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 cell lines (FOCUS, SNU-449, SK-HEP-1, SNU-475, SNU-387, SNU-423, MAHLAVU, Hep-3B, HuH-7 and SNU-398). HOTAIR expression was only abundant in HuH-7 and SNU-398 cell lines which are also known to have low or no c-Met expression, respectively (Figure 1a-1b). To evaluate the potential relation between HOTAIR and c-Met protein expressions, we selected two cell lines with constitutive c-Met activation (SNU-449 and SK-HEP-1) and two with low c-Met expression (HuH-7 and Hep-3B). RT-qPCR analysis of HOTAIR mRNA expression levels and western blot analysis of c-Met protein expression revealed that HOTAIR expression is low in HCC cell lines with high c-Met protein expression (Figure 1c). To understand the relationship between HOTAIR and c-Met expression in HCC, we analyzed HOTAIR and MET gene expression in a microarray dataset, GSE89377, which comprise gene expression analysis in patient tissues from normal liver, dysplasia, early and late HCC stages (35). Expression analysis of the dataset showed that HOTAIR expression decreases through HCC progression whereas MET gene expression increases (Figure 1d).
To evaluate the effect of ectopic HOTAIR expression on c-Met expressing HCC cells, we overexpressed HOTAIR in SNU-449 cell line which has constitutive c-Met activation. Overexpression of HOTAIR (HOTAIR OE, presented in figures as HOTAIR clone) and its effect on c-Met expression were analyzed and confirmed with confocal microscopy imaging of fluorescent in-situ hybridization (FISH) of HOTAIR, immunofluorescent labeling of c-Met (Figure 1e), and also with RT-qPCR (Figure 1f). HOTAIR overexpression suppressed both mRNA and protein levels of c-Met, dramatically.
To understand the possible interplay between two molecules in detail, we knocked down HOTAIR expression with siRNA transfection in HuH-7 cell line and then analyzed mRNA and protein expression of c-Met. Analysis of three independent experiments showed that average of 45% knock-down of HOTAIR (Figure 1g) induced c-Met mRNA (Figure 1h) and protein expression (Figure 1i), significantly. Uncut membrane images and expression levels are presented in Supplementary Figure 1a).
lncRNA HOTAIR overexpression suppresses c-Met downstream signaling and modulates its cellular localization via suppressing Caveolin-1 expression
To understand the effects of HOTAIR on c-Met signaling, we analyzed activation of c-Met and its downstream signaling pathways Akt, MAPK and STAT3. Western blot analysis showed that HOTAIR OE suppressed c-Met protein expression and activation (Figure 2a). Repression of c-Met activation was a result of diminished c-Met protein expression and there was not a significant inhibition on c-Met phosphorylation (p-cMet) when normalized to total c-Met protein expression (Supplementary Figure 1b). HOTAIR OE caused a modest decrease in Akt activation (Figure 2b), and suppressed MAPK (Figure 2c) and STAT3 (Figure 2d) activations, dramatically. We confirmed these results by analyzing expression and activation of c-Met by immunofluorescent microscopy (Figure 2e and 2f). Macroscopic analysis of the cells revealed that HOTAIR OE cells were smaller with roundish morphology and even the immunofluorescence staining intensities of c-Met seem to be higher in HOTAIR OE cells, analysis of immunofluorescence intensity per cells showed that is an optical illusion caused by size (smaller) and morphological (rounder) attributes of HOTAIR OE cells (Figure 2e). Furthermore, immunofluorescent labeling revealed a differential organization and localization of c-Met in HOTAIR OE cells (Figure 2f). In our previous studies, we reported that Caveolin-1 (CAV1), a membrane protein enriched in lipid-rafts, enhances c-Met activation and modulates its membrane localization in HCC cells (27). To investigate the possible role of CAV1 in HOTAIR/c-Met interaction, we analyzed expression and activation of CAV1 and its activating kinase, Src, in HOTAIR OE cells. HOTAIR OE suppressed CAV1 mRNA level (Figure 2g), protein expression and activation (Figure 2h, Supplementary Figure 1c) as well as Src kinase expression and activation (Figure 2i). Moreover, immunofluorescent staining revealed that membrane organization of Caveolin-1 was disrupted in HOTAIR OE cells which was compatible with disorganization of c-Met (Figure 2j).
Overexpression and/or activation of c-Met decreases HOTAIR expression in HCC cell lines
To understand the interplay between HOTAIR and c-Met further, we applied an opposite approach by overexpressing c-Met in SNU-398 HCC cell line which lacks c-Met protein expression and activation in wild-type (Figure 1a). Overexpression and activation phosphorylation of c-Met were confirmed by western blot (Figure 3a), immunofluorescent labeling and RT-qPCR analysis (Figure 3b). Analysis of HOTAIR expression in MET-OE-SNU 398 (presented in figures as MET-GFP) cells by FISH and RT-qPCR showed that HOTAIR mRNA expression was significantly suppressed by c-Met overexpression (Figure 3c). To investigate HOTAIR/c-Met/Caveolin-1 axis further, we analyzed Caveolin-1 expression in c-MET OE cell lines and found that Caveolin-1 expression was significantly upregulated, consistent with HOTAIR down-regulation (Figure 3d).
