Roles and Regulation of Long Noncoding Rnas in Hepatocellular Carcinoma Lee Jin Lim1, Samuel Y.S.Wong2, Feiyang Huang3, Sheng Lim1,2,4, Samuel S

Published OnlineFirst July 23, 2019; DOI: 10.1158/0008-5472.CAN-19-0255 Cancer Review Research

Roles and Regulation of Long Noncoding RNAs in Hepatocellular Carcinoma Lee Jin Lim1, Samuel Y.S.Wong2, Feiyang Huang3, Sheng Lim1,2,4, Samuel S. Chong5, London Lucien Ooi6,7,OiLianKon2, and Caroline G. Lee1,2,6,8

Abstract

Next-generation sequencing has uncovered thousands of ulated gene/protein expression. Notably, 63 deregulated long noncoding RNAs (lncRNA).Manyarereportedtobe lncRNAs are signiﬁcantly associated with clinicopathologic aberrantly expressed in various cancers, including hepato- features of HCC. Twenty-three deregulated lncRNAs associ- cellular carcinoma (HCC), and play key roles in tumori- ated with both tumor and metastatic clinical features were genesis. This review provides an in-depth discussion of the also tumorigenic and prometastatic in experimental models oncogenic mechanisms reported to be associated with of HCC, and eight of these mapped to known cancer path- deregulated HCC-associated lncRNAs. Transcriptional ways. Fifty-two upregulated lncRNAs exhibit oncogenic expression of lncRNAs in HCC is modulated through tran- properties and are associated with prominent hallmarks of scription factors, or epigenetically by aberrant histone acet- cancer, whereas 22 downregulated lncRNAs have tumor- ylation or DNA methylation, and posttranscriptionally by suppressive properties. Aberrantly expressed lncRNAs in lncRNA transcript stability modulated by miRNAs and RNA- HCC exert pleiotropic effects on miRNAs, mRNAs, and binding proteins. Seventy-four deregulated lncRNAs have proteins. They affect multiple cancer phenotypes by altering been identiﬁed in HCC, of which, 52 are upregulated. This miRNA and mRNA expression and stability, as well as review maps the oncogenic roles of these deregulated through effects on protein expression, degradation, struc- lncRNAs by integrating diverse datasets including clinico- ture, or interactions with transcriptional regulators. Hence, pathologic features, affected cancer phenotypes, associated these insights reveal novel lncRNAs as potential biomarkers miRNA and/or protein-interacting partners as well as mod- and may enable the design of precision therapy for HCC.

Introduction stage HCC (2). Potentially curative options like surgical resection and liver transplantation are only indicated for early stage disease As the sixth most common and fourth most fatal cancer in the and are possible in only 10% to 20% of patients who present with world (1), hepatocellular carcinoma (HCC) is a major global HCC, with expected 5-year overall survival rates of 40% to health problem. The high mortality is due to the late stage of 70% (3). However, the risk of recurrences within 5 years is high, presentation in the majority of cases and the limited treatment both for patients who undergo resection (70%) and also liver options in that situation where curative surgery is no longer transplantation (10%–60%) due to the underlying disease caus- possible. This late presentation is due in a large part to the absence ing HCC formation in the first instance (3–5). The majority of symptoms in the early stages of disease, and the lack of (80%) of patients present with advanced HCC, which are not diagnostic biomarkers and other methods for detecting early amenable to surgery, and for which other treatment options are essentially palliative. These treatment options can be locoregional or systemic methods. Locoregional therapies include ablation 1 Department of Biochemistry, Yong Loo Lin School of Medicine, National (radiofrequency, microwave, cryotherapy) or catheter-based University of Singapore, Singapore, Singapore. 2Division of Cellular and Molec- ular Research, Humphrey Oei Institute of Cancer Research, National Cancer transhepatic arterial embolization with chemotherapeutic agents Centre Singapore, Singapore, Singapore. 3NUS High School of Math and Science, (transarterial chemoembolization) or radionuclear agents like Singapore, Singapore. 4Raffles Institution, Singapore, Singapore. 5Department Yttrium 90 (selective internal radiation therapy; ref. 3). For of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, systemic treatment, there are chemotherapy and targeted therapy 6 Singapore, Singapore. Duke-NUS Graduate Medical School, Singapore, options, although only sorafenib (6–9) and regorafenib (10, 11), 7 Singapore. Department of Hepato-Pancreato-Biliary and Transplant Surgery, both multityrosine kinase inhibitors, are clinically approved for Singapore General Hospital, Singapore, Singapore. 8NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singa- treating locally advanced or metastatic HCC, which extend sur- pore, Singapore. vival by only a few months (3). Given the dismal landscape of therapeutic options if diagnosis is made late, there is, therefore, an Note: Supplementary data for this article are available at Cancer Research fi Online (http://cancerres.aacrjournals.org/). urgent need for a ne-grained molecular landscape of HCC from which to discover clinically relevant early diagnostic and prog- Corresponding Author: Caroline G. Lee, National University of Singapore, 8 nostic biomarkers, and to develop curative precision therapies. Medical Drive, Singapore 117596, Singapore. Phone: 011-65-6516-3251; Fax: 011-65-6436-8353; E-mail: [email protected] Risk factors for HCC include viral infections with hepatitis B (HBV) or C virus (HCV), alcoholism, immune-related liver dis- Cancer Res 2019;XX:XX–XX eases, nonalcoholic fatty liver disease, obesity, and aflatoxin doi: 10.1158/0008-5472.CAN-19-0255 exposure, and these factors vary with gender, geographic region, 2019 American Association for Cancer Research. and ethnicity (12). HBV and HCV infections are the most strongly

