [116]. Overall, gene clustering analysis of the 29 UCA1-related miRNA targets with mirPath v.3 [139] showed a significant implication in cancer signaling pathways such as TGFβ, mTOR and WNT signaling. We confirmed the UCA1/miRNA-regulated genes in these pathways with the miRNA network analysis tool ONCO.IO ( Figure 2). These analyses indicate that UCA1 regulates genes that play a central role in CRC.

Role of UCA1-Mediated Regulation in Colorectal Cancer
Reciprocal to the regulation of UCA1 transcript expression by oncogenic pathways, UCA1 may regulate oncogenic pathways. UCA1 expression has been shown to stimulate factors of the WNT signaling pathway in diverse cancer cell types [78,[327][328][329][330]. In CRC, WNT signaling is correlated to 5-FU chemoresistance [331] and UCA1 is induced by 5-FU treatment [332], but no direct correlation is described for UCA1 and WNT signaling in these cells. This also holds true for UCA1 regulating mediators of AKT signaling and its downstream targets in diverse cancer cells [46,108,120,333,334]. In CRC cells UCA1 was implicated in the induction of KRAS expression through the regulation of miR143 [138]. Although no direct evidence of UCA1 regulation for the TGFβ pathway in colorectal cells was shown, UCA1 acts as a competitor RNA for several miRNAs that affect this pathway. Recently Li et al. showed that the interaction of UCA1 with miR-1 and miR203a stabilized the expression of SNAI2, mediating the effects of TGFβ signaling in breast cancer cells [122]. In addition, UCA1 was shown to stimulate the ERK-MMP9 signaling in gastric cancer cells by interacting with G protein-coupled receptor kinase 2 (GRK2), stimulating its ubiquitination and degradation [335].

UCA1-Mediated Regulation of the Cell Cycle
UCA1 stimulates cell proliferation, and silencing its expression in cancer cells has been shown to arrest the cell cycle in the G0/G1 phase (in CRC [114,115] and other cancer cells [46,47,254,[336][337][338]).
Several key players in cell cycle progression are regulated by UCA1 ( Figure 3A). Cell cycle progression from the G1 to S phase relies on the activation of E2F transcriptional regulation. Activation is mediated by dissociation of E2F from the Rb-complex after the phosphorylation of Rb by CDK-cyclin. From this phosphorylation complex, UCA1 increases cyclin D1 expression [46,337] and maintains CDK phosphorylation activity through the repression of its inhibitors p21CIP and p27Kip [45,47,57,133,138,211]. Moreover, during the G1-phase, the cyclin D1 expression and its binding to the CDK inhibitors increased, resulting in less binding of these inhibitors to cyclin E/Cdk2 complexes and acceleration of the cell cycle progression. This mechanism is further stimulated by the fact that UCA1 can upregulate cMYC, either by binding to cyclin D1 [339] or by sequestering miR-135 [177], respectively, in the liver and in thyroid cancer cells. This evidence was obtained in different types of cancer cells, but overall these studies show that UCA1 interferes at different levels with cell cycle regulation.

Role of UCA1-Mediated Regulation in Colorectal Cancer
Reciprocal to the regulation of UCA1 transcript expression by oncogenic pathways, UCA1 may regulate oncogenic pathways. UCA1 expression has been shown to stimulate factors of the WNT signaling pathway in diverse cancer cell types [78,[327][328][329][330]. In CRC, WNT signaling is correlated to 5-FU chemoresistance [331] and UCA1 is induced by 5-FU treatment [332], but no direct correlation is described for UCA1 and WNT signaling in these cells. This also holds true for UCA1 regulating mediators of AKT signaling and its downstream targets in diverse cancer cells [46,108,120,333,334]. In CRC cells UCA1 was implicated in the induction of KRAS expression through the regulation of miR143 [138]. Although no direct evidence of UCA1 regulation for the TGFβ pathway in colorectal cells was shown, UCA1 acts as a competitor RNA for several miRNAs that affect this pathway. Recently Li et al. showed that the interaction of UCA1 with miR-1 and miR203a stabilized the expression of SNAI2, mediating the effects of TGFβ signaling in breast cancer cells [122]. In addition, UCA1 was shown to stimulate the ERK-MMP9 signaling in gastric cancer cells by interacting with G protein-coupled receptor kinase 2 (GRK2), stimulating its ubiquitination and degradation [335].

