The vast majority of breast cancer-associated deaths are due to metastatic spread of cancer cells, a process aided by epithelial-to-mesenchymal transition (EMT). Mounting evidence has indicated that long non-coding RNAs (lncRNAs) also contribute to tumor progression. We report the identification of 114 novel lncRNAs that change their expression during TGFβ-induced EMT in murine breast cancer cells (referred to as EMT-associated transcripts; ETs). Of these, the ET-20 gene localizes in antisense orientation within the tenascin C (Tnc) gene locus. TNC is an extracellular matrix protein that is critical for EMT and metastasis formation. Both ET-20 and Tnc are regulated by the EMT master transcription factor Sox4. Notably, ablation of ET-20 lncRNA effectively blocks Tnc expression and with it EMT. Mechanistically, ET-20 interacts with desmosomal proteins, thereby impairing epithelial desmosomes and promoting EMT. A short transcript variant of ET-20 is shown to be upregulated in invasive human breast cancer cell lines, where it also promotes EMT. Targeting ET-20 appears to be a therapeutically attractive lead to restrain EMT and breast cancer metastasis in addition to its potential utility as a biomarker for invasive breast cancer.

While localized primary breast cancer often has a good prognosis and can be treated effectively, 90% of cancer-related deaths are due to the systemic dissemination of cancer cells and the outgrowth of metastases in distant organs (Chaffer and Weinberg, 2011). To metastasize, however, epithelial cells must unzip their epithelial adhesion complexes, including tight junctions, adherens junctions and desmosomes, and pass through a multistage invasion-metastasis cascade, a process believed to be facilitated by an epithelial-to-mesenchymal transition (EMT) (Dongre and Weinberg, 2019; Huang et al., 2012; Lamouille et al., 2014; Lu and Kang, 2019; Yang et al., 2020). EMT is an evolutionarily conserved, reversible and dynamic cellular process, during which epithelial cells dramatically reprogram their gene expression and undergo morphogenic changes to lose their epithelial characteristics and gain mesenchymal, migratory and invasive capabilities (Nieto et al., 2016; Thiery et al., 2009). EMT has been implicated in several processes associated with the malignant progression of cancer cells by bestowing them with the ability to resist cell death, immune surveillance and chemotherapy; they thereby become competent to disseminate throughout the body and seed deadly metastases (Aiello et al., 2017; Brabletz et al., 2018; Fischer et al., 2015; Krebs et al., 2017; Tiwari et al., 2012; Ye et al., 2017; Zheng et al., 2015). Importantly, EMT is a highly plastic process and, during cancer progression, it can be activated to various degrees resulting in a continuum of EMT hybrid states that might rapidly interconvert between the epithelial and mesenchymal extremes (Yang et al., 2020).

In response to inductive stimuli, EMT is brought about by specific transcription factors (TFs), including Twist1, Snail1 and Snail2 (Snai1 and Snai2), and Zeb1 and Zeb2, which act as transcriptional repressors of epithelial genes, such as the prototype epithelial adherens junctions protein E-cadherin, and of desmosomal components, such as plakophilin and desmoplakin (Aigner et al., 2007; Lu and Kang, 2019; Savagner et al., 2005; Vandewalle et al., 2005). Accompanying the loss of apical-basal polarity and epithelial markers, the cells undergoing EMT reorganize their actin cytoskeleton to gain a front-rear polarity and start expressing mesenchymal markers, such as vimentin, fibronectin, N-cadherin and Ncam1, and also the extracellular matrix (ECM) glycoprotein tenascin C (TNC). Interestingly, TNC has also been shown to actively regulate EMT in different cancers by binding to αvβ6, αvβ1 or α9β1 integrins (Katoh et al., 2013; Lowy and Oskarsson, 2015; Nagaharu et al., 2011; Sun et al., 2018; Takahashi et al., 2013) or by activating JNK/c-Jun signaling (Cai et al., 2017). Furthermore, in addition to creating a fertile soil in the pre-metastatic niche, TNC also enables survival and proliferation of metastasis initiating cells (Brellier and Chiquet-Ehrismann, 2012; Oskarsson et al., 2011). In fact, strong expression of TNC at the tumor invasive front is linked to higher risk of distant metastasis and local recurrence in breast cancer and to poor clinical outcome in melanoma and in lung and liver cancer (Aishima et al., 2003; Jahkola et al., 1998a, 1996; Kaarteenaho-Wiik et al., 2003; Lowy and Oskarsson, 2015). The TF Sox4 has been identified as a direct inducer of Tnc gene expression and a master regulator of EMT, where it acts near the top of the epistatic hierarchy of the EMT interactome (Meyer-Schaller et al., 2019; Tiwari et al., 2013).

Whereas previous studies have shown the involvement of microRNAs (miRNAs) in regulating EMT, only a few studies have reported a role for long non-coding RNAs (lncRNAs) in the EMT process (De Craene and Berx, 2013; Dongre and Weinberg, 2019). lncRNAs have recently been shown to add a new layer of regulation in processes spanning normal physiology to pathological states, including cancer (Fatica and Bozzoni, 2014; Morris and Mattick, 2014; Rinn and Chang, 2012; Schmitt and Chang, 2016). They are transcripts ranging from 200 nt to over 100 kb in length with no obvious protein-coding potential (Quinn and Chang, 2016; Yao et al., 2019). They are mainly transcribed by RNA Pol II and can be m7G-capped at the 5′ end and polyadenylated at the 3′ end. Unlike mRNA, however, lncRNAs are poorly conserved from an evolutionary perspective (Iyer et al., 2015; Ulitsky, 2016). They can either function in cis by affecting the expression and/or chromatin state of nearby genes or in trans by operating at distant genomic or cellular locations (Gil and Ulitsky, 2020; Kopp and Mendell, 2018). Furthermore, lncRNA can mediate their functions by binding DNA, RNA or proteins (Kopp and Mendell, 2018). Hence, lncRNAs are essential epigenetic, transcriptional, post-transcriptional and post-translational regulators that control a variety of cellular processes in a spatial, temporal and cell context-dependent manner (Evans et al., 2016; Mercer et al., 2009). Indeed, lncRNAs have also been reported to exert critical functions during EMT. For instance, the lncRNA ATB is upregulated in response to TGFβ in hepatocellular carcinoma (HCC) cells, and facilitates EMT, cellular invasion and organ colonization by interacting with miR-200 and IL-11 (Yuan et al., 2014). The lncRNA PNUTS is highly expressed in breast tumor cells, where it affects EMT by neutralizing miR-205 (Grelet et al., 2017). H19 has been shown to mediate EMT by differentially binding miR-200b and c, Let7, Snail2 and Ezh2 (Kallen et al., 2013). Finally, HIT has been identified to regulate E-cadherin expression, EMT, cell migration and invasion of murine breast cancer cells (Richards et al., 2015).

Here, we report the identification and functional characterization of the novel lncRNA EMT-associated transcript 20 (ET-20) in EMT of murine and human breast cancer cells. The ET-20 gene is transcribed in antisense to and co-regulated with the Tnc gene by the Sox4 EMT master transcription factor. ET-20 binds to desmosomal proteins at the cell membrane, where it impairs desmosomal junctions and thus promotes EMT.

