TGFβ-induced long non-coding RNA LINC00313 activates Wnt signalling and promotes cholangiocarcinoma progression

doi:10.21203/rs.3.rs-1967984/v1

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TGFβ-induced long non-coding RNA LINC00313 activates Wnt signalling and promotes cholangiocarcinoma progression

https://doi.org/10.21203/rs.3.rs-1967984/v1

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Background: Cholangiocarcinoma (CCA) is a devastating liver cancer characterized by high aggressiveness and resistance to therapy, which results to poor prognosis. Long non-coding RNAs (lncRNAs) and signals imposed by oncogenic pathways, such as transforming growth factor β (TGFβ) frequently contribute to CCA development. Here, we explored novel effectors of TGFβ signalling in CCA.

Methods: We performed gene expression profiling to identify TGFβ-regulated genes in CCA cell lines. RNA-sequencing and ATAC-sequencing after LINC00313 gain-of-function were used to identify transcriptional targets of LINC00313, in vitro. We evaluated the impact of LINC00313 on TCF/LEF signalling by luciferase assays. Mouse xenograft models were used to evaluate the effects of LINC00313 on CCA progression, in vivo. Integrative analysis revealed the clinical relevance of LINC00313 in CCA. We identified LINC00313-interacting proteins by RNA-pull down, followed by mass spectrometry.

Results: LINC00313 was identified as a novel target of TGFβ signalling in CCA cells. TGFβ induced LINC00313 expression in a TβRI/Smad-dependent manner. LINC00313 regulated genes involved in Wnt signalling. LINC00313 gain-of-function increased TCF7 expression. Of note, LINC00313 enhanced TCF/LEF-dependent transcriptional responses, promoted colony-forming capacities of CCA cells in vitro and accelerated tumour growth in vivo. Genes associated with LINC00313 over-expression in CCA tumours were characterized by KRAS and TP53 mutations and reduced patient’s overall survival. Mechanistically, actin-like 6A (ACTL6A), a subunit of the SWI/SNF chromatin remodelling complex, interacted with LINC00313 and impacted on TCF7 and SULF2 transcription and TCF/LEF signalling output.

Conclusions: We propose a model whereby TGFβ induces LINC00313 in order to regulate expression of a subset of target genes, possibly in co-operation with SWI/SNF. By regulating key genes of the Wnt pathway, LINC00313 fine-tunes Wnt/TCF/LEF-dependent transcriptional responses and boosts cholangiocarcinogenesis.

Cholangiocarcinoma

TGFβ signalling

lncRNA

LINC00313

Wnt pathway

chromatin remodelling

Cholangiocarcinoma (CCA) is an aggressive cancer from the biliary tree characterized by resistance to chemotherapies and poor prognosis. CCAs are categorized into intrahepatic (iCCA) and extrahepatic (eCCA), the later including perihilar (pCCA) and distal (dCCA) CCAs [1]. CCA subtypes exhibit high tumour cell heterogeneity, as well as specific genetic and epigenetic alterations, including frequent KRAS, TP53 and IDH1/2 mutation and FGFR2 gene fusions with enhanced tumourigenic activity [2]. Accordingly, signals imposed by oncogenic pathways, such as fibroblast growth factor (FGF), phosphatidylinositol-3-kinase (PI3K)-AKT and transforming growth factor β (TGFβ) frequently contribute to CCA development and/or progression [3].

TGFβ signals through type I and type II TGFβ receptors (TβRI and TβRII) that activate SMAD2, SMAD3 and SMAD4 which modulate gene expression [4]. TGFβ-responsive targets include protein-coding and non-coding genes, which participate in a wide range of processes, such as liver fibrosis, tumour progression and metastasis [5]. TGFβ exerts both anti- and pro-tumourigenic functions. In normal epithelium, TGFβ restricts tumour initiation by promoting cell cycle arrest and apoptosis. However, in advanced malignancies, cancer cells become unresponsive to its tumour-suppressive actions, but still obey to its tumour-promoting functions, such as epithelial-to-mesenchymal transition (EMT) [6]. In addition, TGFβ in the stroma facilitates cancer cell invasion and induces tumour immune evasion [7]. In CCA, SMAD4 is subjected to mutations, leading to gene inactivation. Loss of SMAD4 is capable of nullifying the tumour suppressive axis of TGFβ. In addition, TGFβ is able to induce EMT and favour a fibrotic tumour microenvironment, thereby promoting metastasis [8]. Notably, enhanced secretion of TGFβ2 in the stroma of iCCAs correlates with poor patient’s prognosis [9].

Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nucleotides that lack protein-coding capacities. Long intergenic non-coding RNAs (lincRNAs) do not overlap with protein-coding genes. They form autonomous transcriptional units under the control of their own promoters. Although they structurally resemble messenger RNAs (mRNAs), functional open reading frames (ORFs) are not inherent to lncRNAs, rendering them incapable of encoding polypeptides. However, lncRNAs play crucial roles in physiological and pathological processes, including cancer. They do so by regulating molecular processes, such as gene transcription, splicing and translation, via interactions with DNA, proteins, mRNAs or microRNAs (miRNAs) [10]. Several lncRNAs promote the proliferative, migratory and invasive properties of CCA cells [11]. Notably, lncRNAs with oncogenic functions, such as lncRNA-ATB [12], CCAT1 [13] and TLINC [14] are over-expressed in CCA. Here, we focus on the role and the clinical relevance of LINC00313 in CCA.

Cell culture

Primary cultures of normal human cholangiocytes (NHC) were established, characterized and cultured as previously described [15]. HuCCT1 and Huh28 CCA cell lines were from RIKEN BioResource Center (Tsukuba-shi, Japan) and CCLP1, Egi-1, SG231, TFK1, Sk-ChA-1 and Mz-ChA-1 were provided by Laura Fouassier (Paris, France). HepG2/C3A and HEK293T were from ATCC (www.lgcstandards-atcc.org). Serum-starved cells were stimulated with human recombinant TGFβ1 (R&D Systems) for 16h.

Mass spectrometry

The analysis was performed by the Clinical Proteomics Mass Spectrometry facility (Karolinska Institutet, Karolinska University Hospital, Science for Life Laboratory, Stockholm, Sweden), as previously described [16].

In vivo xenografts

HuCCT1 cells over-expressing empty vector or LINC00313 were implanted on the flanks of immunodeficient NSG male mice (Charles River, USA). All animal procedures met the European Community Directive guidelines (Agreement B35-238-40; DIR #7163) and were approved by the local ethics committee and ensuring the breeding and daily monitoring of animals in the best conditions of wellbeing according to the law and the 3R rule.

Statistical analysis

Comparisons were performed using a two-tailed paired Student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001). The results are shown as mean ± SD from 3 independent experiments.

Details on other materials and methods are provided in the supplementary materials.

LINC00313 is a novel TGFβ target in CCA

By gene expression profiling, we identified 103 non-redundant genes differentially expressed by TGFβ in both HuCCT1 and Huh28 CCA cell lines (Fig. 1A), including known (SERPINE1) and novel coding and non-coding TGFβ targets (Table S1). Notably, the long intergenic non-protein coding RNA 313 (LINC00313) was greatly induced by TGFβ in HuCCT1, Huh28 and NHC (Fig. 1A, B). LINC00313 was up-regulated by TGFβ in a dose-dependent manner (Fig. 1C) and appeared to be an intermediate-to-late and early responsive gene in HuCCT1 and Huh28 cells, respectively (Fig. 1D). Using exon-specific primers, we further confirmed the induction of LINC00313 by TGFβ in CCA cells (Fig. S1A). LINC00313 was also highly expressed in hepatocellular carcinoma (HCC) but TGFβ had no impact on its expression in HepG2 and Hep3B HCC cell lines (Fig. S1B). Finally, subcellular fractionation demonstrated that LINC00313 was mainly a nuclear lncRNA, similar to MALAT1 and SNORD48 (Fig. 1E).