To understand the role of c-Met activation in HOTAIR expression, we analyzed the effect of HGF induced c-Met activation on HCC cell lines with different c-Met and HOTAIR expression levels such as HuH-7, HEP-3B, SNU-449, MAHLAVU and SK-HEP-1. Ligand-dependent c-Met activation by 10 ng/ml HGF for 1 hour suppressed HOTAIR expression significantly in all tested cell lines (Figure 3e). In addition to ligand-dependent activation, we examined the role of ligand-independent c-Met activation on HOTAIR transcription. In our previous studies, we showed that heparin treatment (36), acquired sorafenib resistance (5), high glucose induction (37) or fluidic shear stress treatment (38) induces ligand independent c-Met activation in HCC cell lines. Similar to HGF, heparin (Figure 3f), sorafenib (Figure 3g), high-glucose (Figure 3h) and fluidic shear stress (Figure 3i) treatments induced c-Met activation resulted in a substantial decrease in HOTAIR transcription. The maintenance of the interplay between HOTAIR and c-Met in different contexts supports the hypothesis that there is a reciprocal crosstalk between these two molecules in HCC cell lines. We further confirmed the rescue of HGF-suppressed HOTAIR expression after c-Met tyrosine kinase inhibition by SU11274 in HCC cell lines HuH-7, SNU-449, MAHLAVU and SK-HEP-1. c-Met tyrosine kinase inhibition rescued HGF-induced HOTAIR suppression in HCC cell lines (Figure 3j).
To understand the biological significance of this interaction, we investigated the phenotypic and biological results of HOTAIR overexpression in SNU-449 HCC cell line.
HOTAIR OE induced c-Met inhibition decreases adhesion, proliferation and colony formation of SNU-449 cells
HOTAIR OE cells showed reduced adhesion capacity to cell culture-treated (Figure 4a) and basal membrane extract coated cell culture surfaces (Figure 4b) in xCELLigence real time cell analysis system (RTCA). To understand the cause of decreased extracellular matrix (ECM) attachment capacity, we analyzed the expression of cell surface integrins that contribute to matrix attachment. ITGA6, ITGB1, ITGA4 and ITGB4 expressions were down-regulated in HOTAIR OE cells, significantly (Figure 4c).
Proliferation of the cells were evaluated with both xCELLigence RTCA system (Figure 4d) and MTT analysis (Figure 4e). HOTAIR OE cells showed significant reduction in proliferation rate in both techniques. Compatible with proliferation assays, 2D colony forming potential of HOTAIR OE cells were dramatically low. Colony counts are normalized to number of cells plated for evaluation of colonization percentage. HOTAIR OE cells showed significantly lower colonization ability in comparison to MOCK cells (Figure 4f). Coverage area of colonies in well-plates, staining intensity of colonies and average colony sizes were also significantly lower in HOTAIR OE cells (Figure 4g-4j). These results reflect lower capacities of adhesion and proliferation.
HOTAIR OE induced c-Met inhibition decreases motility and invasion capacity of SNU-449 cells
Motility and invasion experiments were performed in parallel by both xCELLigence RTCA system and trans-well cell culture systems. Cell index of motile (Figure 5a) and invasive (Figure 5c) cells in HOTAIR OE clones were significantly lower in xCELLigence RTCA (p≤ 0.001). Consistent with real-time motility and invasion analysis; the number of motile and invasive cells migrated through trans-well cell culture inserts were reduced in HOTAIR OE cells (Figure 5b, 5d), significantly (p≤0.05).
Since trans-well systems especially tests individual cell movement ability, we further analyzed collective migration ability of clones in scratch (wound-healing) assay. Scratch assay enables cells to move collectively, without losing cell-cell attachments. Even though HOTAIR OE cells have reduced ability to migrate individually, they were still motile. Rather than migrating individually, HOTAIR OE cells migrated without losing cell-cell contacts, collectively (Figure 5e).
lncRNA HOTAIR inhibits c-Met induced cell scattering and promotes metastasis in zebrafish embryos
Decrease in attachment capacity to ECM led us to investigate spheroid formation capacity of HOTAIR OE cells. We analyzed spheroid formation ability of those cells with hanging-drop spheroid formation assay. SNU-449 cells have a complete mesenchymal phenotype, as a result of scattering effect of constitutive c-Met activation, and they are not inclined to form spheroids in hanging-drops. Due to their reduced ability to form cell-cell contacts maintained by constitutive c-Met activation; MOCK cells formed loose, scattered and wide clusters whereas HOTAIR OE cells formed more tight, stacked and small spheroids (Figure 6a). Significant ability to form intact spheroids led us to investigate their survival capacity under fluidic shear stress (FSS). To test their survival ability, cells sheared for 1 hour under FSS of 0.5 dyn/cm2, mimicking the sinusoidal shear stress in liver (39). HOTAIR OE improved survival ability under sinusoidal FSS. Alive cell numbers after one hour of FSS was significantly higher (p≤ 0.05) in HOTAIR OE clones (Figure 6b).