Identification and characterization of the novel lncRNA ET-20

To identify novel lncRNAs that could play a functionally important role in the regulation of EMT, we treated normal murine mammary gland (NMuMG) cells with TGFβ to induce EMT and performed RNA sequencing over a detailed time course starting from 2 h and lasting until 10 days (Fig. S1Ai,ii). The analysis of the RNA sequencing data revealed the differential regulation of 4289 non-coding transcripts throughout the EMT time course, belonging to lncRNA, lincRNA, miscRNA, snRNA, snoRNA and processed transcript biotypes. K-means clustering revealed nine clusters of which two were unregulated. Removal of the unregulated clusters in addition to transcripts belonging to classes of snoRNA, snRNA and miRNA yielded 224 lncRNA transcripts. Since many studies have demonstrated cis-regulating functions of lncRNAs (Gil and Ulitsky, 2020; Kopp and Mendell, 2018), we focused on transcripts whose genes lay close to genes that were known to play a part in processes associated with EMT, such as cell adhesion, migration and proliferation, and whose expression had also changed during the EMT time course. Such curation reduced the list to 149 EMT-regulated potential lncRNA transcripts. Finally, removal of transcripts with a residual coding potential, as assessed by using the CPC and the CPAT coding potential calculator tools (Kong et al., 2007; http://lilab.research.bcm.edu/cpat/), of transcripts with an inconsistent expression pattern during the EMT time course, or of transcripts withdrawn from Ensemble yielded a final list of 114 lncRNAs which we named ‘EMT-associated transcripts’ (ET-1, 2, 3 etc.) (Fig. 1A; Table S1). Of these, 75 transcripts were upregulated and 39 were downregulated in their expression during an EMT (Fig. 1A). Of the 114 regulated transcripts, only seven lncRNA transcripts had previously been functionally annotated, namely mir155 (ET-15), Has2as (ET-19), Igf2as (ET-75), H19 (ET-95), Rian (ET-92), Dio3os (ET-104) and Dleu (ET-123) (Table S1).

Fig. 1.

Identification and characterization of the novel lncRNA ET-20 during TGFβ-mediated EMT. (A) Heatmap representing the expression of 114 novel lncRNA of which 75 were found to be upregulated (in red) and 39 downregulated (in blue) during a time course of TGFβ-induced EMT of NMuMG cells. ET-20 is highlighted with an arrow and red text label. FC, fold change. (B) The scheme depicts the genomic locus of ET-20 with respect to the Tnc gene on chromosome 4 in mice. The ET-20 gene (Ensembl gene ID: ENSMUSG00000073821) has four transcript variants that we named ET-20 long (ET-20l; 2618 bp; transcript ID: ENSMUST00000141428.7), ET-20 medium (ET-20m; 969 bp; transcript ID: ENSMUST00000135042.1), ET-20 short (ET-20s; 671 bp; ENSMUST00000138411.1) and ET-20 non-specific (ET-20ns; 393 bp; ENSMUST00000166003.1). Gm11216 is ET-58 in our dataset (refer to Table S1). The scheme depicted here is based on genome annotations available on Ensembl and UCSC genome browser. (C) The graph represents log2 fold changes in expression of ET-20 and tenascin C (Tnc) over 10 days of TGFβ-induced EMT of NMuMG cells normalized to the values in untreated samples at 0 h. The values were obtained from two biological replicates of the RNA sequencing data. (D) NMuMG (i) and PyMT-1099 (ii) cells were treated or not with TGFβ for 4 days followed by RNA isolation and analysis by quantitative RT-PCR for ET-20l, ET-20s and Tnc expression. Open white bars depict untreated cells and solid black bars represent TGFβ-treated cells. Data are represented as mean±s.e.m., n=3. **P<0.01; ***P<0.001; ****P<0.0001 (paired two-tailed t-test). (E) PyMT-1099 cells were treated with TGFβ for more than 20 days followed by single-molecule RNA-FISH analysis for ET-20l and ET-20s using specific fluorophore-tagged probes. Nuclei were counterstained with DAPI (blue). ET-20l and ET-20s were detected as bright red spots, and white arrows indicate the cellular localization of the lncRNAs. Representative of n=3. Scale bar: 10 µm. (F) Total RNA was isolated from either normal mammary glands (NMG) of FVB/N mice or mammary gland tumors formed in MMTV-PyMT (PyMT) transgenic mice in FVB/N genetic background mice and subjected to RT-qPCR analysis for ET-20s expression. ΔCt values in the NMG were averaged and normalized to 1. Fold-change in expression was calculated over NMG. Open white bars depict untreated cells and solid black bars represent TGFβ-treated cells. Data are represented as mean±s.e.m., n=6 for NMG and n=5 for PyMT tumors. ****P<0.0001 (non-parametric t-test).

Fig. 1.

Identification and characterization of the novel lncRNA ET-20 during TGFβ-mediated EMT. (A) Heatmap representing the expression of 114 novel lncRNA of which 75 were found to be upregulated (in red) and 39 downregulated (in blue) during a time course of TGFβ-induced EMT of NMuMG cells. ET-20 is highlighted with an arrow and red text label. FC, fold change. (B) The scheme depicts the genomic locus of ET-20 with respect to the Tnc gene on chromosome 4 in mice. The ET-20 gene (Ensembl gene ID: ENSMUSG00000073821) has four transcript variants that we named ET-20 long (ET-20l; 2618 bp; transcript ID: ENSMUST00000141428.7), ET-20 medium (ET-20m; 969 bp; transcript ID: ENSMUST00000135042.1), ET-20 short (ET-20s; 671 bp; ENSMUST00000138411.1) and ET-20 non-specific (ET-20ns; 393 bp; ENSMUST00000166003.1). Gm11216 is ET-58 in our dataset (refer to Table S1). The scheme depicted here is based on genome annotations available on Ensembl and UCSC genome browser. (C) The graph represents log2 fold changes in expression of ET-20 and tenascin C (Tnc) over 10 days of TGFβ-induced EMT of NMuMG cells normalized to the values in untreated samples at 0 h. The values were obtained from two biological replicates of the RNA sequencing data. (D) NMuMG (i) and PyMT-1099 (ii) cells were treated or not with TGFβ for 4 days followed by RNA isolation and analysis by quantitative RT-PCR for ET-20l, ET-20s and Tnc expression. Open white bars depict untreated cells and solid black bars represent TGFβ-treated cells. Data are represented as mean±s.e.m., n=3. **P<0.01; ***P<0.001; ****P<0.0001 (paired two-tailed t-test). (E) PyMT-1099 cells were treated with TGFβ for more than 20 days followed by single-molecule RNA-FISH analysis for ET-20l and ET-20s using specific fluorophore-tagged probes. Nuclei were counterstained with DAPI (blue). ET-20l and ET-20s were detected as bright red spots, and white arrows indicate the cellular localization of the lncRNAs. Representative of n=3. Scale bar: 10 µm. (F) Total RNA was isolated from either normal mammary glands (NMG) of FVB/N mice or mammary gland tumors formed in MMTV-PyMT (PyMT) transgenic mice in FVB/N genetic background mice and subjected to RT-qPCR analysis for ET-20s expression. ΔCt values in the NMG were averaged and normalized to 1. Fold-change in expression was calculated over NMG. Open white bars depict untreated cells and solid black bars represent TGFβ-treated cells. Data are represented as mean±s.e.m., n=6 for NMG and n=5 for PyMT tumors. ****P<0.0001 (non-parametric t-test).

Based on its intriguing chromosomal localization, we focused on the lncRNA ET-20 locus (Ensembl accession code ENSMUSG00000073821), which maps in antisense orientation to the gene encoding tenascin C (Tnc), an extracellular protein known to play a key role in EMT and also in breast cancer invasion and metastasis (Brellier and Chiquet-Ehrismann, 2012; Lowy and Oskarsson, 2015; Nagaharu et al., 2011; Oskarsson et al., 2011) (Fig. 1B; Fig. S1B–E). ET-20 is transcribed from the forward strand on mouse chromosome 4 and has three alternative spliced transcripts with eight exons, represented by four cDNAs and some expressed sequence tags (ESTs) (Fig. S1B–E). The ET-20 locus comprises three alternatively spliced transcripts that share only the first exon. Several cDNAs define these transcripts, and additional EST reads confirm them. Fantom 5 CAGE sequence data show a distinct transcription start site (TSS) at the 5′ end of exon 1 (Lizio et al., 2019, 2015), indicating that exon 1 of ET-20 is the start of the locus. The shortest transcript (named ET-20s) is comprised of three exons (designated 1, 2 and 3) and overlaps exon 16 of Tnc on the antisense strand. An intermediate transcript (ET-20m) is also comprised of three exons (1, 4 and 5), while the longest transcript (ET-20l) is comprised of five exons (1, 4, 6, 7 and 8), two of which (1 and 4) are shared with ET-20m. ET-20l spans a large genomic region, where the 3′ exon 8 lies over 100 kb 5′ of the start of Tnc (Fig. 1B).