TGFβ-induced LINC00313 expression requires TβRI/Smad- and p38-dependent pathways

TGFβ signals through Smad-dependent (canonical) or Smad-independent (non-canonical) pathways [4]. TβRI inhibitor LY2157299 abolished TGFβ-induced LINC00313 expression in TGFβ-responsive cells (Fig. 2A, S1A, S2A). Similarly, blocking SMAD3 activation using SIS3 inhibitor completely abolished the TGFβ-mediated up-regulation of LINC00313 in HuCCT1 and Huh28 cells (Fig. 2B). In HuCCT1, SMAD4 silencing reduced baseline and TGFβ-induced LINC00313 expression similar to the combined SMAD2/3/4 silencing (Fig. 2C, S2B). In Huh28, SMAD2 silencing also prevented TGFβ-induced LINC00313 expression (Fig. 2D, S2B).

As regard to the non-canonical TGFβ pathway, p38 inhibition decreased, while MEK inhibition increased LINC00313 expression. Inhibition of c-Jun N-terminal kinase (JNK) induced LINC00313 in the presence of TGFβ in HuCCT1 cells (Fig. 2E). The efficiency of MAPKs inhibition was verified (Fig. S3A). The enhanced LINC00313 expression upon MEK inhibition correlated with an enhanced TGFβ/SMAD response, as revealed by a SMAD-binding element (SBE) reporter assay (Fig. S3B). In Huh28 cells, LINC00313 remained unchanged after blocking MAPKs (Fig. 2E). Thus, both SMAD3 and SMAD4 and the kinase activity of p38 are required for the TGFβ-mediated up-regulation of LINC00313.

LINC00313 modulates expression of genes involved in Wnt pathway

Based on its nuclear localization, we hypothesized that LINC00313 acts at the transcriptional level to regulate gene expression (Fig. 3A). Gain-of-function tools were developed in HuCCT1 cells. An efficient LINC00313 over-expression and a nuclear abundance similarly to parental HuCCT1 cells was confirmed (Fig. S4A). RNA-seq analysis identified 334 up- and 316 down-regulated genes after LINC00313 gain-of-function (Fig. 3B, S4B, Table S2). Up-regulated genes were related to signalling pathways regulating pluripotency of stem cells (e.g. WNT5A, TCF7, AXIN2, FZD2 and ID1) (Fig. 3C). Gene Set Enrichment Analysis (GSEA) further highlighted signatures of Hippo, Wnt and TGFβ pathways in the gene expression profile of LINC00313 overexpressing cells (Fig. 3D, S4C). Induction of WNT5A, AXIN2 and SULF2 following LINC00313 gain-of-function was also validated (Fig. 3E, S4D).

Next, gene expression profiling after LINC00313 silencing was performed in the presence or absence of TGFβ (Fig. S5A, S5B and Table S3). GO analysis confirmed that TGFβ-induced genes participated in cell migration, extracellular matrix organization and cell differentiation (Fig. S5C). KEGG pathway analysis revealed that many genes repressed after LINC00313 silencing are associated with cancer signalling pathways regulating pluripotency of stem cells, including the Wnt signalling (Fig. S5D). In agreement with LINC00313 gain-of-function experiments, WNT5A, AXIN2 and SULF2 expression levels were decreased upon LINC00313 silencing in HuCCT1 cells (Fig. 3F). Expression of TCF7, SULF2 and WNT5A was also diminished in LINC00313-silenced Huh28 and TFK1 cells (Fig. S6A). These observations were confirmed in Huh28 using another siRNA in order to exclude possible siRNA off-target effects (Fig. S6B).

