lncRNA HOTAIR overexpression promotes hybrid epithelial/mesenchymal phenotype through suppression of c-Met Receptor Tyrosine Kinase Signaling in Hepatocellular Carcinoma Cells

non-coding Abstract Background: Epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) are both reversible processes, and regulation of phenotypical transition is very important for progression of several cancers including hepatocellular carcinoma (HCC). Recently, it is defined that cancer cells can attain a hybrid epithelial/mesenchymal (hybrid E/M) phenotype. Cells with hybrid E/M phenotype comprise mixed epithelial and mesenchymal properties, they can be more resistant to therapeutics and also more capable of initiating metastatic lesions. However, the mechanisms regulating hybrid E/M in HCC are not well described yet. In this study, we investigated the role of the potential crosstalk between lncRNA HOTAIR and c-Met receptor tyrosine kinase, which are two essential regulators of EMT and MET, in acquiring of hybrid E/M phenotype in HCC. Methods: Expression of c-Met and HOTAIR were defined in HCC cell lines and patient tissues through HCC progression. lncRNA HOTAIR was overexpressed in SNU-449 cells and its effects on c-Met signaling were analyzed. c-Met was overexpressed in SNU-398 cells and its effect on HOTAIR expression was analyzed. Biological significance of HOTAIR/c-Met interplay was defined in means of adhesion, proliferation, motility behavior, invasion, spheroid formation and metastatic ability. Effect of ectopic HOTAIR expression on phenotype was defined with investigation of molecular epithelial and mesenchymal traits. Results: In vitro and in vivo experiments verified the pivotal role of lncRNA HOTAIR in acquisition of hybrid E/M phenotype through modulating c-Met and its membrane co-localizing partner Caveolin-1 expression, activation and membrane organization to cope with the rate limiting steps of metastasis such as survival in adhesion independent microenvironment, escaping from anoikis and resisting to fluidic shear stress (FSS) in HCC. Conclusions: Our work provides the first evidence suggesting a role for lncRNA HOTAIR in the modulation of c-Met to promote hybrid E/M phenotype. The balance between lncRNA

incident between 2007 and 2017 by Global Burden of Disease (GBD) 2017 study. Hepatocellular Carcinoma (HCC) accounts more than 80% of liver cancer cases and it is estimated to be the 4th most common cause of cancer related deaths worldwide (1). HCC is reported to have complex and heterogeneous molecular features which is previously stressed out by integrative studies combining genomic characterization, exome sequencing, transcriptome analysis and clinical trials. Molecular heterogeneity and etiological complexity of HCC makes it unlikely for one treatment or agent to effectively target all or most HCCs (2,3).
c-Met, a receptor tyrosine kinase, is known to be upregulated in liver diseases favoring hepatocyte proliferation. Besides potential benefits in chronic liver diseases, c-Met contributes to initiation, development and progression of HCC. c-Met activation in HCC is mostly driven by molecular networks instead of activating mutations and it is activated by non-canonical signaling mechanisms as well as canonical activation by its ligand, HGF. c-Met is regarded as one of the most promising targets for HCC treatment and c-Met targeted clinical trials are being conducted, currently (4). In addition to its contribution to HCC development and progression, c-Met is also considered to be a key player in drug resistance (5).
HOX transcript antisense intergenic RNA, HOTAIR, is a 2,148-nt-long spliced and polyadenylated long non-coding RNA (lncRNA) encoded within HOXC cluster. HOTAIR is first defined to take role in epidermal tissue development by recruiting chromatin remodeling complexes to its epigenetic targets (6,7). Overexpression of HOTAIR has been associated with poor prognosis, invasiveness and aggressiveness of various cancer types (8)(9)(10)(11). Complex secondary structure and ability to form independent structural domains ensure the multi-acting nature of HOTAIR and it has been defined to contribute to various cellular mechanisms via different molecular interactions such as scaffolding protein complexes, decoying microRNAs, epigenetically targeting genes and enabling RNAprotein/DNA-protein interactions (7,(12)(13)(14).
Transcription factors, non-coding RNAs and epigenetic regulators play critical roles in cell-fate determination such as transition between epithelial and mesenchymal phenotypes. Starting from the origin of a tumor, cancer cells go through complex and dynamic phenotypical changes -epithelial to 4 mesenchymal transition (EMT), or its reverse mesenchymal to epithelial transition (MET)-to cope with metastasis rate-limiting steps. It has been reported that, some cancer cells display both epithelial and mesenchymal markers and this phenotype referred as hybrid, intermediate, partial, metastable or incomplete EMT phenotype (15)(16)(17)(18). The tumor cells simultaneously expressing both mesenchymal and epithelial markers may be more plastic and most likely to contribute to metastatic outgrowth.
Cells bearing this hybrid phenotype are first defined in circulating tumor cells (CTCs) of patients with both epithelial and mesenchymal traits (19,20). Hybrid cells that co-express mesenchymal and epithelial markers migrate collectively, more resistant to fluidic shear stress damage in circulation, exit from anoikis and have enhanced metastatic ability than cells with complete epithelial or mesenchymal phenotypes (21). That is why, cancer cells that attain a hybrid E/M (epithelial/mesenchymal) phenotype pose greater risk for metastasis in cancer patients (22). c-Met is reported to play an essential and complex role in modulation of transitional states within the broad spectrum of cellular phenotypes (23). Although HOTAIR is also reported to contribute attainment of cellular phenotypes, its role is generally defined in means of correlations with aggressiveness traits of cancer cells (10,11,24,25). As the number of the studies reporting induced expression and critical roles of these two molecules in HCC are increasing, the likelihood of their direct or indirect molecular interaction becomes non-negligible. In this study, we aimed to investigate the nature of their interaction and here, we describe the interplay between HOTAIR and c-Met in HCC context. Our data present strong evidence that HOTAIR ensures hybrid E/M phenotype and its downregulation is required for c-Met induced complete mesenchymal phenotype in HCC cells.