All ET-20 variants combined and Tnc displayed a similar temporal pattern of expression during EMT, as observed in the EMT time course RNA sequencing data (Fig. 1C). ET-20l and ET-20s showed a significant increase in their expression upon treatment with TGFβ in several cell lines, including immortalized NMuMG cells and the murine breast cancer cell lines PyMT-1099 and Py2T (Fig. 1Di,ii; Fig. S2Ai). The expression of Tnc was also found upregulated with comparable kinetics upon TGFβ treatment in these cells (Fig. 1Di,ii; Fig. S2Ai). ET-20m was not further pursued, since ET-20m had a very low level of expression in these cells, even upon TGFβ treatment. Interestingly, ET-20l and ET-20s were also found to be upregulated in the highly metastatic 4T1 murine breast cancer cells as compared to the less aggressive cell lines 67NR, 168FARN and 4TO7 (Fig. S2Aii). However, Tnc expression was not found to be increased in 4T1 cells (Fig. S2Aii).

The fact that ET-20 expression was detected at comparable levels by RNA sequencing after rRNA depletion but also by RNA sequencing after oligo(dT) priming (Meyer-Schaller et al., 2019), suggested that ET-20 is a poly-adenylated lncRNA (Fig. S2Bi,ii). Indeed, one ET-20s cDNA (AK020205) does have a short poly(A) tail, and the 3′ ends of all three isoforms of ET-20 have AAUAAA polyadenylation signals. RNA-fluorescence in situ hybridization (FISH) experiments suggested that ET-20l and ET-20s were localized both in the nucleus and the cytoplasm (Fig. 1E; Fig. S2C). Finally, whereas ET-20s was expressed at very low levels in normal mammary glands of FVB/N wild-type mice, compared to its expression in other organs (Fig. S2D), its expression was significantly increased in primary mammary tumors obtained from the MMTV-PyMT mouse model of metastatic breast cancer (Fig. 1F). In contrast, ET-20l levels were not significantly different between normal mammary glands and tumors (data not shown).

Expression of ET-20 is regulated by TGFβ-Smad signaling and Sox4

Since ET-20 was upregulated in response to TGFβ treatment, we asked whether ET-20 expression was regulated by canonical TGFβ-Smad signaling. Indeed, siRNA-mediated knockdown of Smad4 in NMuMG cells prevented TGFβ-induced EMT, as exemplified by a decrease in the expression of the epithelial adhesion molecule E-cadherin (E-cad; also known as CDH1) and an increase in mesenchymal markers, such as N-cadherin (N-cad; also known as CDH2) and Ncam1 as well as Tnc (Fig. S2E). Notably, the expression of ET-20l and ET-20s was also not increased by TGFβ treatment in the absence of Smad4, suggesting that ET-20 is indeed regulated by canonical TGFβ signaling (Fig. S2E).

TGFβ-induced EMT has previously been shown to increase the activity of several EMT-inducing transcription factors (EMT-TFs), of which Sox4 has been demonstrated to be a master transcription factor that functions upstream in the epistatic hierarchy of EMT regulation (Tiwari et al., 2013). Furthermore, Tnc gene expression has previously been shown to be regulated by Sox4 (Scharer et al., 2009). Hence, we asked whether ET-20 gene expression was also regulated by Sox4. As expected, siRNA-mediated ablation of Sox4 expression in PyMT-1099 murine breast cancer cells prevented TGFβ-induced EMT, as manifested by a maintenance of E-cad expression and a compromised ability to induce the expression of the mesenchymal markers N-cad, fibronectin1 (Fn1) and, notably, also of Tnc, ET-20l and ET-20s (Fig. 2A). Ablation of Sox4 expression also prevented EMT and an increase in ET-20l and ET-20s expression in short-term TGF-β-treated NMuMG cells (Fig. S2F) and in long-term TGFβ-treated (>20 days), highly mesenchymal Py2T-LT cells (Fig. 2B). Conversely, the forced expression of Sox4 in PyMT-1099 and Py2T cells increased the expression of its direct target genes Ezh2 and Tnc and, with it, the expression of the mesenchymal markers N-cad and Fn1 and also of ET-20l and ET-20s, although ET-20l was without statistical significance (Fig. 2C; Fig. S2G).

Fig. 2.

Regulation of ET-20 expression. (A) PyMT-1099 cells were transiently transfected with control siRNA (siCtrl) or siRNA against Sox4 (siSox4) and were treated or not with TGFβ for 2 days followed by RNA isolation and RT-qPCR for the genes indicated. Open white bars depict control siRNA-transfected cells and solid black bars represent Sox4 siRNA-transfected cells. The left two bars depict untreated and the right two bars depict TGFβ-treated cells. Knockdown efficiency of Sox4 was >80%. Data are represented as mean±s.e.m., n=3. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 (one-way ANOVA with Tukey's multiple comparison post-hoc test). (B) Long-term TGFβ-treated (>20 days) Py2T cells (Py2T-LT) were transiently transfected with siCtrl or siSox4 followed by RNA isolation and RT-qPCR for the expression of the genes indicated. Open white bars depict control siRNA-transfected cells and solid black bars represent Sox4 siRNA-transfected cells. Knockdown efficiency of Sox4 was >80%. Data are represented as mean±s.e.m., n=3. *P<0.05; **P<0.01 (paired two-tailed t-test). (C) PyMT-1099 cells were transiently transfected with either a pcDNA3.1 empty vector (ev) or with pcDNA3.1 carrying the cDNA for murine Sox4 cDNA (Sox4). At 3 days post transfection, cells were harvested for RNA isolation and subsequent RT-qPCR analysis for expression of the genes indicated. Open white bars depict pcDNA3 empty vector-transfected cells and solid black bars represent pcDNA3-Sox4-transfected cells. Data are represented as mean±s.e.m., n=3. *P<0.05; **P<0.01 (ratio two-tailed paired t-test). (D) (i) The scheme depicts the promoter region [2500 bp upstream and 200 bp downstream of the transcriptional start site (TSS)] of the ET-20 gene. Red vertical lines highlight the positions for putative Sox4 (SRY)-binding motifs, and red arrows mark the primer pairs (1–3) that were designed to amplify the regions containing the SRY motifs. (ii) The graph represents the fold enrichment of chromatin immunoprecipitation (ChIP) of Sox4 occupancy at the ET-20 gene promoter regions 1–3 in Py2T cells stably expressing HA–Sox4. Data were normalized to ChIP with control IgG and are presented as mean fold enrichment above background. Occupancy of Sox4 at Ezh2 and Tnc promoters served as positive controls. Intergenic 1 (IG1) served as a negative control. Open white bars depict IgG control and solid black bars represent pull down with anti-HA-tag antibody. Data are represented as mean±s.e.m., n=3. *P<0.05; **P<0.01 (ratio two-tailed paired t-test).

Fig. 2.