Nuclear LINC00313 alters chromatin accessibility at genomic loci of Wnt-related genes

ATAC-seq identified genome-wide alterations in chromatin accessibility, including 23,628 unique peaks in LINC00313 over-expressing cells (pcLINC00313) (Fig. 4A, S7A, S7B). No global alteration of the location of peaks, relative to genomic annotations was observed (Fig. S7C). Differential region analysis revealed 1657 genomic regions with increased accessibility and 2090 regions with decreased accessibility in pcLINC00313 cells (Fig. 4B and Table S4). HOMER motif enrichment analysis highlighted Fos-related antigen 1 (Fra1), an AP-1 transcription factor subunit, as the most significant enriched motif (Fig. S7D).

We then integrated ATAC-seq and RNA-seq data and pinpointed the common genes. We found 44 genes that show both altered chromatin accessibility and altered expression after LINC00313 over-expression (Fig. 4C). Interestingly, these genes were associated with the Wnt signalling pathway (Fig. 4D), known to play a key role in CCA progression [17]. Thus, we decided to investigate in deep the regulation of selected genes of the pathway, such as TCF7. Notably, we observed increased peak signal around the transcription start site and in the gene body of TCF7 locus (Fig. 4E), as well as elevated TCF7 mRNA and protein levels in LINC00313 over-expressing cells (Fig. 4F). Interestingly, TGFβ induced TCF7 expression whereas silencing LINC00313 reduced TCF7 mRNA and protein levels (Fig. 4G) in both control and TGF-β stimulated cells, suggesting that LINC0313 may promote chromatin opening and enhanced transcriptional activity at the TCF7 locus.

LINC00313 potentiates TCF/LEF transcriptional responses

Then, we investigated whether LINC00313 could modulate Wnt/β-catenin-dependent transcription. We first validated luciferase reporter assays in HuCCT1 cells treated with CHIR99021 (CHIR), a glycogen synthase kinase 3 inhibitor, which activates the Wnt signalling. In HuCCT1 cells stably expressing a TCF/LEF reporter, CHIR increased reporter activity but TGFβ had no impact (Fig. S8A). Silencing LINC00313 repressed CHIR-induced TCF/LEF reporter activity (Fig. 5A) and the associated target genes (Fig. 5B). LINC00313 over-expression further enhanced the baseline and CHIR-induced TCF/LEF-luciferase activity (Fig. 5C). Administration of CHIR increased the viability of control HuCCT1 cells, an effect that was more pronounced in cells over-expressing LINC00313 (Fig. S8B). Moreover, CHIR boosted colony formation of HuCCT1 control cells and to a larger magnitude of LINC00313 over-expressing cells (Fig. S8C).

XAV939, a Wnt pathway inhibitor, did not influence TCF/LEF responses in control cells, but reduced it in pcLINC00313 cells, which exhibit enhanced Wnt activity (Fig. 5D). Interestingly, XAV939 reduced colony formation (Fig. S8C). We confirmed the efficiency of the inhibitors by measuring AXIN2 expression, as a typical Wnt-target gene. Indeed, CHIR strongly induced AXIN2, an effect that was enhanced in LINC00313 over-expressing cells, whereas XAV939 modestly reduced AXIN2 (Fig. S8D). Also, CHIR potentiated TCF7 but not LINC00313, while XAV939 had no effect on their expression (Fig. S8D). β-catenin nuclear import is the major event that drives Wnt transcriptional responses. LINC00313 over-expression did not change the subcellular localization of β-catenin (Fig. S8E). Collectively, the data support a model whereby LINC00313 acts as a positive regulator of TCF/LEF-mediated signalling, but is dispensable for the initial steps of Wnt activation.

LINC00313 promotes CCA colony-forming capacities in vitro and tumour growth in vivo

Wnt signalling regulates cancer stem cell maintenance, cancer cell proliferation and migration [18]. In this context, we showed that LINC00313 boosted the ability of single cells to form colonies in vitro (Fig. 5E). Moreover, LINC00313 over-expression neutralized the TGFβ-mediated decrease in HuCCT1 cell viability (Fig. S9A). However, LINC00313 did not influence cell migration (Fig. S9B).

