The crosstalk between lncRNAs and the Hippo signalling pathway in cancer progression

Abstract LncRNAs play a pivotal role in the regulation of epigenetic modification, cell cycle, differentiation, proliferation, migration and other physiological activities. In particular, considerable studies have shown that the aberrant expression and dysregulation of lncRNAs are widely implicated in cancer initiation and progression by acting as tumour promoters or suppressors. Hippo signalling pathway has attracted researchers’ attention as one of the critical cancer‐related pathways in recent years. Increasing evidences have demonstrated that lncRNAs could interact with Hippo cascade and thereby contribute to acquisition of multiple malignant hallmarks, including proliferation, metastasis, relapse and resistance to anti‐cancer treatment. Specifically, Hippo signalling pathway is reported to modulate or be regulated by widespread lncRNAs. Intriguingly, certain lncRNAs could form a reciprocal feedback loop with Hippo signalling. More speculatively, lncRNAs related to Hippo pathway have been poised to become important putative biomarkers and therapeutic targets in human cancers. Herein, this review focuses on the crosstalk between lncRNAs and Hippo pathway in carcinogenesis, summarizes the comprehensive role of Hippo‐related lncRNAs in tumour progression and depicts their clinical diagnostic, prognostic or therapeutic potentials in tumours.

prognosis prediction and therapeutic targets are still lacking, which may hinder the effective monitoring as well as treatment of cancer. 5,7,8 Long non-coding RNAs (lncRNAs) are a large and heterogeneous class of endogenous lncRNA family that are generally greater than 200 nucleotides (nts) in length. 9 Previously, lncRNAs were characterized as transcriptional noise since they exhibit no or limited protein-coding capacity. 10,11 Recently, owing to the advancement of next-generation sequencing-based transcriptome profiling, tremendous lncRNAs were identified and functionally annotated. 7,12,13 LncRNAs are found to execute a wide spectrum of biological processes, 14 such as alternative splicing, chromatin modification, sponging microRNAs (miRNAs) as competing endogenous RNA (ceRNAs), nuclear-cytoplasmic trafficking or interaction with genes, and thereby involve in crucial regulation of various human diseases including cancer. 15 Compelling experimental evidences indicate an engagement of lncRNAs in pleiotropic pathophysiological functions related to tumorigenesis, like the cell growth, invasion, metastasis, apoptosis and chemo-resistance, 16 by interaction with other macromolecules. 17 Accumulating studies have shown that lncRNAs could be considered as a promising candidate in cancer prognosis and diagnosis. 1,4,7,18 Accordingly, lncRNAs have attracted great attention due to their multifaceted modulatory functions and the capacity as predictive biomarkers in cancers. 1,4,7 Hippo signalling pathway consists of a broad range of proteins and controls lots of molecular and cellular processes. 12 It is reported that Hippo pathway could be activated or suppressed by genetic or epigenetic regulation, leading to a plethora of pathological disorders including cancers. 11 Notably, advanced studies have demonstrated that the crosstalk between lncRNAs and Hippo pathway may contribute to cancer occurrence and progression in recent years. For instance, YAP (or YAP1), a major transducer in downstream of Hippo pathway, is amplified and nuclear accumulated in a variety of cancers. 19 LncRNA TNRC6C antisense RNA 1 (TNRC6C-AS1) was reported to be abundantly expressed in thyroid carcinoma and could regulate the subcellular localization and activation of YAP, leading to the promotion of cell proliferation and tumorigenicity. 20 In this review, we systematically summarize the up-to-date insights provided by studies regarding the crosstalk between ln-cRNAs and Hippo signalling pathways in cancers. In addition, we provide a brief overview of the Hippo-related lncRNAs as clinicopathological biomarkers and highlight its potential role as therapeutic targets in cancers. The interplay between Hippo and lncRNA may shed light on the role of underlying mechanisms in carcinogenesis.