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associated risk factors; epidemiologic studies have shown that mRNAs, the functions of lncRNAs are less understood as they 50% to 55% of HCC cases globally are associated with HBV (2, 13) cannot be inferred due to low sequence conservation (34). None- and 25% to 30% with HCV infections (13). In highly endemic theless, increasing evidence implicates lncRNAs in every aspect in regions like Southeast Asia, HBV infection can account for approx- the life cycle of a gene, including transcription, splicing, RNA imately 80% of HCC (13, 14). The link between HBV infection decay, and translation (35). and HCC is one of the closest relationships identified between an Expression of lncRNAs is deregulated in various pathologic environmental agent and a cancer, as exemplified by the 20-fold conditions including HCC (22) and other cancers (27, 36). to 100-fold higher risk for developing HCC among chronic HBV In recent years, several lncRNAs were reported to be signifi- carriers (14–17). cantly deregulated in HCC (see 22, 37–42). HBV (43–45), The progression from normal liver tissue to HCC (18) is a the etiological agent most commonly associated with HCC, multistep process involving repeated cycles of hepatocyte and its key gene, HBx (37, 46, 47), deregulate the expression damage, followed by regeneration leading to chronic liver disease. of cellular lncRNAs and modulate the hallmarks of can- In chronic liver disease, regenerating hepatocytes are present in cer (48, 49). Significantly, the HBx-LINE1 fusion transcript is cytologically normal hyperplastic nodules, signaling an initial an lncRNA that activates Wnt signaling, promotes HCC devel- first step toward HCC formation. Progression to premalignant opment and progression, and correlates with shorter patient dysplastic nodules is visualized by abnormal liver architecture survival (50). These observations point to the considerable with thickened trabeculae and abnormal cytological features such potential of lncRNAs as a source of novel targetable molecules as clear cell changes and nuclear crowding. Dysplastic nodules for HCC precision therapy and for discovering new diagnostic may then evolve into overt HCC having invasive and metastatic biomarkers. capabilities. Accumulation of numerous aberrations and dysre- Recent reviews of lncRNAs in HCC (37–42) have focused gulations of the genome and epigenome affecting both mRNAs mainly on the functions and regulatory mechanisms of indi- and noncoding RNAs (ncRNA), including long noncoding RNAs vidual deregulated lncRNAs in isolation from other deregu- (lncRNA), are key events in this multistep progression and con- lated lncRNAs, and were necessarily fragmentary snapshots tribute to both tumorigenicity and the invasive behavior of of the field (37–42). The primary focus of this review is on HCC (19). 74 deregulated lncRNAs in HCC and their mechanisms of Advances in molecular and cell biology, detailed characteriza- action in hepatocarcinogenesis. By integrating diverse datasets, tions of genomes, epigenomes, proteomes, and metabolomes in including clinicopathologic associations, cancer phenotypes, HCC have added new insights into hepatic carcinogenesis (see miRNA- and protein-interacting partners, altered transcrip- reviews; refs. 20, 21), but with scant clinical benefits as much more tomes, and protein expression, this review attempts to provide remains unknown. The advent of state-of-the-art next-generation a perspective of 74 lncRNAs known to be deregulated in HCC, sequencing (NGS) of the human transcriptome has uncovered a with an emphasis on integrating their oncogenic functions vast number of transcripts, of which, only approximately 2% are into a coherent model. Signaling and other functional path- protein-coding, whereas the majority is actively transcribed novel ways of mRNAs and proteins associated with deregulated ncRNAs (22). Many ncRNAs are differentially expressed in various lncRNAs are inferred to advance understanding of the role of disease states (23). Once regarded as "junk RNA," there is increas- lncRNAs in hepatocellular carcinogenesis and influencing clin- ing evidence that many ncRNAs are functionally important ical outcomes of HCC patients. and actively participate in various physiologic and pathologic processes (24, 25). Deregulation of LncRNAs in HCC To date, a total of 74 lncRNAs have been reported to be LncRNAs: A Distinct Subclass of ncRNAs deregulated in HCC (Supplementary Table S1). This section Among ncRNAs are lncRNAs, which are defined as RNAs reviews the growing body of evidence regarding transcriptional longer than 200 nucleotides (26) and undergo similar proces- and posttranscriptional mechanisms that deregulate lncRNAs in sing as mRNAs such as splicing, capping, polyadenylation, and HCC (Fig. 1). editing (22). Unlike mRNAs, lncRNAs lack significant open reading frames (<100 aa; ref. 27). Thus far, >58,000 lncRNAs Aberrant transcriptional regulation of lncRNAs have been identified (28), and approximately 30,000 have been Epigenetic mechanisms are known to regulate lncRNA tran- curated in GENCODE v29. Like protein-coding genes, most scription. Three independent studies reported abnormal his- lncRNAs are transcribed by RNA polymerase II, capped at the 50 tone acetylation of the promoters of H19, lncRNA-p21, and end, and polyadenylated at the 30end (29). Despite being more lncRNA-LET in HCC (Fig. 1A,i; refs. 51–53). LncRNA-LET diverse than mRNAs in human cells, lncRNAs are often expression was repressed during hypoxia through reduced expressed at lower levels (30). Nonetheless, their expression H3/H4 acetylation effected by HIF1a-induced HDAC3 activa- is more tissue-specific than mRNAs, suggesting more tightly tion (53). Downregulated lncRNA-LET in turn enhanced regulated transcription (31, 32). HIF1a expression, generating a positive feed-forward loop LncRNAs are classified with reference to their relation to pro- driving robust HIF1a expression in hypoxia (53, 54). Besides tein-coding genes, namely their relative genomic locations, dis- aberrant acetylation, epigenetic regulation through miRNAs tance from protein-coding genes, direction of lncRNA transcrip- and methylation also occurs. MEG3 is a tumor-suppressor tion, and their origin from introns or exons. Some lncRNAs are lncRNA whose expression is profoundly decreased in a major- transcribed from intergenic regions of the genome (Supplemen- ity of HCCs by methylation of its promoter (55). MEG3 tary Fig. S1; ref. 33). Unlike the well-studied miRNAs, which have expression in normal hepatocytes and HCC is correlated with defined regulatory functions based on predicted interactions with expression of miR-29, which directly blocks DNMT mRNA.