Interestingly, LINC00313 accelerated tumour growth in vivo when cells were xenografted in nude mice (Fig. 5F, S10A). Expression analysis revealed a positive correlation between LINC00313 and TCF7, SULF2 and AXIN2 mRNA levels in resected tumours (Fig. 5G). Moreover, we measured increased SULF2 and AXIN2 expression in LINC00313 xenograft tumours, compared to control (Fig. 5H). Although TCF7 mRNA levels did not change (Fig. 5H), we observed increased TCF7 protein expression in LINC00313 tumours (Fig. S10B). LINC00313 had no impact on the development of spontaneous metastases in mice (Fig. S10C) in agreement with the absence of effects on cell migration in vitro.

The SWI/SNF complex subunit ACTL6A is an interactor of LINC00313 lncRNA

RNA pull-down assay followed by mass spectrometry identified LINC00313 partners possibly involved in chromatin remodelling. In total, 1538 proteins were identified to interact with LINC00313 and 1840 proteins with the negative control firefly luciferase (f-luc) mRNA (Fig. 6A, Table S5). From the above, 121 proteins were specifically bound to LINC00313 (Fig. 6B). In order to single out interesting candidates, we set three sorting layers (Fig. 6C). First, we focused on nuclear proteins (51/121), considering the nuclear localisation of LINC00313. Protein network analysis revealed multiple physical and functional associations and clustered them in four groups. The largest cluster contained proteins involved in cell cycle regulation and the rest members of the transcription factor TFIID complex, the integrator complex and proteins with RNA helicase activity (Fig. 6D). Second, we predicted the binding of LINC00313 to 51 nuclear proteins using catRAPID [19]. Thirty two proteins were predicted to interact with LINC00313 lncRNA (interaction propensity ≥ 75, discriminative power ≥ 98%) (Table S5). Third, among the 32 proteins we searched for transcription factors or chromatin modifiers (Fig. 6E), including actin-like 6A (ACTL6A) (Fig. 6F), a subunit of the SWI/SNF chromatin remodeling complex [20]. Given that LINC00313 modulates chromatin state of certain loci, we investigated the physical and functional association between LINC00313 and ACTL6A deeper. Initially, we verified the nuclear localization of ACTL6A. TGFβ stimulation did not alter the nuclear abundance of ACTL6A (Fig. 6G). A specific interaction between LINC00313 lncRNA and ACTL6A was demonstrated in HuCCT1, especially in the presence of TGFβ (Fig. 6H). Similarly, in HA-tagged ACTL6A over-expressing HEK293T cells, an even stronger interaction between LINC00313 and HA-ACTL6A was observed (Fig. 6I). We also confirmed this interaction by RNA immunoprecipitation (RIP) assays. Interestingly, TGFβ stimulation increased enrichment of endogenous LINC00313 to immunoprecipitated ACTL6A (Fig. 6J). Overall, LINC00313 forms ribonucleoprotein complexes with several nuclear proteins that belong to transcriptional regulatory networks.

ACTL6A silencing or pharmacological inhibition of SWI/SNF complex diminishes TCF/LEF-mediated gene expression

Next, we evaluated the effects of ACTL6A on TCF/LEF-dependent transcription. Silencing ACTL6A attenuated TCF7 mRNA and protein levels but did not affect TGFβ-induced LINC00313 (Fig. 7A). ACTL6A silencing also resulted in decreased TCF/LEF luciferase activity and a drop in TCF7 and AXIN2 mRNA levels (Fig. 7B). Rescue experiments demonstrated that ACTL6A silencing dampened the LINC00313-mediated up-regulation of SULF2 mRNA (Fig. 7C) and TCF7 mRNA and protein levels (Fig. 7D).