Cell Culture
All Human HCC cell lines were kindly provided by Prof. Dr. Mehmet Öztürk (Izmir Biomedicine and Genome Center, Izmir, Turkey). FOCUS, SNU-449, SK-HEP-1, SNU-475, SNU-387, SNU-423, SNU-398, MAHLAVU, HEP-3B and HuH-7 cell lines were cultured as described previously (26). All cell lines were tested for mycoplasma infection and confirmed as negative before the experiments. SU11274 (Calbiochem, 448101) solubilized with DMSO used with 2500 nM concentration. c-Met kinase activity was inhibited with SU11274 (2500 nM in 2% FBS complete medium). Ligand dependent c-Met activation was induced by 10 ng/ml Hepatocyte Growth Factor (HGF) in 2% FBS complete medium Labeling with c-Met antibody and hybridizing HOTAIR mRNA with fluorescent-labeled probes were performed sequentially. For sequential labeling, HCC cells were firstly fixed with 3.7% (vol./vol.) formaldehyde in 1X-PBS for 10 minutes at room temperature. Fixed cells were incubated with primary antibody against c-Met protein (Cell Signaling, #3127) for 2 hours at room temperature. After couple of washes with 1X-PBS, cells were incubated with secondary antibody (Alexa488-conjugated goat antimouse secondary antibody, Invitrogen, A-11001) for 1 hour at RT. After immunofluorescent labeling, cells were fixed again with 3:1 methanol-acetic acid (MeOH-AcOH) fixation solution for 10 minutes.
Following hybridization was performed according to the manufacturer's instructions with 500 nM HOTAIR targeting probes for 4 hours at 37 o C. Cell nucleus was stained with DAPI and cells were analyzed with Zeiss LSM 880-Confocal Laser Scanning Microscopy with Airyscan in IBG Optic Imaging Core Facility.

Immunoblotting
The cells were lysed using RIPA lysis buffer containing 1 mM Na3VO2, 1 mM NaF, and 1% protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA), and the lysates were subjected to Western blot analysis as described previously (26). Primary antibodies are described in Immunofluorescent analysis of the cells HCC cells were grown on glass cover-slips and after experimental conditions set, cells were immunofluorescently labeled as described in previous study (27). Imaging was performed with upright fluorescence microscope (Olympus -BX61) and Zeiss LSM 880-Confocal Laser Scanning Microscopy with Airyscan at IBG Optic Imaging Core Facility.