Regulation of ET-20 expression. (A) PyMT-1099 cells were transiently transfected with control siRNA (siCtrl) or siRNA against Sox4 (siSox4) and were treated or not with TGFβ for 2 days followed by RNA isolation and RT-qPCR for the genes indicated. Open white bars depict control siRNA-transfected cells and solid black bars represent Sox4 siRNA-transfected cells. The left two bars depict untreated and the right two bars depict TGFβ-treated cells. Knockdown efficiency of Sox4 was >80%. Data are represented as mean±s.e.m., n=3. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 (one-way ANOVA with Tukey's multiple comparison post-hoc test). (B) Long-term TGFβ-treated (>20 days) Py2T cells (Py2T-LT) were transiently transfected with siCtrl or siSox4 followed by RNA isolation and RT-qPCR for the expression of the genes indicated. Open white bars depict control siRNA-transfected cells and solid black bars represent Sox4 siRNA-transfected cells. Knockdown efficiency of Sox4 was >80%. Data are represented as mean±s.e.m., n=3. *P<0.05; **P<0.01 (paired two-tailed t-test). (C) PyMT-1099 cells were transiently transfected with either a pcDNA3.1 empty vector (ev) or with pcDNA3.1 carrying the cDNA for murine Sox4 cDNA (Sox4). At 3 days post transfection, cells were harvested for RNA isolation and subsequent RT-qPCR analysis for expression of the genes indicated. Open white bars depict pcDNA3 empty vector-transfected cells and solid black bars represent pcDNA3-Sox4-transfected cells. Data are represented as mean±s.e.m., n=3. *P<0.05; **P<0.01 (ratio two-tailed paired t-test). (D) (i) The scheme depicts the promoter region [2500 bp upstream and 200 bp downstream of the transcriptional start site (TSS)] of the ET-20 gene. Red vertical lines highlight the positions for putative Sox4 (SRY)-binding motifs, and red arrows mark the primer pairs (1–3) that were designed to amplify the regions containing the SRY motifs. (ii) The graph represents the fold enrichment of chromatin immunoprecipitation (ChIP) of Sox4 occupancy at the ET-20 gene promoter regions 1–3 in Py2T cells stably expressing HA–Sox4. Data were normalized to ChIP with control IgG and are presented as mean fold enrichment above background. Occupancy of Sox4 at Ezh2 and Tnc promoters served as positive controls. Intergenic 1 (IG1) served as a negative control. Open white bars depict IgG control and solid black bars represent pull down with anti-HA-tag antibody. Data are represented as mean±s.e.m., n=3. *P<0.05; **P<0.01 (ratio two-tailed paired t-test).

Analysis of the ET-20 gene promoter revealed the presence of several motifs/binding sites for the Sox (SRY) family of transcription factors (Fig. 2Di). To assess whether Sox4 could regulate expression of ET-20 by directly binding its promoter, we performed chromatin immunoprecipitation (ChIP) with antibodies against HA in Py2T cells stably expressing HA-tagged Sox4. Specific binding of Sox4 to the Ezh2 and Tnc promoters, well-established Sox4 target genes (Scharer et al., 2009; Tiwari et al., 2013), served as positive controls (Fig. 2Dii). Importantly here, Sox4 occupancy was enriched at all three regions of the ET-20 promoter containing SRY-binding motifs, as compared to the IgG control (Fig. 2Dii).

Collectively, these results suggest that ET-20 gene expression is regulated like the anti-sense oriented Tnc gene, by Sox4, which itself is activated by canonical TGFβ-Smad signaling (Tiwari et al., 2013).

ET-20 is required for TGFβ-induced EMT

To assess whether ET-20 is required for EMT, the expression of ET-20l and ET-20s was ablated in PyMT-1099 and NMuMG cells by specific siRNAs against each ET-20 variant, followed by TGFβ treatment. In the absence of TGFβ (untreated), ET-20 ablation did not have any significant effect on cell morphology (Fig. 3A; Fig. S3A). However, whereas TGFβ treatment of control siRNA-transfected cells led to a change from epithelial to mesenchymal cell morphology, the ablation of ET-20l and ET-20s prevented this change in cell morphology in response to TGFβ treatment (Fig. 3A; Fig. S3A). Although the siRNAs were designed specifically against the ET-20l and ET-20s variants, real-time quantitative PCR (RT-qPCR) analysis revealed that the ablation of ET-20l also decreased the expression of ET-20s and vice versa in PyMT-1099 cells (Fig. 3B) and in NMuMG cells (Fig. S3B). Knockdown of ET-20l and ET-20s also abrogated EMT by preventing an increase in the expression of the mesenchymal markers Tnc, Fn1 and Ncam1 in TGFβ-treated PyMT-1099 cells (Fig. 3B) and NMuMG cells (Fig. S3B). The decrease in expression of Tnc in TGFβ-treated PyMT-1099 and NMuMG cells upon ET-20 knockdown was further confirmed by immunoblotting analysis (Fig. S3C,D).

Fig. 3.

ET-20 is required for EMT. (A) Photomicrographs representing the morphology of PyMT-1099 cells transfected with control siRNA (siCtrl) or siRNA against ET-20l and ET-20s (siET-20l and siET-20s), respectively, followed by 2 days of treatment or not (UT, untreated) with TGFβ. Images representative of three experiments. Scale bar: 100 µm. (B) PyMT-1099 cells transiently transfected with siCtrl or siET-20 were treated with TGFβ for 2 days followed by RNA isolation and RT-qPCR for the expression of the genes indicated. Gray bars depict control siRNA-transfected cells, red bars depict ET-20l siRNA-transfected cells and orange bars represent ET-20s siRNA-transfected cells. The left three bars depict untreated and the right three bars depict TGFβ-treated cells. Knockdown efficiency of ET-20l and ET-20s was between 50 and 60%. Data are represented as mean±s.e.m., n=3. **P<0.01; ****P<0.0001; ns, not significant (one-way ANOVA with Tukey's multiple comparison post-hoc test). (C) (i) PyMT-1099 cells were transiently transfected with control or ET-20 siRNA and treated or not with TGFβ for 2 days followed by immunofluorescence analysis for the epithelial marker E-cad (green) and for actin (stained with phalloidin; red). Nuclei were counterstained with DAPI (blue). (ii) PyMT-1099 cells transiently transfected with siCtrl or siET-20 were treated with TGFβ for 2 days followed by immunofluorescence analysis for the mesenchymal markers Tnc (green) and fibronectin (Fn; red). Nuclei were counterstained with DAPI (blue). Images representative of n=3. Scale bars: 50 µm. (D) Py2T-LT cells were transiently transfected with siCtrl or siRNA against ET-20l or ET-20s and then subjected to a modified transwell Boyden chamber migration assay (i) or a transwell Boyden chamber Matrigel invasion assay (ii). The graph represents the number of cells migrated per field upon ET-20 knockdown compared to siCtrl-transfected cells. Gray bars depict control siRNA-transfected cells, red bars depict ET-20l-siRNA transfected cells and orange bars represent ET-20s siRNA-transfected cells. Data are represented as mean±s.e.m., n=3. *P<0.05; **P<0.01; ****P<0.0001 (one-way ANOVA).

Fig. 3.