Since ACTL6A is an accessory subunit of SWI/SNF, we hypothesized that blocking the catalytic activity of the complex may yield effects similar to ACTL6A silencing. Thus, we utilized two inhibitors, targeting different domains of the core catalytic subunits SMARCA2/SMARCA4. Notably, the bromodomain inhibitor PFI-3 did not affect SULF2 or TCF7 mRNA expression. In contrast, administration of BRM/BRG1 ATPase inhibitor significantly reduced SULF2 mRNA (Fig. 7E) and TCF7 expression both at the mRNA and protein levels (Fig. 7F), implying that these two genes are targets of SWI/SNF. Consistent with the effects on the individual genes, BRM/BRG1 ATPi, but not PFI-3 treatment resulted in a modest, but significant decrease of CHIR-induced TCF/LEF-luciferase reporter activity (Fig. 7G). Overall, we suggest a mechanism, whereby increased LINC00313 levels promote chromatin accessibility in an ACTL6A/SWI/SNF-dependent manner and facilitate transcription of TCF7 and SULF2, resulting in enhanced Wnt activation (Fig. 7H).

LINC00313 signature predicts poor prognosis in patients with CCA

From the TCGA dataset, LINC00313 was neither overexpressed in CCA tissues nor correlated with overall or disease-free survival in patients with CCA (Fig. S11A). On the other hand, the expression levels of ACTL6A, TCF7, WNT5A, AXIN2 and SULF2 were all increased in CCA human tumours (Fig. S11B). Interestingly, LINC00313 was significantly up-regulated in pancreatic ductal adenocarcinoma and correlated with lower disease-free survival (Fig. S11C).

Next, we hypothesized that if LINC00313 expression may not be a prognostic factor in CCA, its activity could better reflect its clinical relevance. Thus, we performed RNA-seq analysis in control and LINC00313 xenograft tumours to establish a signature reflecting LINC00313 activity. In total, 347 genes were up-regulated and 327 genes were down-regulated in LINC00313-overexpressing tumours (Fig. 8A). Then, we integrated this signature with the gene expression profiles of 255 cases of clinically annotated human CCA. Integrative transcriptomics using principal component analysis (PCA) showed that the four control samples clustered together and were distinct to the four LINC00313 samples (Fig. 8B). Dimension 2 of the PCA was related to LINC00313 activity and identified two main clusters of human CCA (Fig. 8B). Interestingly, the cluster associated with LINC00313 activity correlated with lower overall survival (Fig. 8C). Furthermore, IDH1 and IDH2 mutations were more prevalent in control cluster, while KRAS and TP53 mutations were significantly enriched in LINC00313 cluster (Fig. 8D). We also observed increased levels of carbohydrate antigen 19 − 9 and γ-glutamyltransferase and decreased levels of albumin, markers of hepatobiliary disease and of unfavourable prognosis in CCA, in the LINC00313 cluster (Fig. 8E). In addition, the LINC00313 cluster was characterized by significantly higher perineural invasion and regional lymph node metastasis (Fig. 8E). Collectively, we conclude that although LINC00313 expression per se is not a prognostic factor in CCA, the in vivo gene signature that reflects its activity exhibits a strong prognostic value in terms of survival outcome.

By exploring the TGFβ-regulated transcriptome in CCA cell lines, we identified LINC00313 as a TGFβ-responsive gene, a finding that expands the growing list of TGFβ-regulated lncRNAs [5]. An interesting observation is that TGFβ is able to induce LINC00313 only in a subset of CCA cell lines, as well as in normal cholangiocytes. A possible explanation is that some CCA cell lines are not responsive to TGFβ stimulation. Since most of the CCA cell lines have not, yet, been fully characterized, concerning the responsiveness to TGFβ, a deeper investigation on TGFβ family members is required. On the other hand, TGFβ did not affect LINC00313 expression in HepG2 and Hep3B, two HCC cell lines known to respond to TGFβ signalling, suggesting that TGFβ may selectively regulate this gene in certain cancer cells.