Trans-well motility and invasion assays
Trans-well inserts with 8µm pore size (SPL Life Sciences, #37224, Korea) were used to analyze motility and invasion ability of HCC cells. The mix of 0.25 mg/ml basal membrane extract (Corning Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix, Phenol Red-Free, #356231) was coated on invasion inserts. Trans-well motility and invasion experiments performed as described in previous study (26). Motile and invasive cells located at the exterior surface of the inserts were counted with bright field inverted microscope and statistical analysis was performed via Prism 8.

Real-time adhesion, proliferation, motility and invasion assays
Real-time cell growth monitoring was performed with the Real-Time Cell Analyzer, xCELLigence System (Roche, Germany) as described previously (28). 5000 cell/well was plated to analyze proliferation and adhesion to cell-culture treated wells of E-Plates. For motility and invasion assays, Phalloidin Staining HCC cells were seeded on glass cover slips (22x22 cm) and incubated overnight for adhesion.
MTT Assay SNU-449 HOTAIR OE and SNU-449 MOCK 1,000 cells per well were seeded on 96-well plate and incubated for 24, 48 and 72 hours. MTT analysis performed as described previously (31). Measured absorbance was analyzed by Microsoft Excel 2016 and plotted by Prism 8.

Analysis of TCGA and GEO microarray datasets
Normalized gene expression data from dataset GSE89377 was analyzed with GEO2R. Expression 9 values plotted and statistically analyzed with Prism 8.

Zebrafish Xenograft Assay
Zebrafish xenograft assay was performed with SNU-449 MOCK and SNU-449 HOTAIR cell clones. The experiment was performed as described in a previous study (30). Briefly, 48hpf embryos were anesthetized and DiI labeled cells were injected to the yolk sack.100 cells per embryo were injected, Analysis of variance (ANOVA), Student's t-test and linear regression. Zebrafish xenograft data was statistically analyzed with chi-square with Yates correction as described in previous study (32).
Results with p<0.05 were considered as statistically significant.

Expression of HOTAIR is low in HCC cells with high c-Met expression and activation
To examine expression levels of HOTAIR and c-Met, we analyzed their mRNA expression in 10 HCC  (Figure 7a). To test this hypothesis, we examined previously defined EMT markers in HOTAIR OE SNU-449 cells (36)(37)(38). First, we found that in contrary to the mesenchymal and elongated phenotype, HOTAIR OE cells were round-shaped with thinner F-actin stress fibrils indicated with phalloidin staining (Figure 7b). Then, we performed a detailed investigation of molecular biomarkers of hybrid E/M phenotype. In HOTAIR OE cells, beta-catenin was enriched in the plasma membrane (Figure 7c)  in previous definitive studies (40,41). In our study, we report that HCC cells can be divided into two clusters which show differential expression tendency for HOTAIR and c-MET genes. Expression of these two molecules among 10  Indicated studies suggested HOTAIR as a competing long non-coding RNA decoying microRNAs targeting c-Met in different cancer contexts, and those microRNAs are reported to have different molecular targets in HCC rather than c-Met (45,46).
In addition to suppression of c-Met, HOTAIR weakens downstream Akt1, MAPK and STAT3 signaling pathways not only by decreasing c-Met expression but also by modulating its membrane organization.
In our previous studies, we showed that Caveolin-1 enhances c-Met signaling by co-localizing in plasma membrane (27). Src kinase, a well-identified downstream effector of c-Met, phosphorylates Caveolin-1 from Tyr-14 residue to induce lipid-raft enrichment of c-Met with Caveolin-1 and formation of caveola structures (27,47). HOTAIR over-expression reduced Caveolin-1 expression and activation via suppressing activation of c-Met and its downstream effector Src kinase. In parallel to c-Met, Caveolin-1 organization in plasma membrane was also disrupted in HOTAIR OE cells. Tsai et. al defined function of HOTAIR in epigenetic regulation of its target genes and they reported Caveolin-1 as one of the most differentially expressed genes in response to HOTAIR knock-down in foreskin fibroblasts (12). We confirmed the interaction axis of c-Met, HOTAIR and Caveolin-1 is conserved in c-Met over-expression model of SNU-398 cell line its wild type is lack of c-Met expression but expresses HOTAIR, abundantly. In addition to the lack of c-Met expression, SNU-398 cells also do not express Caveolin-1 (26). As a consequence of c-Met overexpression and HOTAIR suppression, Caveolin-1 expression was induced in those cells. In addition to the over-expression studies of c-Met and HOTAIR, we showed that c-Met and HOTAIR reverse interaction is conserved in both ligand-dependent and independent c-Met activation contexts. These results support the defined interplay between two molecules.
To understand the biological significance of HOTAIR/c-Met interaction, we analyzed the results of HOTAIR overexpression on c-Met related biological responses which are clearly defined in the literature as contributing to aggressive phenotype of HCC cells (48). HOTAIR over-expressing cells showed delayed adhesion to surfaces and suppressed expression of adhesion-related integrins (49).
In addition to decreased attachment to surface, proliferation and colony formation ability of HOTAIR OE cells were decreased. Suppression of individual motility and invasion of HOTAIR OE cells were consistent with suppressed scattering effect of c-Met signaling. HOTAIR OE cells were migrating collectively rather than individually which is compatible with previous studies explaining migration behavior of cells with hybrid E/M phenotype (15). Increased expression of cell adhesion molecules such as CDH-1 and JAM-2 ( Supplementary Fig. 1a) were consistent with the evidence of collective migration that requires preservation of cell-to-cell interactions (50,51).