ET-20 is required for EMT. (A) Photomicrographs representing the morphology of PyMT-1099 cells transfected with control siRNA (siCtrl) or siRNA against ET-20l and ET-20s (siET-20l and siET-20s), respectively, followed by 2 days of treatment or not (UT, untreated) with TGFβ. Images representative of three experiments. Scale bar: 100 µm. (B) PyMT-1099 cells transiently transfected with siCtrl or siET-20 were treated with TGFβ for 2 days followed by RNA isolation and RT-qPCR for the expression of the genes indicated. Gray bars depict control siRNA-transfected cells, red bars depict ET-20l siRNA-transfected cells and orange bars represent ET-20s siRNA-transfected cells. The left three bars depict untreated and the right three bars depict TGFβ-treated cells. Knockdown efficiency of ET-20l and ET-20s was between 50 and 60%. Data are represented as mean±s.e.m., n=3. **P<0.01; ****P<0.0001; ns, not significant (one-way ANOVA with Tukey's multiple comparison post-hoc test). (C) (i) PyMT-1099 cells were transiently transfected with control or ET-20 siRNA and treated or not with TGFβ for 2 days followed by immunofluorescence analysis for the epithelial marker E-cad (green) and for actin (stained with phalloidin; red). Nuclei were counterstained with DAPI (blue). (ii) PyMT-1099 cells transiently transfected with siCtrl or siET-20 were treated with TGFβ for 2 days followed by immunofluorescence analysis for the mesenchymal markers Tnc (green) and fibronectin (Fn; red). Nuclei were counterstained with DAPI (blue). Images representative of n=3. Scale bars: 50 µm. (D) Py2T-LT cells were transiently transfected with siCtrl or siRNA against ET-20l or ET-20s and then subjected to a modified transwell Boyden chamber migration assay (i) or a transwell Boyden chamber Matrigel invasion assay (ii). The graph represents the number of cells migrated per field upon ET-20 knockdown compared to siCtrl-transfected cells. Gray bars depict control siRNA-transfected cells, red bars depict ET-20l-siRNA transfected cells and orange bars represent ET-20s siRNA-transfected cells. Data are represented as mean±s.e.m., n=3. *P<0.05; **P<0.01; ****P<0.0001 (one-way ANOVA).

We next employed immunofluorescence microscopy analysis of various EMT-related markers in PyMT-1099 cells transfected with siRNAs against ET-20l or ET-20s to further validate the requirement of ET-20 for TGFβ-induced EMT. ET-20 knockdown in the absence of TGFβ did not affect the cellular localization or levels of actin, E-cad, Tnc or Fn1 (Fig. 3Ci,ii). In contrast, the ablation of ET-20 in TGFβ-treated cells prevented the reorganization of cortical actin to stress fibers and the full re-localization of E-cad from the cell membrane to the cytoplasm, and prevented an increase in the levels of Tnc and Fn1 (Fig. 3Ci,ii; Fig. S3E).

The ablation of ET-20 expression also repressed the migration of long-term TGFβ-treated mesenchymal Py2T-LT and PyMT-1099-LT cells (Fig. 3Di; Fig. S3F) and invasion of Py2T-LT cells (Fig. 3Dii).

As expected from previous studies (Katoh et al., 2013; Nagaharu et al., 2011; Sun et al., 2018), siRNA-mediated knockdown of Tnc in Py2T cells prevented a TGFβ-mediated increase in expression of the mesenchymal markers Fn1 and N-cad and, importantly, of ET-20l and ET-20s (Fig. S4A). The loss of Tnc expression also prevented the TGFβ-induced increase of the migratory and invasive capacity of Py2T-LT cells (Fig. S4Bi,ii), demonstrating that Tnc is indeed required for mediating EMT, but also for the expression of ET-20l and ET-20s. However, the forced expression of ET-20s did not induce the expression of Tnc or of mesenchymal markers, such as N-cad, Ncam, fibronectin and vimentin (Fig. S4C).

Together, the results suggest that ET-20 and Tnc regulate each other's expression and are critically required for EMT, cell migration and invasion.

ET-20 mediates EMT by disrupting desmosomal integrity

To gain mechanistic insights into how ET-20 regulates EMT, we performed chromatin isolation by RNA purification (ChIRP) followed by mass spectrometry to identify the proteins associated with ET-20 during EMT. Tiling biotinylated DNA probes were generated for ET-20l and ET-20s. We could only pulldown ET-20l successfully along with associated proteins in NMuMG cells treated with TGFβ for 4 days (Fig. S5A), presumably because ET-20s is very short and an insufficient number of tiling probes could be made. RT-qPCR on RNA isolated from the streptavidin beads confirmed an efficient and specific pulldown of ET-20l (Fig. S5B). Actin and Tnc mRNAs were not amplified, demonstrating the specificity of the ChIRP approach and also suggesting that ET-20l does not physically interact with Tnc mRNA (Fig. S5B). The proteins eluted from the beads after ET-20l pulldown were separated by PAGE and visualized by silver staining. Additional bands could be observed only in the ET-20l pulldown lanes as compared to the negative control lanes, including RNase A treatment before pulldown or pulldown with LacZ-specific DNA probes (Fig. S5C). The eluted proteins were then subjected to quantitative mass spectrometry analysis which revealed, among a list of various peptides, a significant detection of four desmosomal proteins, namely plakoglobin (PG, also known as JUP), desmoplakin (DP, also known as DSP), desmoglein 1 (DSG1) and plakophilin (PKP) (Table S2). With the exception of PKP, the specific interaction of desmosomal proteins was confirmed by additional ChIRP experiments followed by non-quantitative mass spectroscopy, a method with less sensitivity (Fig. 4A).

Fig. 4.

Mechanisms of ET-20 action in EMT. (A) The list represents the number of mass spectrometric runs out of which the indicated desmosomal proteins were pulled down with ET-20l in chromatin isolation by RNA purification mass spectrometry (ChIRP-MS) in PyMT-1099 cells after treatment with TGFβ for 4 days. (B) PyMT-1099 cells were treated with TGFβ for 2 days and plakoglobin (PG) was then immunoprecipitated with specific antibodies from cell lysates. RNA bound to plakoglobin was subjected to RT-qPCR for ET-20l. Pulldown of U1 snRNA with an anti-SNRNP70 antibody served as a positive control. Data are represented as mean±s.e.m., n=3. (C) PyMT-1099 cells transiently transfected with control or ET-20 siRNA were treated with TGFβ for 2 days followed by immunofluorescence microscopy analysis for the expression of plakoglobin (PG; green) and E-cadherin (E-cad; red) (i) and for desmoplakin 1 (DP; green) (ii). Nuclei were counterstained with DAPI (blue). (D) PyMT-1099-LT cells were transiently transfected with control siRNA or with siRNAs against ET-20l and ET-20s followed by immunofluorescence microscopy analysis for plakoglobin (PG; green) and E-cadherin (E-cad); red. Nuclei were counterstained with DAPI (blue). (E) PyMT-1099 cells were transiently transfected with either pcDNA3.1 empty vector control (pEV) or pcDNA3.1 encoding ET-20l (pET-20l) (i) or ET-20s (pET-20s) (ii). Cells were then subjected to a co-staining for ET-20l and ET-20s, respectively, by RNA-FISH (red) and plakoglobin (PG) by immunofluorescence antibody staining (green). Cells overexpressing ET-20l and ET-20s, respectively, are marked with white arrows. Nuclei were counterstained with DAPI (blue). Images in C–E representative of n=3. Scale bars: 50 µm.

Fig. 4.