Finally, embryonic zebrafish xenograft model was used to test in vivo metastatic capacity of HOTAIR OE, further. Average number of 100 HOTAIR OE and MOCK cells were injected to the yolk sac of 48h zebrafish embryos and embryos which bear an intact tumorosphere after 4 hours of injection were raised for 4 days to analyze metastatic capacity. Three independent experiments were performed and in every experiment each group consisted an average number of 50 embryos with intact tumorospheres. Metastatic capacity was calculated by number of xenograft hosts that have metastasized cells at 4 days post injection (dpi). Even though wild-type SNU-449 cells are known to be metastatic in embryonic zebrafish model (30), HOTAIR OE dramatically increased their metastatic capacity when compared to MOCK cells (Figure 6c).
lncRNA HOTAIR maintains hybrid E/M phenotype to promote metastasis
Our experiments with HOTAIR OE cells show morphological and biological properties that are consistent with the hybrid E/M phenotype which can be defined as a fluidic state between epithelial and mesenchymal phenotypes (Figure 7a). To test this hypothesis, we examined previously defined EMT markers in HOTAIR OE SNU-449 cells (40-42). First, we found that in contrast 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), expression of epithelial biomarker E-cadherin was upregulated (Figure 7d) whereas mesenchymal biomarker Vimentin expression was significantly down-regulated and showed polarity (Figure 7e). Consistent with epithelial phenotype, N-Cadherin expression was down-regulated and mostly located in the plasma membrane of HOTAIR OE cells (Figure 7f). Immunofluorescent analysis of cytoskeletal organization of F-actin and Vimentin in MET OE clones showed that ectopic MET expression and its suppressive effect on HOTAIR expression was also resulted in thickening of F-actin stress fibrils, and increase in Vimentin expression and polarity (Supplementary Figure 1d).
In addition to in-vitro experiments, we performed bioinformatic analysis of available data-sets GSE98091 and GSE109483. GSE98091 dataset has an integrated RNA sequencing transcriptomic and quantitative proteomic analysis those performed to explore the regulatory role of HOTAIR in HCC (43). Researchers inhibited HOTAIR expression by siRNA silencing in HepG2 cells and analyzed differentially expressed genes and proteins between control-siRNA and HOTAIR-siRNA HepG2 clones. Even though transcriptional dysregulation of MET was not significant in this study, protein expression of c-Met was significantly higher in HepG2 cells transfected with HOTAIR siRNA (Figure 8a). In GSE109483 dataset, researchers repressed HOTAIR expression in three Ewing sarcoma cell lines (ES2, A673, SK-ES) and overexpressed HOTAIR, alone in hTERT-immortalized human mesenchymal stem cells to evaluate its effects on gene expression (44). They performed RNA-Seq in Ewing sarcoma cell lines (control-GapmeR and HOTAIR repressing GapmeR clones), and hTERT-immortalized hMSCs with expression of control GFP and HOTAIR. MET gene expression was significantly upregulated by HOTAIR repression in ES2 and SK-ES Ewing Sarcoma cell lines and its expression was significantly repressed by HOTAIR over-expression in hTERT-immortalized human mesenchymal stem cells (Figure 8b). The data of these datasets supports proposed interaction of HOTAIR and MET and very important to show similar results in HCC and a different cancer context.
Finally, we analyzed TCGA datasets of various cancers to have a foresight about the possible relationship of two molecules (3). HOTAIR and MET gene transcripts in TCGA datasets were analyzed by GREIN and LncTarD (32, 33). Despite the limitations in RNAseq sample counts among TCGA datasets that comprise RNA sequencing of long non-coding RNAs, HOTAIR and c-Met were differentially expressed between normal and primary tumor tissues in various cancer types (Figure 8c-d). While MET expression was significantly suppressed in primary tumor tissues in breast invasive carcinoma (BRCA) and upregulated in kidney chromophobe (KIHC), HOTAIR expression was significantly higher and lower, respectively (Figure 8c-d). Analysis of the correlation of these two molecules among TCGA datasets showed that and they were reversely correlated with each other in mesothelioma (MESO) but positively correlated in kidney renal papillary cell carcinoma (KIRP), brain lower grade glioma (LGG), ovarian serous cystadenocarcinoma (OV) and testicular germ cell tumors (TGCT) with statistical significance. Even lack of significance, tendency of reverse regulation was significance in adrenocortical carcinoma (ACC), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), lymphoid neoplasm diffuse large b-cell lymphoma (DLBC), glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), kidney renal clear cell carcinoma (KIRC), acute myeloid leukemia (LAML), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD) and ulveal melanoma (UVM), (Figure 8e). Although lncRNA HOTAIR, one of the most studied long non-coding RNAs, is reported to be elevated in patients with metastasis and poor prognosis in many cancer types, analysis of HOTAIR in TCGA datasets revealed that HOTAIR has a context-dependent expression pattern which is not always upregulated through the progression of the disease (Figure 8d-e).