Surprisingly, HOTAIR over-expressing cells showed increased survival ability in adhesion independent
culture and under FSS. Escaping from anoikis and overcoming damage incurred by shear forces are important necessities of survival in circulation to achieve metastasis (52). Response to fluidic shearstress or adhesion-independent conditions are context-dependent processes those depend on the molecular pool of the cell; HOTAIR over-expressing cells resisted those unfavorable conditions via formation of spheres (35). Aggregation into spheroids to overcome the stress of unfavorable microenvironment, enhancing cell-cell adhesion and surviving under damage of shear forces are well-defined requirements of metastasis (35,52). As expected, by attainment of those abilities, HOTAIR over-expression increased metastatic ability of SNU-449 cells which was defined to be highly metastatic in our previous studies in zebrafish xenograft model (30). Our study is the first in the literature defining the molecular basis of HOTAIR overexpression mediated increase in the metastatic ability of HCC cells.
Since the definition of EMT has been changed and broadened, transitional states between epithelial and mesenchymal phenotypes are called "partial EMT". Existence of intermediate hybrid phenotypes have been defined in different molecular and biological processes such as fibrosis, development, wound healing and cancer (15). In our study, we found that HOTAIR acts as a fine tuner of maintenance of hybrid E/M phenotype by modulating c-Met signaling. Compatibly with the literature, we analyzed defined morphological and molecular markers of epithelial and mesenchymal phenotypes in HOTAIR over-expressing cells (15,16,37,38). HOTAIR OE cells were round-shaped and had thinner F-actin stress fibrils. beta-Catenin was enriched in membrane compatible with increased E-cadherin expression and hybrid E/M phenotype characteristics (18). In comparison with MOCK cells, mesenchymal biomarkers Vimentin and N-cadherin expression was suppressed in HOTAIR OE cells (Fig. 7). Changes in cytoskeletal components and dis-/re-assembling of cell-cell contacts regulates gene expression, motility and cell-cycle. Cells placed in spectrum between epithelial and mesenchymal phenotypes are reported to arrest cell cycle and decrease their proliferative activity (15,17). Consistent with the literature, proliferation rate of HOTAIR OE cells were decreased (Fig. 4) and cell cycle was arrested ( Supplementary Fig. 1e).
Decrease in biological responses such as individual cell motility and invasion are consistent with suppressed scattering effect of c-Met signaling activity. Even individual cell motility was decreased, HOTAIR OE clones have ability to enclose the scratch via collective migration as fast as MOCK clones in wound-healing assay (Fig. 5). Collective migration is not only important for maintenance of invading front of tumors but also it is an emerging mechanism for seeding of secondary tumors (53).
Taken together, our data shows that decrease in HOTAIR expression and its effects on c-Met and Caveolin-1 activation is crucial through the completion of mesenchymal phenotype.
Our study clearly defines the fine tuning of c-Met signaling by HOTAIR to maintain hybrid E/M phenotype which ensures flexibility of commitment and enhances metastatic ability under unfavorable conditions through rate-limiting steps of metastasis. Further studies are needed to address the molecular cascade between HOTAIR, c-Met and Caveolin-1 in detail.

Conclusions
In conclusion, HOTAIR over-expression suppresses c-Met expression, activation and also disrupts its

Consent for Publication
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