LINC00313 is a lincRNA that possesses oncogenic properties and is a marker of poor prognosis in papillary thyroid cancer [21, 22]. Also, a pro-tumorigenic role of LINC00313 has been described in osteosarcoma [23] and cervical carcinoma [24]. Moreover, LINC00313 is up-regulated in glioma and promotes tumorigenesis, via enhancing cell proliferation, migration and invasion [25]. The functional role of LINC00313 in CCA remained unknown but our data support a pro-oncogenic role. We demonstrated that LINC00313 favours CCA tumour growth in vivo and colony formation in vitro. Although we did not observe up-regulation of LINC00313 in human CCA, we showed that in vivo gene signature associated with LINC00313 gain-of-function correlated with poor overall survival. We speculate that LINC00313 activity, rather than its expression, is of paramount importance for the stratification of CCA patients based on survival outcomes.

Integration of ATAC-seq and RNA-seq data allowed us to select genes involved in Wnt signalling. The Wnt pathway plays crucial roles in CCA progression and is often activated in CCA tissues. CCA tumours are usually characterized by enhanced expression of several Wnt ligands and increased nuclear accumulation of β-catenin [26]. We focused on TCF7 because it encodes one of the main transcription factors of Wnt/β-catenin pathway [27]. Furthermore, TCF7 reinforces CCA progression by positively regulating MYC and FOSL1 [28] and induces CCA proliferation and drug resistance, via regulating SOX9/FGF7/FGFR2 axis [29]. SULF2 is the second interesting transcriptional target of LINC00313. It encodes a heparan sulfate-editing enzyme that activates Wnt signalling, by facilitating the bioavailability of Wnt ligands to their receptors [30]. SULF2 is up-regulated in human CCA and is associated with enhanced PDGFRβ-YAP signalling, tumour progression and chemoresistance. Importantly, targeting SULF2 protein with a monoclonal antibody abolishes tumour growth in a mouse CCA xenograft model [31]. We also identified additional genes induced by LINC00313, such as ID1 and ID3, which inhibit cell differentiation. Thus, we suggest that LINC00313 positively regulates genes related to cell proliferation and stemness that could explain its effects on colony formation and CCA xenograft tumour growth.

Our proteomic analysis revealed numerous nuclear proteins that associate with LINC00313. We focused on ACTL6A, which is frequently over-expressed in cancer. For instance, ACTL6A is up-regulated in HCC and promotes migration and invasion in vitro, as well as tumour growth and metastasis in vivo, via activating Notch signalling [32]. The role of ACTL6A in CCA is still unknown. However, the SWI/SNF catalytic subunit BRG1 is over-expressed and predicts a poor prognosis in iCCA. More importantly, it has been demonstrated that BRG1 activates the Wnt pathway through transcriptional regulation of Wnt receptor and target genes, but also via binding to β-catenin/TCF4 transcription complex, in HuCCT1 [33]. These observations are in line with our data indicating decreased activation of Wnt transcriptional responses upon ACTL6A silencing or BRG1 inhibition.

At the molecular level, TGFβ promotes ribonucleoprotein complexes between LINC00313 and ACTL6A without changing ACTL6A expression. This finding opens up the possibility that TGFβ may engage LINC00313 to bring the chromatin remodelling machinery, in close proximity to Smads, at the regulatory regions of selected targets. Although ACTL6A is not a classical RNA-binding protein, it was previously reported to interact with the lncRNA uc.291 to regulate epidermal differentiation [34].

Several lncRNAs establish interactions with SWI/SNF subunits. For example, SWINGN lncRNA binds to SMARCB1 and facilitates transcription of target genes [35]. In HCC cells, lncTCF7 interacts with BRG1, BAF170 and SNF5 subunits and recruits SWI/SNF to TCF7 promoter, thereby activating TCF7 transcription in cis [36]. Our mechanistic evidence propose a model, whereby LINC00313 acts in trans to regulate TCF7 and SULF2 transcription, through binding ACTL6A and possibly via increased loading of SWI/SNF to TCF7 and SULF2 loci, setting an additional layer of regulation of TCF/LEF signalling. Undoubtedly, additional modes of action for LINC00313 cannot be excluded. For example, nuclear lncRNAs frequently form triplex formation with DNA at chromatin regions, through direct base pairing [37]. It is worth to investigate whether LINC00313 participates in these loops, in order to alter chromatin accessibility.