Mechanisms of ET-20 action in EMT. (A) The list represents the number of mass spectrometric runs out of which the indicated desmosomal proteins were pulled down with ET-20l in chromatin isolation by RNA purification mass spectrometry (ChIRP-MS) in PyMT-1099 cells after treatment with TGFβ for 4 days. (B) PyMT-1099 cells were treated with TGFβ for 2 days and plakoglobin (PG) was then immunoprecipitated with specific antibodies from cell lysates. RNA bound to plakoglobin was subjected to RT-qPCR for ET-20l. Pulldown of U1 snRNA with an anti-SNRNP70 antibody served as a positive control. Data are represented as mean±s.e.m., n=3. (C) PyMT-1099 cells transiently transfected with control or ET-20 siRNA were treated with TGFβ for 2 days followed by immunofluorescence microscopy analysis for the expression of plakoglobin (PG; green) and E-cadherin (E-cad; red) (i) and for desmoplakin 1 (DP; green) (ii). Nuclei were counterstained with DAPI (blue). (D) PyMT-1099-LT cells were transiently transfected with control siRNA or with siRNAs against ET-20l and ET-20s followed by immunofluorescence microscopy analysis for plakoglobin (PG; green) and E-cadherin (E-cad); red. Nuclei were counterstained with DAPI (blue). (E) PyMT-1099 cells were transiently transfected with either pcDNA3.1 empty vector control (pEV) or pcDNA3.1 encoding ET-20l (pET-20l) (i) or ET-20s (pET-20s) (ii). Cells were then subjected to a co-staining for ET-20l and ET-20s, respectively, by RNA-FISH (red) and plakoglobin (PG) by immunofluorescence antibody staining (green). Cells overexpressing ET-20l and ET-20s, respectively, are marked with white arrows. Nuclei were counterstained with DAPI (blue). Images in C–E representative of n=3. Scale bars: 50 µm.

To further validate the specific interaction of ET-20 lncRNA with desmosomal proteins, we immunoprecipitated PG with specific antibodies and analyzed, by means of RT-qPCR, whether ET-20l was co-immunoprecipitated with the desmosomal proteins. Indeed, although with variable efficiency, ET-20l was found bound to PG as compared to an immunoprecipitation with unrelated IgG (Fig. 4B). Co-immunoprecipitation of U1 snRNA with antibodies against the snRNP U1 protein SNRNP70 was used to validate functionality of the RNA immunoprecipitation (RIP) protocol (Fig. 4B).

Desmosomes are epithelial junction complexes that keep epithelial cells juxtaposed to each other and maintain epithelial cell layer integrity. Together with a disruption of tight and adherens junctions (Yang et al., 2020), they are readily dissolved during EMT. We hence speculated that, during EMT, the increased levels of ET-20 lncRNA bind to desmosomal proteins to either destabilize and dismantle existing desmosomal complexes or to prevent the de novo formation of desmosomal complexes, thus enabling EMT to occur. To test this hypothesis, we ablated ET-20 expression during TGFβ-induced EMT. Indeed, siRNA-mediated ablation of ET-20l or ET-20s prevented the dissociation of PG and DP from desmosomal junctions upon TGFβ treatment in PyMT-1099 cells (Fig. 4Ci,ii). Ablation of ET-20 in TGFβ-treated cells in parts also repressed the relocalization of E-cad from the cell membrane to the cytoplasm (Fig. 4Ci), as observed above (Fig. 3Ci). Similar results were also obtained in NMuMG cells, where ET-20 ablation prevented the dissociation of PG and DP from the cell membranes upon TGFβ treatment (Fig. S5Di,ii). Ablation of ET-20 expression in long-term TGFβ-treated PyMT-1099-LT cells caused a re-localization of PG from the cytoplasm to the cell membrane (Fig. 4D). However, E-cad did not fully relocalize to the cell membranes upon ET-20 knockdown in these highly mesenchymal cells (Fig. 4D), suggesting that ET-20 affects the integrity of desmosomes more than that of adherens junctions.

Since an ET-20-mediated dissolution of desmosomes would indicate a function in trans, we next assessed whether the forced expression of ET-20 may be sufficient to dissolve desmosomes and to even induce an EMT. Indeed, the transient expression of ET-20l and ET-20s in PyMT-1099 cells seemed sufficient to dissociate PG from the membranes of ET-20-expressing cells, as determined by anti-PG immunofluorescence staining and by RNA-FISH for ET-20 (Fig. 4Ei,ii). However, the forced expression of ET-20 was not sufficient to induce a full EMT, since adherens junctions were unaffected, and the cells did not start to express mesenchymal markers (Fig. S4C).

Collectively, these results indicate that the Sox4-mediated and EMT-related upregulation of ET-20 is not only required but also sufficient for the disruption of desmosomes, yet by itself it is not sufficient to induce a full EMT.

ET-20 expression is upregulated in human breast cancer cells

In order to see whether a homolog of ET-20 exists in the human genome, we conducted BLAT and blastn searches at NCBI and in the UCSC genome browser using the mouse ET-20 transcripts as query. Significant matches could be found to exons 2 and 3 (ET-20s), as well as exon 8 (ET-20l) in the human genome and in other mammalian species (Fig. S6A–D). No similarity in humans was found for ET-20m. Exons 2 and 3 overlap exon 16 of TNC on the antisense strand, explaining in part the sequence conservation. However, the 3′ region of exon 3 (∼430 bp) with no overlap shows 81% sequence identity between mouse and human. Furthermore, short insertions and deletions that are not multiples of three exist in the multiple sequence alignment, indicating that the conservation is not due to a coding region. In the case of ET-20l, sequence conservation was found in the region of exon 8 over a length of ∼800 bp (Fig. S6A–C). This region lies about 100 kbps 5′ of the TNC gene. In the case of ET-20s, the splice sites of the intron between exons 2 and 3 are conserved (Fig. S6E). These sequence conservations suggest that a functional ET-20 ortholog may exist in other species as well.

To determine whether we could detect transcriptional activity in these conserved regions, we designed qPCR primers for exon 3 (ET-20s) and exon 8 (ET-20l). For exon 8 we did not detect significant signals, and we did not pursue this further. However, for exon 3 we were able to detect reliable RT-qPCR signals revealing expression of ET-20s across a panel of human cell lines, including immortalized MCF10A, non-invasive MCF7, T47D, invasive Hs-578T, BT-549 and MDA-MB-231 cells, and the circulating tumor cell (CTC) cluster-derived cell lines Brx16 and Brx50. We refer to this as human ET-20s (hET-20s). Interestingly, hET-20s expression was specifically high in the invasive cell lines Hs578T, BT-549 and MDA-MB-231, and in the CTC cluster-derived cell lines Brx16 and Brx50 (Fig. 5A). In addition, we examined TNC expression and found it to be elevated only in BT-549 and MDA-MB-231 cells (Fig. 5A). To further assess whether ET-20s expression also induced during EMT in human cells, we treated MCF10A cells with TGFβ for 2 days. Notably, during the induction of an EMT-like process, together with the increase in the expression of the mesenchymal markers N-cad and TNC, hET-20s was also found to be upregulated (Fig. 5B).

Fig. 5.

ET-20s is expressed and functional during EMT in human cells. (A) A panel of human breast cancer cell lines, as indicated in the graphs, were tested for the mRNA expression of human tenascin C (TNC) and human ET-20s (ET-20s). Data are represented as mean±s.e.m., n=3. (B) MCF10A cells were treated with TGFβ for two days followed by RNA isolation and RT-qPCR for the expression of the genes indicated. Black bars depict untreated cells and gray bars depict TGFβ-treated cells. Data are represented as mean±s.e.m., n=3. (C) MCF10A cells were transiently transfected with control siRNA (siCtrl) or an siRNA against human ET-20s (sihET-20s) and treated or not with TGFβ for 2 days followed by immunofluorescence analysis for the epithelial marker E-cadherin (E-cad; green) and the desmosomal marker plakoglobin (PG; green). Nuclei were counterstained with DAPI (blue). Two microscopy images per treatment are shown. Knockdown efficiencies for ET-20s and TNC were 80%. Images representative of n=2. Scale bar: 50 µm

Fig. 5.