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A. Transcriptional level HBx – Epigenetic and transcription factors H3 : Linc-p21 miR-29 HBx: DBH-AS1S1 Ac H4 : H19 Sp faSS family: HEIH, MALAT1, HULC i) H3 & H4 : lncRNA-LET ii) iii) CREB + HBx : HULC MEG3 P CREB: HULC Me Me Me Ac DNMT CREB

B. Post-transcriptional level HULC – miRNA and RNA binding proteins

HULC HOTTIP HULC IGF2BP HULC ii) i) v) iv) RXRA HOTTIP miR-372 HULC AGO Stimulate IGF2BP CCR4-NOT activity ACSL1 miR-192/204/125b/29a PRKACB Cholesterol

LncRNA Expression in T vs NT Protein Histone miRNA Methylation Acetylation Phosphorylation Viral protein Indirect Interaction MEG3 effect Downregulated Me Ac P HULC Upregulated

Figure 1. Summary of regulation of lncRNAs at transcriptional level (A) and posttranscriptional level (B). A, i, Epigenetic modiﬁcations such as acetylation of histone proteins (H3 or/and H4) could lead to aberrant expression of lncRNAs (Linc-p21, H19, and LET). ii, miRNA can regulate lncRNAs indirectly through regulating transcription. For example, miR-29 downregulates DNMT expression and leads to MEG3 promoter hypomethylation, thereby increasing MEG3 expression. iii, An lncRNA can be transcribed by different transcription factors such as CREB, Sp4, Sp3, and Sp1. HBx alone or interacting with CREB activates lncRNA expression. iv, Positive feedback loop is shown in this example, HULC-miR372-PRKACB-Creb. HULC acts as miRNA sponge to sequester miR-372, which in turn increases the expression of PRKACB and phosphorylation of CREB, resulting in increased HULC expression. v, Another positive feedback loop of HULC is shown in this example. HULC upregulates ACSL1 and a by-product of ACSL1, cholesterol, stimulates activity of RXRA that acts on HULC promoter and enhances its transcription. B, i, RNA-binding protein such as IGF2BP also recruits lncRNA HULC to CCR4-NOT deadenylase complex and accelerates HULC degradation. ii, miRNA can directly bind lncRNA and leads to its degradation. miR-192/204/125b/29a were reported to degrade HOTTIP and decreased HOTTIP expression.