Overall, our study highlights an important and new role for the TGFβ-induced LINC00313 as a modulator of Wnt/TCF signalling, that affects tumour progression and with clinical implications in CCA.

ACTL6A, actin-like 6A; CCA, cholangiocarcinoma; CHIR, CHIR99021; dCCA, distal cholangiocarcinoma; eCCA, extrahepatic cholangiocarcinoma; EMT, epithelial to mesenchymal transition; FGF, fibroblast growth factor; GSEA, Gene Set Enrichment Analysis; HCC, hepatocellular carcinoma; iCCA, intrahepatic cholangiocarcinoma; LINC00313, long intergenic non-protein coding RNA 313; lincRNAs, long intergenic non-coding RNAs; lncRNAs, long non-coding RNAs; miRNAs, microRNAs; mRNAs, messenger RNAs; NHC, normal human cholangiocytes; ORFs, open reading frames; PCA, principal component analysis; pCCA, perihilar cholangiocarcinoma; PI3K, phosphatidylinositol-3-kinase; RIP, RNA immunoprecipitation; SBEs, SMAD-binding elements; TGFβ, transforming growth factor β; TβRI, type I TGFβ receptor; TβRII, type II TGFβ receptor

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

In vivo and in vitro RNA-seq data, as well as microarray and ATAC-seq data are available at ArrayExpress under the accession number E-MTAB-12030. The ATAC-seq peaks can be visualized in the UCSC Genome Browser following the URL: https://genome.ucsc.edu/s/ppapouts/ATAC%2Dseq%20HuCCT1%20LINC00313%20overexpression.

Competing interests

The authors declare that they have no competing interests.

Funding

The authors are supported by Inserm, Université de Rennes 1, Région Bretagne, Ministère de l’Enseignement Supérieur de la Recherche et de l’Innovation, Ligue Nationale Contre le Cancer, Ligue Contre le Cancer (CD22, CD35, CD44, CD85), Fondation ARC, INCa and ITMO Cancer AVIESAN (Alliance Nationale pour les Sciences de la Vie et de la Santé) dans le cadre du Plan cancer (Non-coding RNA in cancerology: fundamental to translational). This work was supported by a grant from the French Ministry of Health and the French National Cancer Institute, PRT-K20-136, CHU Rennes, CLCC Eugène Marquis, Rennes. JMB is supported by Spanish Carlos III Health Institute (ISCIII) [(FIS PI18/01075, PI21/00922, and Miguel Servet Programme CPII19/00008) cofinanced by “Fondo Europeo de Desarrollo Regional” (FEDER)] and CIBERehd (ISCIII); La Caixa Scientific Foundation (HR17-00601); AMMF-The Cholangiocarcinoma Charity (EU/2019/AMMFt/001); PSC Partners US and PSC Supports UK (06119JB); European Union’s Horizon 2020 Research and Innovation Program [grant number 825510, ESCALON].

Authors’ contributions

CC conceived the project, provided funding and supervision. PP and CC designed the experiments. PP, CL, and RP acquired the data. PP, CL, RP, ALH and MA analysed the data. PP, CL, RP, ALH, MA and CC interpreted the data. JMB and DG contributed resources and editing. PP drafted the article. All authors critically revised the article.

Acknowledgements

We thank Dr Yutaro Tsubakihara and Prof. Aristidis Moustakas (Uppsala University, Sweden) for providing the pcDNA3.1/Hygro (+) and the pGL4.74 TK-Renilla luciferase reporter expression vectors. We thank Dr Laura Fouassier for providing CCA cell lines and for helpful discussion. Gaëlle Angenard, Raffaële Leroux, Léa Chailloux and Matthis Desoteux are acknowledged for their technical help.

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No competing interests reported.

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TGFβ-induced long non-coding RNA LINC00313 activates Wnt signalling and promotes cholangiocarcinoma progression

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ACTL6A silencing or pharmacological inhibition of SWI/SNF complex diminishes TCF/LEF-mediated gene expression

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