| C ANONIC AL HIPP O S IG NALLING PATHWAY IN TUMORI G ENE S IS
The Hippo signalling pathway is initially characterized as a critical signalling cascade that regulates organ size in fruit fly (drosophila melanogaster) in 1995. 21 It is an evolutionarily ancient and conserved network among different species, 22 and its homology molecules in mammals have been subsequently identified. A growing number of studies have highlighted a critical role of Hippo pathway in the regulation of organ size, tissue homeostasis, cell proliferation, apoptosis, metastasis, autophagy, angiogenesis and stem cell self-renewal. 23,24 The misregulation of Hippo signalling pathway can cause many disease conditions. 25 In tumorigenesis, Hippo pathway is wellestablished as a tumour-suppressive cascade due to its proliferation restriction and apoptosis induction. 26,27 In mammals, the central axis of the Hippo signalling pathway comprises two serine/threonine kinases: mammalian sterile 20-like kinase 1/2 (MST1/2) and its homolog large tumour suppressor 1 and 2 (LAST1/2); two adaptor/scaffold protein: WW45 for MST1/2 and Mps one binder kinase activator-like 1 (MOB1) for LAST1/2; downstream transcriptional co-regulators: yes-associated protein (YAP) and its paralog transcriptional co-activator with PDZ-binding motif (TAZ, also known as WWTR1); and various nuclear transcriptional factors: transcriptional enhancer-associated domain (TEAD1/2/3/4). 26,28 Of them, YAP and TAZ are key intracellular messengers, whose localizations are critical in Hippo pathway. 11 YAP/ TAZ could be positively or negatively modulated by phosphorylation at different sites by upstream kinases, elicit target gene expression signature through forming complexes with TEAD family, the major nuclear partner, and thereby play a prominent role in cellular plasticity, lineage differentiation during development, tumour initiation, progression and metastasis. 29,30 In canonical Hippo signalling, the cascade is on ('Hippo On') when the upstream Hippo pathway is activated by stimuli or regulators, such as mechanical stress, cell polarity determinants and increased cell-cell contact. 28,31 Then, MST1/2 kinase is phosphorylated and subsequently phosphorylates salvador homolog 1 (SAV1) to form a heterotetramer to further promote the LATS1/2 phosphorylation. Activated LATS1/2 could result in inactivation of YAP/TAZ through sequestering its cytoplasmic localization by binding to 14-3-3 protein or degradation via ubiquitination, and thereby dampen the transcription of downstream genes. 11,31 Conversely, when the Hippo pathway is inactivated ('Hippo off'), YAP/TAZ translocates to the nucleus and binds primarily to enhancer elements by using TEAD as DNA-binding sites, 11,32 thereby driving target gene (AREG, CTGF, Cyr61, ANKRD1, AXL, etc) transcription and promoting cell tissue growth, survival, proliferation and self-renewal, 28,33,34 as presented in Figure 1.

| REG UL ATORY NE T WORK OF LN CRNA S AND HIPP O S I G NALLING PATHWAY IN C AN CER
Overall, considerable crosstalk between lncRNAs and Hippo signalling pathway has been revealed in several tumours as demonstrated in Tables 1-3. A vast majority of lncRNAs were discovered in the regulation of Hippo signalling pathways. Conversely, Hippo pathways were also reported to modulate expression of lncRNAs. 31 These bilateral regulations ultimately impact target gene expressions in cancer progression, indicating a close relationship and complexity between lncRNAs and Hippo signalling cascades.

| LncRNAs regulate members of Hippo pathway
Recently, lncRNAs are emerging as a critical mediator in a wealth Further mechanism assay revealed that B4GalT1-AS1 could directly bind to YAP. B4GALT1-AS1 silencing could sequester YAP in cytoplasm and decrease YAP transcriptional activity, while overexpression of YAP attenuated the inhibition effect caused by B4GAlT1-AS1 knockdown. 35 In similar, another study showed that B4GALT1-AS1 was expressed in osteosarcoma tissues as well as cell spheres at an enhanced level. 38 Functionally, B4GALT1-AS1 acted as an oncogene to enhance YAP mRNA stability and transcriptional activity by recruiting HuR, and in turn maintain osteosarcoma cells stemness, and promote migration and chemo-resistance. 38 Conclusively, these studies clarified an obvious association of B4GALT1-AS1 and Hippo pathway, which may contribute to the malignant properties of tumour. 35,38 GHET1, located in chromosome 7q36.1, was firstly identified as an overexpressed lncRNA in gastric cancer. 39  In addition to YAP, other components of Hippo cascade including TAZ, LATS1/2 and MST1/2 were also found involved in crosstalk with a variety of lncRNAs in carcinogenesis. For example, both LINC00174 and TAZ showed an upregulated expression pattern in human primary colorectal cancer (CRC) tissues as compared to corresponding normal tissues. 32 Overexpression of LINC00174 or TAZ could enhance CRC cell proliferation motility. Bioinformatics and luciferase reporter assays revealed that LINC00174 may competitively bind to miR-1910-3p to increase TAZ expression in CRC carcinogenesis. 32 MiR-125a-5p, an important endogenous tumour suppressor, 40 was reported to target TAZ and inhibit EGFR pathway to repress retinoblastoma progression. 41   In summary, these findings help to illuminate the role of lncRNA in the regulation of Hippo signalling to subsequently control cell proliferation and tumorigenesis.