Decreased miR-29, levels lead to aberrant hypermethylation of HULC acts as a sponge for miR-372 causing PRKACB dere- the MEG3 promoter through high expression of DNMT (Fig. 1A, pression (Fig. 1A,iv; ref. 60). PRKACB phosphorylates CREB, ii; ref. 55). which activates HULC expression, leading to a positive feed- LncRNAs share transcription factors with protein-coding genes. forward cycle, which increases HULC expression. Interestingly, The Sp family of transcription factors is one of many that is another feedback loop regulating HULC expression in human overexpressed in various cancers (56). Sp transcription factors hepatoma cell lines involves cholesterol (Fig. 1A,v; ref. 61). mediate the upregulation of several lncRNAs in HCC, e.g., HULC expression is positively correlated with ACSL1 expres- lncRNA-HEIH, MALAT1, and HULC (Fig. 1A,iii; refs. 57–59). sion in HCC. HULC activates the ACSL1 promoter through Conversely, a single lncRNA may be regulated by multiple upregulating its transcription factor, PPARA, at both transcript transcription regulators. For example, HULC expression is and protein levels. Cholesterol, a byproduct of ACSL1, also regulated not only by Sp1, Sp3, and Sp4, but also by phos- upregulates HULC through activating RXRA, a transcription phorylated CREB, which helps to maintain an open chromatin factor at the HULC promoter, forming a second positive structure for active transcription (Fig. 1A,iii; ref. 60). In studies feedback loop. of HBV-associated HCC, the HBV protein, HBx, deregulates lncRNAs expression by itself (41) or by interacting with tran- Aberrant posttranscriptional regulation of LncRNAs scription factors, e.g., CREB, leading to activation of the HULC HULC is also posttranscriptionally regulated (Fig. 1B,i). promoter(Fig.1A,iii;ref.56). IGF2BP1 serves as an adaptor protein that speciﬁcally binds and Remarkably, two independent studies demonstrated that recruits HULC to the CCR4-NOT deadenylase complex, leading to HULC regulation in liver cancer involves two feedback loops. degradation of HULC (62).

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Like mRNAs, lncRNAs are also regulated by multiple miRNAs. Fifty-two upregulated lncRNAs in HCC mediate enhanced cell HOTTIP lncRNA is upregulated in HCC, and its expression is proliferation, cell-cycle progression, anchorage-independent negatively correlated with miR-125b, miR-192, and miR-204 growth, cell migration or invasion (Supplementary Table S3A, expression (Fig. 1B,ii; refs. 63, 64). miR-192 and miR-204 sup- pink/red), reduced apoptosis (Supplementary Table S3A, green), press HOTTIP expression through the Argonaute 2–mediated as well as increased tumor size (Supplementary Table S3A, RNAi pathway. The miR-192/-204–HOTTIP axis is postulated orange) in mouse models, whereas 22 downregulated lncRNAs to interfere with glutaminolysis in HCC through GLS1 inhibit various cancer phenotypes (Supplementary Table S3A). inhibition (64). Notably, a single lncRNA can modulate multiple hallmarks of In summary, the foregoing examples reveal highly complex cancer, as exemplified by UCA1, ANRIL, MALAT-1, CASC15, FTX, regulatory mechanisms in HCC that affect the expression of and PTENP1 lncRNAs, which are involved in seven different even a single lncRNA (e.g., HULC). These consist of intricate cancer phenotypes (Supplementary Table S3A, #1, 2, 4, 41, 53, and interlocking feedback loops operating at both transcrip- and 54). tional and posttranscriptional levels involving numerous As illustrated in the Venn diagram (Supplementary Fig. S2), molecular classes, including transcription factors, miRNAs, and most HCC-deregulated lncRNAs have oncogenic or tumor- RNA-binding proteins. Disruption of any one of these complex suppressive properties by positively or negatively modulating regulatory systems can deregulate lncRNA expression with cancer pathways (Supplementary Table S3B) leading to altered downstream effects that alter the expression of multiple cellular phenotypes (Supplementary Table S3A) through inter- mRNAs and proteins associated with that specificlncRNAand acting with miRNAs (Supplementary Table S3C) or proteins their cognate functional pathways. Such cascading events ulti- (Supplementary Table S3D). The ultimate result is deregulating mately manifest in HCC and other cancers. This may explain the expression of 104 mRNAs (Supplementary Table S3E), of why abnormally high expression of oncogenic lncRNAs like which, 