ET-20s is expressed and functional during EMT in human cells. (A) A panel of human breast cancer cell lines, as indicated in the graphs, were tested for the mRNA expression of human tenascin C (TNC) and human ET-20s (ET-20s). Data are represented as mean±s.e.m., n=3. (B) MCF10A cells were treated with TGFβ for two days followed by RNA isolation and RT-qPCR for the expression of the genes indicated. Black bars depict untreated cells and gray bars depict TGFβ-treated cells. Data are represented as mean±s.e.m., n=3. (C) MCF10A cells were transiently transfected with control siRNA (siCtrl) or an siRNA against human ET-20s (sihET-20s) and treated or not with TGFβ for 2 days followed by immunofluorescence analysis for the epithelial marker E-cadherin (E-cad; green) and the desmosomal marker plakoglobin (PG; green). Nuclei were counterstained with DAPI (blue). Two microscopy images per treatment are shown. Knockdown efficiencies for ET-20s and TNC were 80%. Images representative of n=2. Scale bar: 50 µm

To assess whether hET-20s could play a conserved function in EMT regulation also in human cells, we designed siRNAs against the human ET-20s sequence and assessed the effect of hET-20s knockdown in TGFβ-treated MCF10A human breast cancer cells. Interestingly, similar to our results with mouse cell lines (Fig. 4C,D; Fig. S5D), knockdown of hET-20s in MCF10A cells prevented the TGFβ-induced dislocation of PG and E-cad from cell junctions and the subsequent junctional dissolution (Fig. 5C). These results indicate that a human counterpart of murine ET-20s, here designated hET-20s, exerted functions comparable to murine ET-20l and ET-20s in the impairment of desmosomal junctions during TGFβ-induced EMT of breast cancer cells.

lncRNAs have been shown to play crucial roles in cell physiology as well as in numerous pathophysiologies, including cancer (Batista and Chang, 2013; Fatica and Bozzoni, 2014; Schmitt and Chang, 2016; Slack and Chinnaiyan, 2019). In fact, lncRNAs can serve as drivers of cancer phenotypes affecting each of the hallmarks of cancer, such as proliferation, apoptosis, angiogenesis, invasion and metastasis (Batista and Chang, 2013; Gutschner and Diederichs, 2012). In recent years, several examples have also highlighted that lncRNAs might function in EMT (Grelet et al., 2017; Hao et al., 2019; Kallen et al., 2013; Richards et al., 2015; Yuan et al., 2014). Using whole transcriptome sequencing, we have identified 114 lncRNAs [which we refer to as EMT-associated transcripts (ETs)], whose expression is significantly regulated during EMT of normal and transformed mammary gland epithelial cells. In this study, we have focused on the novel lncRNA ET-20 (ENSMUSG00000073821) due to its chromosomal localization within the tenascin C (TNC) gene, which encodes for an ECM protein that is critical for EMT and metastatic outgrowth of breast cancer cells (Aishima et al., 2003; Cai et al., 2017; Jahkola et al., 1998a,b; Katoh et al., 2013; Lowy and Oskarsson, 2015; Nagaharu et al., 2011; Oskarsson et al., 2011; Sun et al., 2018; Takahashi et al., 2013). ET-20 was found to be upregulated in response to TGFβ in both immortalized and transformed murine and human cell lines, suggesting that it could be a novel marker of EMT in breast cancer cells. Notably, the transcript for ET-20 has been previously listed as upregulated in dedifferentiated ovarian granulosa cells that have been depleted of estrogen receptor expression in mice (Binder et al., 2013).

Interestingly, the expression of ET-20 and Tnc was concomitantly upregulated very early during a TGFβ-induced EMT time course by the transcriptional activity of Sox4 (between 2 and 6 h) and its expression was maintained until 10 days of TGFβ treatment. Furthermore, ET-20 was found to be localized both in the nucleus and the cytoplasm, suggesting that ET-20 could play functions in cis and in trans. In fact, siRNA-mediated ablation of ET-20 was found to prevent a TGFβ-mediated increase in Tnc levels suggesting that ET-20 transcripts are required for regulating Tnc expression. However, transient overexpression of ET-20 neither increased the expression of Tnc mRNA levels nor overcame the repression of TGFβ-induced EMT by Tnc ablation. Conversely, Tnc knockdown also prevented the TGFβ-mediated increase in ET-20 expression, suggesting that ET-20 and Tnc regulate each other's expression (Fig. 6). The detailed mechanisms of how Sox4, ET-20 and Tnc gene elements and mRNAs interact to mediate this regulatory feedback loop will need further investigation. Finally, ET-20 expression does not always correlate with Tnc expression in murine breast cancer cells; for example, in highly metastatic 4T1 cells, which represent a hybrid EMT state, raising the possibility that with higher cell plasticity in the EMT continuum, the co-expression of the early EMT markers Tnc and ET-20 may be uncoupled.

Fig. 6.

Working model of the functional contribution of ET-20 to EMT. TGFβ treatment of epithelial mammary cancer cells leads to the activation of Smad signaling and the activation of the transcription factor Sox4. By binding the promoter regions of the tenascin C (Tnc) and ET-20 (ET-20) genes, Sox4 causes their transcriptional activation. On one hand, ET-20 and Tnc reciprocally promote each other's expression. On the other hand, ET-20 binds desmosomal proteins and dismantles desmosomes at the cell membrane thereby paving the way for an EMT to occur.

Fig. 6.

Working model of the functional contribution of ET-20 to EMT. TGFβ treatment of epithelial mammary cancer cells leads to the activation of Smad signaling and the activation of the transcription factor Sox4. By binding the promoter regions of the tenascin C (Tnc) and ET-20 (ET-20) genes, Sox4 causes their transcriptional activation. On one hand, ET-20 and Tnc reciprocally promote each other's expression. On the other hand, ET-20 binds desmosomal proteins and dismantles desmosomes at the cell membrane thereby paving the way for an EMT to occur.

Most importantly here, we have discovered that ET-20 interacts with desmosomal proteins and thereby impairs desmosomal junctions (Fig. 6). Notably, ablation of ET-20 expression prevented TGFβ-induced EMT. Conversely, the forced expression of ET-20 caused the dissociation of the desmosomal protein PG from cell membranes, yet it was not sufficient to induce a full EMT. ET-20 may impair desmosomal integrity in several ways, including affecting the relocalization of desmosomal proteins from the plasma membrane to the cytosol and thereby dissolving desmosomal junctions, or by repressing the initial formation of desmosomal junctions, or by affecting the stability of desmosomal proteins. Interestingly, in long-term TGFβ-treated cells, which were fully mesenchymal, knockdown of ET-20 caused the reassembly of PG into desmosomal complexes, whereas E-cad was still retained in the cytoplasm. Hence, ET-20-mediated loss of desmosomes might be an early event in EMT that further enables the unzipping of the other epithelial junctions and allows progression through a complete EMT.

In the past, EMT research has mainly focused on the loss of E-cad in adherens junctions as a hallmark of EMT. However, some studies suggest that dislocation of desmosomal proteins precedes that of E-cadherin (Dusek and Attardi, 2011). In fact, downregulation of desmosomes with the retention of adherens junctions has been observed in squamous cell carcinoma upon Perp depletion and in pancreatic cancer upon desmoplakin ablation (Beaudry et al., 2010; Chun and Hanahan, 2010). It has hence been suggested that reduced expression of desmosomal proteins could be an early driver of local invasion, and subsequent loss of adherens junctions could promote later stages, including widespread invasion and metastatic dissemination of cancer cells (Dusek and Attardi, 2011). In fact, a recent report indicates that E-cadherin expression is required for efficient metastasis formation (Padmanaban et al., 2019). Loss of desmosomes has also been associated with advanced epithelial tumor grade and increased metastasis, suggesting that desmosomal components suppress both early and late stages of carcinogenesis (Johnson et al., 2014).