| LncRNAs induced by Hippo pathway
Several studies demonstrated that the core components in Hippo pathways could also exert functions in the regulation of the expression as well as functions of lncRNAs, such as lncRNA breast cancer antiestrogen resistance 4 (BCAR4) 26 and metallothionein 1D, pseudogene (MT1DP) 47 ( Figure 3). LncRNA BCAR4 is an upregulated lncRNA in multiple cancers with clinicopathological significance in prognosis. 4  sponging to miR-632 and miR-185-3p, and thereby promoted the occurrence and metastasis of CRC. 51 Besides, another study showed that NORAD, a unique kind of lncRNA that responds to DNA damage and maintains genome integrity and stability in cancers, [55][56][57] was synergistically transcriptionally inhibited by the YAP/TAZ-TEAD and the NuRD complex, which in turn affected the development and metastasis of lung and breast cancer via sequestration of S100P. 52 Moreover, lncRNA H19, a well-characterized oncogenic lncRNA in tumour progression, metastasis and chemo-resistance, 58-60 was also found abnormally expressed in osteosarcoma and could be upregulated by overexpression of YAP. 53 To summarize, it is clear that Hippo pathway could intimately modulate certain lncRNA to engage in multiple processes of cancer development.

| THE THER APEUTI C P OTENTIAL OF LN CRNA S INVOLVED IN HIPP O PATHWAY
As mentioned above, the Hippo pathway comprises multiple downstream signalling proteins, such as YAP/TAZ, whose activation can endow cells with several hallmarks of cancer, 103

| CON CLUS I ON S AND PER S PEC TIVE S
Hippo pathway is one of the most complicated signalling pathways with multiple downstream effectors that respond to extracellular and intracellular stimuli to coordinately govern cell differentiation, migration and proliferation. 25 Genetic or epigenetically provoked disruption of Hippo pathway leads to imbalanced regulation of these mechanisms, resulting in tumorigenesis. 25,121 Targeting Hippo signalling may provide novel approaches in treatment of cancer. However, given the fact that Hippo pathway has striking tumour regulatory activity in various contexts, the factors and concise regulation mechanisms for activation or inactivation of Hippo signalling are still poorly understood. 11 LncRNAs are a subclass of ncRNAs with growing recognition for their role in diverse cellular activities. Altered expression and mutation of lncRNAs are reported to drive multifaceted cancer phenotypes by regulating gene expression and signalling pathways at various levels. 96 Nowadays, a group of lncRNAs have been delineated to directly or indirectly target the core components of Hippo cascade, such as YAP, TAZ, LATS1/2 and MST1. 36,58,84,122 By contrast, Hippo can also modulate certain lncRNAs by affecting their transcriptional activity. 31 The expression of lncRNAs is closely correlated with tumorigenesis and tumour aggressiveness. Importantly, lncRNAs related to Hippo signalling may be useful as predictive indicators for diagnosis and prognosis in cancers. Researches on the interaction between lncRNAs and Hippo signalling pathway may potentially offer us a more comprehensive understanding in cancer occurrence and progression.
However, it should be noticed that the link between lncRNAs and Hippo pathways may be cell type-, context-and even tumour stage-specific. 31,52 Thus, more studies are still warranted to further elucidate their detailed structures and functions for developing biomarker and individualized therapy. 80 Besides the canonical Hippo pathway, there are studies reporting the non-canonical Hippo signalling axis in the regulation of tumorigenesis. 123,124 Currently, the crosstalk between lncRNAs and non-canonical Hippo pathway has not been elucidated yet, which may merit further exploration.
Moreover, despite our understanding of lncRNA has been expanding in past decades, the discovery and functional annotation of lncRNAs still remain just the tip of an iceberg. 125 Furthermore, in order to promote efficient therapeutic interventions in cancers by targeting lncRNAs and Hippo pathway, further in-depth pre-clinical and clinical studies are urgently needed.

CO N FLI C T S O F I NTE R E S T S
The authors have no conflicts of interest to declare.

AUTH O R CO NTR I B UTI O N S
CT, KXY and JYH wrote the manuscript; LW, LQ, WCW, QL and ZHL reviewed and edited the manuscript before submission; CT and ZHL prepared the figures; and all authors read and approved the final version of the manuscript as submitted.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.