43 map to 27 different cancer-related pathways. Only HULC, which is most highly expressed in HCC, is also highly seven of these pathways have been reported previously (Supple- deregulated in many other cancers (65). mentary Table S3B and S3E), raising intriguing opportunities to investigate the other 20 predicted pathways. Deregulation of HCC-associated lncRNAs in other cancers To survey deregulation of the 74 HCC-associated lncRNAs in fi other cancers, we compared their expression in HCC with 37 Clinical Signi cance of LncRNAs in HCC non-HCC cancers and cancers grouped by anatomical site of Several lncRNAs have been associated with clinicopathologic origin (10 cancer groups; Supplementary Table S2). Fifty-three characteristics and prognosis in various cancers (68–73), of the 74 deregulated lncRNAs in HCC are also differentially highlighting the potential of lncRNAs as prognostic biomarkers. expressed in tumor versus nontumor tissues in other cancers Sixty-three of the 74 HCC-deregulated lncRNAs have been (Supplementary Table S2). Strikingly, 35 of these lncRNAs, reported in different studies to be significantly associated comprising 27 oncogenic and 8 tumor-suppressive lncRNAs, with clinicopathologic features of HCC, such as primary tumor are consistently deregulated across all 37 cancers surveyed. characteristics (size, focality, differentiation, and encapsula- Similar trends of lncRNA expression were observed within tion), invasion and metastasis, stage of disease and survival, cancer groups except for gastrointestinal cancers, thoracic, as well as nontumor characteristics (e.g., cirrhosis), serum urological cancers, and gynecologic cancers. SPRY4-IT1 is the alpha-fetoprotein (AFP), and HBV infection status (Supplemen- only lncRNA that exhibits inconsistent differential expression tary Table S4A). Notably, a majority of lncRNAs were associated among different cancers within the same cancer group, suggest- with overall survival (47/74), cancer stage (43/74), and tumor ing possible involvement of distinct oncogenic pathways even size (32/74), strengthening the potential of lncRNAs as prog- in different cancers that develop in the same anatomical tissue nostic biomarkers because tumor size, cancer stage, and patient (Supplementary Table S2_boxed). Seventeen lncRNAs showed survival are key indicators of prognosis. Of equal interest is the consistent differential expression in nearly all cancers with one observation that several lncRNAs are significantly associated or two exceptions, likely due to differences in lncRNA regula- with multiple clinicopathologic features, ranging from serum tion (Supplementary Table S2_purple words). For example, AFP, HBV status, cirrhosis, tumor invasiveness, metastatic dis- p53 is involved in downregulating TUG1 in non–small cell ease, HCC stage, and survival (Supplementary Table S4A). lung cancer, whereas Sp1 upregulates TUG1 in HCC (66, 67). For example, six upregulated lncRNAs (PANDAR, MALAT-1, Hence, different transcription factors in diverse cancer types GPC3-AS1, CARLo-5, UCA1, and LOC90784) are significantly may modulate lncRNA expression differently such that the associated with six to eight different clinicopathologic features, same lncRNA may be upregulated in one cancer type but suggesting that these six lncRNAs could potentially form a downregulated in another. signature of unfavorable prognosis. Similarly, three downregulated lncRNAs (FTX, AOC4P, and linc-p21) are significantly LncRNAs deregulated in HCC affect cancer phenotypes associated with six different clinicopathologic characteristics through modulating miRNAs and mRNAs in cancer (Supplementary Table S4A) and hence could be a potential pathways signature of favorable prognosis. Given that lncRNAs are important regulators of cellular gene Deeper understanding of these key and other differentially expression, their aberrant expression in HCC may reasonably expressed lncRNAs listed in Supplementary Table S4A will be expected to play key roles in hepatocarcinogenesis and facilitate validation of novel, clinically relevant HCC prognos- metastasis. Functional assays and animal models show that tic biomarkers and provide a scientifically sound basis for the 74 lncRNAs deregulated in HCC modulate various hall- developing superior therapeutic strategies to improve patient marks of cancer (Supplementary Tables S1 and S3A; ref. 48). survival. To this end, we have integrated reports of clinical