UCA1-Mediated Regulation of the Cell Cycle
UCA1 stimulates cell proliferation, and silencing its expression in cancer cells has been shown to arrest the cell cycle in the G0/G1 phase (in CRC [114,115] and other cancer cells [46,47,254,[336][337][338]). Several key players in cell cycle progression are regulated by UCA1 ( Figure  3A). Cell cycle progression from the G1 to S phase relies on the activation of E2F transcriptional regulation. Activation is mediated by dissociation of E2F from the Rb-complex after the phosphorylation of Rb by CDK-cyclin. From this phosphorylation complex, UCA1 increases cyclin D1 expression [46,337] and maintains CDK phosphorylation activity through the repression of its inhibitors p21CIP and p27Kip [45,47,57,133,138,211]. Moreover, during the G1-phase, the cyclin D1 expression and its binding to the CDK inhibitors increased, resulting in less binding of these inhibitors to cyclin E/Cdk2 complexes and acceleration of the cell cycle progression. This mechanism is further stimulated by the fact that UCA1 can upregulate cMYC, either by binding to cyclin D1 [339] or by sequestering miR-135 [177], respectively, in the liver and in thyroid cancer cells. This evidence was obtained in different types of cancer cells, but overall these studies show that UCA1 interferes at different levels with cell cycle regulation.

UCA1 in Colorectal Cancer Diagnosis and Therapy
Initially, UCA1 was proposed as a predictive biomarker for the prognosis and survival of CRC patients [114][115][116][117]. Since UCA1 expression in CRC may depend on primary tumor localization and molecular subtypes, its prognostic value may be restrictive to different CRC subclasses. Interestingly, a recent evaluation of tumor-derived exosomes in cancer diagnosis showed that UCA1 is not only expressed in gallbladder cancer exosomes [125], but also in exosomes isolated from the serum of CRC patients [352]. Whether the implication of UCA1 in several oncogenic pathways makes it a good target for therapy remains to be investigated. Invalidating an lncRNA, like UCA1 in CRC, may have the advantage of both inhibiting epigenetic silencing through chromatin remodeling for several tumor suppressor genes and stimulating the miRNA-mediated mRNA degradation of oncogenes due to a decreased ceRNA level. In that aspect, a recent innovation was the use of an artificial lncRNA targeting multiple miRNAs in hepatocellular carcinoma cells [353].

Conclusions
The lncRNA UCA1 has, like other lncRNAs, diverse functions and can affect both epigenetic and transcriptional gene regulation, as well as posttranscriptional regulation by acting as a ceRNA for diverse miRNAs. UCA1 has been studied in a wide range of cancer cells, including colorectal cancer. Extrapolating the role of UCA1 in different cancer cells to CRC cells suggests a role for UCA1 in cell cycle progression and cell proliferation, which is highly relevant to tumor growth. In addition, UCA1 plays a role in CRC chemoresistance, although the implicated mechanisms remain to be studied. It will also be worthwhile to identify more UCA1/EZH2-silenced target genes, to asses whether UCA1 is a driver of carcinogenesis by silencing key tumor repressor genes. UCA1 is probably not a strong general prognostic marker for CRC as its overexpression is dependent on the primary tumor site (colon vs. rectal) and molecular characteristics, such as the microsatellite stability profile. Nevertheless, studying the UCA1-regulated genes and miRNA decoy function in CRC cells may reveal novel pathways and potential new therapeutic targets for managing CRC. Funding: This research was funded by "Institut National de la Santé et de la Recherche Médicale" (Inserm), "Centre National de la Recherche Scientifique" (CNRS) and "la Ligue Nationale contre le Cancer" (Committees 59 (R17037EE -RAB17005EEA), 62 and 80).