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characteristics (Supplementary Table S4A) with reports of found within paraspeckles, they are thought to modulate gene phenotypes (Supplementary Table S3A) and pathways (Sup- expression within a confined space by sequestering RNA or plementary Table S3B) to identify lncRNAs whose clinical protein molecules. Paraspeckles are also thought to provide an characteristics are congruent with experimentally demonstrat- efficient platform for miRNA-protein binding, thereby enhanced phenotypes and pathways (Supplementary Table S4B; Sup- ing pri-miRNA processing as well as controlling gene expres- plementary Fig. S3). Notably, 16 upregulated and 7 down- sion by directly anchoring transcription factors to transcription regulated lncRNAs that were clinically associated with one or start sites of actively transcribed genes (79). Given the func- more tumor characteristics (tumor size, focality, differentia- tional versatility of lncRNAs, it is perhaps not surprising that tion, invasion, and metastasis) were confirmed experimentally deregulated lncRNAs are linked to cancer development and in in vitro assays and in vivo mouse models to modulate cellular progression. hallmarks of cancers including tumor size, cell proliferation, cell cycle, apoptosis, cell transformation, cell migration, inva- LncRNAs alter miRNA expression and activity in HCC sion, and metastasis (Supplementary Table S4B, boxed in miRNAs are another class of ncRNAs that regulate posttran- blue). Significantly, six (PCAT-14, SNHG12, UCA1, MALAT- scriptional expression of mRNAs (81). LncRNAs can modulate 1, Linc00462, and PDIA3P1) of these 16 upregulated lncRNAs expression of miRNAs at both transcriptional and posttranscrip- demonstrated to play important roles in tumor formation and tional levels, thereby providing an additional layer of control of metastasis were reported to modulate key cancer pathways mRNA expression (Supplementary Fig. S4A). including Hedgehog, NF-kB, FGFR1/ERK, mTOR, PI3K/AKT, In HCC, lncRNAs modulate miRNA transcription by epigenetic and p53 pathways (Supplementary Fig. S3). Similarly, down- mechanisms. Cui and colleagues showed that HULC decreased regulated linc-p21 activates the Notch signaling pathway, miR-9 expression by inducing hypermethylation of its promoter, whereas downregulated uc.134 inactivates the Hippo kinase possibly through upregulating DNMT1 (Supplementary Fig. S4A, pathway(SupplementaryFig.S3).Hence,deregulationof i; ref. 61). Emerging evidence also suggests that lncRNAs modu- these eight clinically relevant, experimentally validated late miRNA activity by acting as miRNA sponges, which sequester deregulated lncRNAs, targeting key cancer pathways, was sig- miRNA posttranscriptionally (Supplementary Fig. S4A,ii). For nificantly associated with tumor characteristics, vascular inva- instance, HULC prevents miR-107 from binding E2F1 transcrip- sion, metastasis, cancer stage, and survival (Supplementary tion factor, thereby enhancing SPHK1 expression and angiogen- Fig. S3), reiterating their promise as potential prognostic esis (82). SNHG6-003, an upregulated lncRNA in HCC, binds biomarkers and for drug development. miR-26a/b directly and prevents miR-26a/b from interacting with and inhibiting TAK1/MAP3K7, leading to activation of the p38 Mechanisms of action of lncRNA pathway (83). Another upregulated lncRNA, lncRNA-ATB, inhi- Growing evidence has demonstrated that lncRNAs regulate bits the miR-200 family, resulting in upregulation of ZEB1 and gene expression via diverse mechanisms. Besides interacting ZEB2, key transcriptional repressors of E-cadherin, which plays an with different intracellular miRNAs, mRNA, and proteins, important role in tumor invasion (84–86). lncRNA transcription itself may regulate the expression of neighboring genes (74, 75). For example, Airn transcription, LncRNAs modulate mRNA stability in HCC which overlaps with the Igf2r promoter locus, interferes with Intriguingly, lncRNAs also bind directly to and stabilize RNA polymerase II recruitment to the promoter, thereby mRNAs (Supplementary Fig. S4B). For instance, lncRNA-ATB, silencing the Igf2r gene (74). Intriguingly, lncRNAs can also which acts as a miR-200 sponge, also plays another role in serve as unique scaffolds, which act as molecular landing pads tumor progression through direct binding to IL11 mRNA and for the assembly of various proteins that mediate cellular abnormally prolonging its expression (Supplementary signaling (76). The advantage of lncRNAs over proteins as Fig. S4B,i; ref. 84). This binding sets up autocrine induction scaffolds is their dynamic and flexible biophysical structure, of IL11 and feed-forward activation of STAT3 signaling, which which allows different structural states with similar energetic promotes colonization of tumor cells during metastasis. properties to be adopted. This confers on lncRNAs the ability PCNA-AS1, an lncRNA whose sequence is antisense to PCNA, to interact with different sets of proteins under varying specific binds PCNA mRNA and forms an lncRNA:mRNA duplex, structural conformational states (76). Moreover, as lncRNAs which promotes tumor growth (Supplementary Fig. S4B,i). are often >100 nucleotides long, they are able to accommodate This hybrid duplex enhances PCNA mRNA stability, possibly simultaneous binding of more than two proteins (77). Fur- by altering its secondary and tertiary structure (87). LncRNAs thermore, lncRNAs are functional immediately upon tran- can also prevent miRNAs from binding to 30 untranslated scription and, unlike mRNAs, do not require the additional region (UTR) of target mRNAs (Supplementary Fig. S4B,ii). process of translation before functioning as scaffolds (77). A For example, DANCR, an upregulated lncRNA in HCC, binds well-known example is HOTAIR, which acts as a scaffold for to the 30UTRofCTNNB1mRNAtoblocktherepressiveeffects two protein complexes, namely PRC2 and LSD1/CoREST/REST of miR-214, miR-320a, and miR-199a, thereby increasing complexes, to modulate gene expression epigenetically (78). stemness-like features of HCC (88). Recent interest in the scaffold role of lncRNAs has extended to their key role in the formation of paraspeckles, dynamic LncRNAs regulate protein stability and activity in HCC membraneless intracellular nuclear organelles that modulate Several experimental methods such as RNA pull-down and gene expression (79). The 23 kb isoform of the lncRNA, RNA immunoprecipitation have been deployed to explore NEAT1, forms the "architectural backbone" of paraspeckles, lncRNA–protein interactions. These have allowed researchers to which brings together >40 different RNA-binding pro- elucidate the role lncRNAs play in modulating protein activity and teins (79, 80). Although no catalytic activity has thus far been stability (Supplementary Fig. S4C).

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Various lncRNAs enhance HCC cancer phenotypes through lncRNA–mRNA interactions. Because lncRNAs can affect mul- stabilizing the expression of target proteins (Supplementary tiple hallmarks of cancers, it will be interesting to explore Fig. S4C,i). The lncRNA, hPVT1, stabilizes the nucleolar protein mechanisms that lncRNAs adopt to target different molecules NOP2, a cell proliferation marker, leading to an increase in cell at different cancer stages. proliferation, cell-cycle progression, and stem-like properties in HCC (89, 90). Similarly, linc-cdh4-2 (lnc-CDH26-2:1) binds Diagnostic and therapeutic potential of lncRNAs in HCC and stabilizes R-cadherin expression, increasing R-cadherin- As one of the most malignant and lethal cancers, diagnosis RAC1 signaling, and potentially enhancing the migratory and of early stage HCC and development of novel therapies are invasive activities of HCC cells (91), because R-cadherin can urgently needed, especially for individuals at high risk. LncRNAs induce cell motility via activating the Rho family GTPases, such have several advantages as potential biomarkers: (i) Huarte as RAC1 (92). highlighted that the highly cell- and tissue-specific expression LncRNAs can also inhibit the expression of target proteins of lncRNAs may facilitate treatment response prediction or the through degradation (Supplementary Fig. S4C,ii). For example, classification of tumor subclasses (26); (ii) lncRNAs are stably lncRNA-LET binds and degrades the RNA-binding protein, expressed and detectable in body fluids. Notable examples NF90 (53), which is important in regulating the stability, trans- include lncRNA PCA3, which is a more specific and sensitive port, and translation of mRNAs (93). Interestingly, the associa- urine biomarker of prostate cancer than the widely used tion of lncRNA-LET/NF90 reduces HCC cell invasiveness by serum PSA (26, 99), and HULC lncRNA, which is highly expressed altering the stability of two different mRNAs in different tumor in the blood of HCC patients (100). As expressions of most of microenvironments (53). Under hypoxic conditions, HIF1a the clinically relevant, experimentally validated, deregulated mRNA expression is reduced by lncRNA-LET/NF90, whereas lncRNAs in HCC were identified in tumor tissues, it is a rational expression of CDC42 mRNA was decreased under normoxic next step to evaluate their levels in minimally invasive, readily conditions (53). accessible body fluids like blood to assess their utility as potential Besides modulating protein expression, lncRNAs can also biomarkers. It is worth exploring if these lncRNAs reside within alter protein structure (Supplementary Fig. S4C,iii). In HBV- exosomes or other membranous vesicles because these vesicles associated HCC, lncRNA Dreh is reported to interact with may convey RNases-protected tumor-specific lncRNAs in body vimentin, resulting in reduced cell migration by extending its fluids (101). helical filament structures from the nuclear membrane to the As discussed above, several lncRNAs are associated with cellular membrane (45). different characteristics spanning the clinicopathologic spec- Finally, lncRNAs can contribute to tumor progression by trum, namely tumor features, angiogenesis, metastasis, cancer modulating the activity of target proteins in HCC through stage, and patient survival (Supplementary Table S4B). These interacting with transcription regulators, including transcrip- clinically relevant lncRNAs also alter the expression and tion factors and chromatin modifying complexes (Supplemen- activity of various miRNAs (Supplementary Table S3C), genes tary Fig. S4C,iv; ref. 94) or other proteins (Supplementary (Supplementary Table S3E), and proteins (Supplementary Fig. S4C,v). LncRNA MEG3, which is downregulated in HCC, Table S3D), which consequently derange multiple signaling interacts with and stabilizes p53. This lncRNA–protein inter- pathways (Supplementary Table S3B), resulting in the man- action alters p53 transcriptional activity by upregulating 21 and ifestation of various tumorigenic and metastatic behaviors in downregulating 13 of 287 p53 target genes (95). A more in vitro and in vivo models (Supplementary Table S3A). Hence, complex example is H19, which affects target proteins via targeting lncRNAs may be more advantageous than targeting different mechanisms. It associates with hnRNPU/PCAF/RNA mRNAs and proteins because lncRNAs simultaneously mod- PolIII complex to upregulate miR-200 levels through histone ulate several molecules and pathways that aggravate multiple acetylation (51). The miR-200 family inhibits epithelial– hallmarks of cancer (from cancer initiation to cancer progres- mesenchymal transition (EMT) by targeting ZEB1 and ZEB2, sion), thus enabling a broader therapeutic effect compared transcriptional repressors of E-cadherin. Decreased H19 expres- with drugs directed at single targets (39, 102). However, it is sion in HCC upregulates ZEB1 and ZEB2 through effects on acknowledged that a multitargeted approach has a higher miR-200, leading to enhanced invasiveness and metastasis likelihood of incurring more adverse side effects due to its in vitro and in vivo, respectively (51, 85, 86, 96). Two lncRNAs pleiotropic nature. As lncRNAs are less abundant than mRNAs discovered in HCC have yet other mechanistic effects on in cells (103), lower doses of lncRNAs may be sufficient to proteins. LncRNA-UFC1 and uc.134 interact with proteins oth- achieve therapeutic effects. Furthermore, the ability of er than transcription regulators to control mRNA expression. lncRNAs to modulate specific protein activities aids the LncRNA-UFC1 interacts with the mRNA-stabilizing protein, design of precision drugs with more refined therapeutic HuR, to enhance b-catenin expression at both transcript and effects and lower toxicity compared with conventional pro- protein levels, accelerating tumor progression in HCC (97). tein-targeting drugs (26). Given that lncRNAs function at the Uc.134, a tumor-suppressing lncRNA, interacts with a nuclear RNA rather than protein level, the effects of lncRNAs can be E3 ubiquitin ligase, CUL4A, preventing its export to the cyto- expected to be more rapid in onset after intracellular delivery plasm to degrade LATS1, an important kinase in the Hippo than drugs that act on protein targets (104). Several strategies signaling pathway (98). This favors LATS1-mediated phosphor- targeting lncRNAs including siRNAs, antisense oligonucleo- ylation and inactivation of the oncogene, YAPS127,consequent- tides, locked nucleic acids, GapmeRs, and ribozymes have ly decreasing cell proliferation and invasion. Among the been explored for various cancers (104, 105), including mechanisms reported in studies of lncRNAs that were first HCC (39, 40, 106). Such efforts are certain to intensify as identified in HCC, lncRNA–protein interactions have led to lncRNAs have emerged as a rich source for achieving precision more diverse effects as compared with lncRNA–miRNA or oncology.

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Conclusion and Future Perspectives ciated with diverse clinicopathologic features, tumor characteristics, angiogenesis, and metastasis leading to altered clin- Recent technical advances have uncovered lncRNAs as a ical outcomes including altered cancer stage and patient class of highly versatile regulators of cellular functions. survival (Supplementary Table S4B). Deregulated lncRNA expression is pervasive across many can- Despite intensive lncRNA research in HCC in recent years, cers, including HCC. LncRNA expression is deregulated at our understanding of lncRNAs remains significantly less transcriptional and posttranscriptional levels in HCC, greatly mature than mRNAs, or even miRNAs. NGS technologies may increasing the complexity of their role in modulating cancer accelerate identification and characterization of important, phenotypes. Hence, a deeper understanding of the complex yet-to-be-annotated functional lncRNAs in HCC. As multiple regulation of lncRNAs during hepatocarcinogenesis and lncRNAs may act synergistically to promote tumorigenesis and metastasis can be expected to advance efforts to effectively metastasis (e.g., HULC and MALAT1 synergistically promote exploit lncRNAs for therapeutic purposes. In this review, we malignancy in liver stem cells; ref. 107), it could be fruitful present a model highlighting eight (six upregulated and two to identify and characterize combinations of deregulated downregulated) lncRNAs that have potential to be clinically lncRNAs that act synergistically in HCC. Another aspect of useful prognostic biomarkers and therapeutic targets to lncRNAs in HCC that remains largely unexplored is the role of improve the prognosis of HCC patients. Not only are these short polypeptides translated from some lncRNAs (108, 109). lncRNAs significantly associated with several clinical charac- As lncRNAs play significant roles in modulating the expression teristics that span the cancer progression spectrum, they have and activities of mRNAs, miRNAs, proteins, and pathways that also been experimentally validated to modulate key genes in play key roles in tumorigenesis, further characterization of this important cancer pathways to affect multiple key hallmarks of category of molecules to uncover their potential roles as cancers (Supplementary Fig. S3). therapeutic targets and biomarkers for HCC is an important Deregulated lncRNAs in HCC alter the expression and priority for the many thousands who die annually from this activities of mRNAs, miRNAs, and proteins through their challenging malignancy. interactions with these molecules, leading to multiple con- sequences such as enhancing or inhibiting expression or stability of these molecules, or even altering protein structure. Disclosure of Potential Conflicts of Interest Intriguingly, a single lncRNA can modulate the expression and No potential conflicts of interest were disclosed. activities of several different molecules through distinct mechanisms during oncogenesis and cancer progression. The Acknowledgments best example is the prometastatic lncRNA-ATB, which displays This work was supported by grants from the Singapore Ministry of Health's pleiotropic properties affecting HCC cell invasion, EMT, tumor National Medical Research Council (NMRC; NMRC/CBRG/0095/2015); National Cancer Center Research Fund; and block funding Duke-NUS cell colonization, as well as metastasis (84). It acts as a miRNA Graduate Medical School to A/P Caroline G. Lee. The funders had no role sponge during tumor invasion and EMT and forms lncRNA: in study design, data collection and analysis, decision to publish, or prep- mRNA duplexes during tumor cell colonization at sites of aration of the article. metastasis, promoting the invasion-metastasis cascade. The pleiotropic nature of lncRNAs may perhaps account for the Received January 29, 2019; revised May 6, 2019; accepted July 19, 2019; observation that a single lncRNA can simultaneously be asso- published first July 23, 2019.

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Roles and Regulation of Long Noncoding RNAs in Hepatocellular Carcinoma

Lee Jin Lim, Samuel Y.S. Wong, Feiyang Huang, et al.

Cancer Res Published OnlineFirst July 23, 2019.

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