ET-20 is found expressed as several alternative transcripts, with ET-20l and ET-20s being significantly expressed during EMT and in murine breast tumors. Although the efficiencies of siRNA-mediated ablation of ET-20l and ET-20s were comparable, and they both seemed to have similar effects on EMT, the depletion of ET-20s seemed to have a stronger effect on the EMT phenotype as compared to that of ET-20l.

lncRNAs are known to have a poor evolutionary conservation across different species, with conservation observed either in micro-homologous regions, in position on the genome (synteny conservation), or only in terms of function (Ulitsky, 2016; Ulitsky and Bartel, 2013). We could not detect human ET-20 expression upon analyzing RNA sequencing data of the TCGA database, which could be due to the depth of the data available or also the fact that the expression of lncRNA is typically very low with only few molecules per cell (Ransohoff et al., 2018). However, BLAT and blastn analysis against the human genome revealed conserved elements in syntenic position to Tnc with high percentage sequence identity in the exons. Especially for ET-20s, the conserved splice structure of exons 2 and 3 and the high sequence similarity of 81%, despite being non-coding, suggests that there has been an evolutionary pressure for functional conservation. The data suggest that a human homolog of ET-20s exists and may also function in EMT. Indeed, hET-20s expression was detected in several human breast cancer cell lines with highest expression in invasive breast cancer cell lines as compared to differentiated breast cancer cells. hET-20s was also found to be upregulated in mammary tumor tissue as compared to normal tissue in mice. Since hET-20s seems to exert a cancer-specific expression, and lncRNAs typically have a stable expression in tissue and plasma (Slack and Chinnaiyan, 2019), investigations into the use of hET-20s as a biomarker for breast cancer diagnosis is warranted.

The sequence similarity between mouse and human is confined to exons 2, 3 and 8. Given that we were not able to find significant amounts of transcript from exon 8, we suspect that the lncRNA isoforms of ET-20m and ET-20l are not conserved. An explanation for the sequence conservation in exon 8 is that it functions as a regulatory region. It is annotated by the ENCODE project as a potential regulatory region and histone H3K27 acetylation also marks this region as an enhancer (Fig. S6C). If this region indeed is a regulatory region and affects the expression of Tnc, it could provide a possible mechanism for the observed cross-regulation in mouse between ET-20 and Tnc that we observe. In such a hypothetical scenario the ET-20l transcript would interfere with the activity of this regulatory element.

Finally, our transcriptome-wide RNA sequencing analysis of EMT kinetics identified a number of additional novel ET lncRNAs whose expression appeared temporally co-regulated with proximally located metastasis genes. For instance, lncRNA ET-1 co-maps with the miR-181 locus, ET-8 to thrombospondin1 (Thbs1), ET-26 to Cyr61, ET-38 to CTGF and ET-122 close to miR-200 family members, all genes known to be essential for various stages of EMT and tumor progression. Further studies may reveal a potential role for these lncRNAs in EMT regulation as well. Owing to its manual curation, the list of 114 lncRNAs certainly does not represent an exhaustive list of all differentially regulated lncRNA in EMT, and it is possible that several potentially interesting lncRNA may have been excluded. The raw data of RNA sequencing can be found at GEO database GSE112797 of which an in-depth analysis has been recently reported (Meyer-Schaller et al., 2019).

In summary, we describe the identification of the novel lncRNA ET-20, which exhibits high expression during a TGFβ-induced EMT in murine mammary tumor cells and in human invasive breast cancer cells. The gene for ET-20 is localized in anti-sense direction within the Tnc gene, and the expression of both ET-20 and Tnc is induced by the TGFβ-Smad signaling-induced transcriptional activity of Sox4. The lncRNA ET-20 regulates EMT by executing its functions both in cis and in trans; ET-20 modulates EMT by regulating Tnc expression. In fact, in a reciprocal feedback loop, Tnc can also regulate the expression of ET-20, and both ET-20 and Tnc are required for TGFβ-induced EMT (Schmitt and Chang, 2016; Slack and Chinnaiyan, 2019) (Fig. 6). Most importantly, our data suggest that ET-20 executes its function in trans by binding desmosomal proteins and impairing desmosomal junctions, thereby initiating the disassembly of other epithelial junctions and promoting an EMT (Fig. 6).

Antibodies and reagents

The following antibodies were used: α-tubulin (Sigma, T-9026, 1:1000), desmoplakin (Abcam, ab16434, for immunofluorescence, 1:50), E-cadherin (BD Transduction Labs, 610182; used for immunoblotting, 1:2000), E-cadherin (Zymed, 13-1900; used for immunofluorescence staining, 1:100), fibronectin (Sigma-Aldrich, F3648, for immunofluorescence, 1:100), N-cadherin (Takara, M142, for immunofluorescence, 1:100), plakoglobin (Cell Signaling, 2309S, for immunofluorescence, 1:50) and tenascin-C (Mtn-12) (Abcam, ab63460, for immunofluorescence, 1:100).

Alexa-Fluor-488 and -568-conjugated secondary antibodies (Molecular Probes) and secondary horseradish peroxidase (HRP)-conjugated antibodies against mouse and rabbit IgG (Jackson ImmunoResearch) were used. Recombinant human TGFβ1 was obtained from R&D Systems (240-B).

Phalloidin–Alexa-Fluor-568 (Molecular Probes, A12380, at 1:400 dilution) was used to mark F-actin and was added along with the secondary antibodies during the immunofluorescence experiment.

siRNA used was against TNC (siTenascin C; Ambion silencer select, catalog no. 4390771; siRNA ID: s75240).

Cell lines and cell culture

Murine breast epithelial cell lines NMuMG (E9- epithelial clone 9) (Maeda et al., 2005), Py2T (Waldmeier et al., 2012), PyMT-1099 (Saxena et al., 2018), the 67NR, 168FARN, 4TO7 and 4T1 cell series (Aslakson and Miller, 1992), and human cell lines HEK-293T, MCF7, T47D, Hs578T and BT459 (obtained from the ATCC) were cultured in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich, D5671) supplemented with 10% fetal bovine serum (Sigma-Aldrich, F7524), 2 mM glutamine (Sigma-Aldrich, G7513), 100 U penicillin (Sigma-Aldrich) and 0.1 mg/ml streptomycin (Sigma-Aldrich). Human MDA-MB-231 cells (ATCC) were cultured in RPMI supplemented with 10% fetal bovine serum (Sigma-Aldrich, F7524), 2 mM glutamine (Sigma-Aldrich, G7513), 100 U penicillin (Sigma-Aldrich) and 0.1 mg/ml streptomycin (Sigma-Aldrich). The human epithelial mammary gland cell line MCF10A (Soule et al., 1990) was cultured in DMEM/F12 medium supplemented with 5% horse serum (Bioconcept Amimed), 10 µg/ml insulin (Sigma), 0.5 µg/ml hydrocortisone (Sigma), 0.02 µg/ml human EGF (Invitrogen) and 0.01 µg/ml cholera toxin (Sigma). All cell lines were grown at 37°C, 5% CO2 and 95% humidity. For EMT experiments, cells were treated with 2 ng/ml TGFβ1 for the time points indicated.

RNA isolation and real-time RT-qPCR

Total RNA from cell lines was isolated using the guanidine isothiocyanate and phenol/chloroform method from cells harvested with TRI reagent (Sigma-Aldrich) according to the manufacturer's instructions. RNA isolation from mouse tissues or tumors was performed using the RNAeasy kit (Qiagen, 74104). Reverse transcription of mRNA or lncRNA was carried out using the ImProm-IITM Reverse Transcription System (Promega, A3803), and mRNA/lncRNA levels were quantified by real-time qPCR using PowerUp SYBR Green Master Mix (ThermoFisher, A25743). Mouse Riboprotein L19 (mRPL19) primers were used for normalization. qPCR assays were performed in duplicates, and fold changes were calculated using the comparative Ct method (ΔΔCt). Sequences of the primers used are listed in Table S3.

Immunoblotting

Cells were lysed on ice for 30 min in RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 10% glycerol, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM MgCl2 and 2 mM CaCl2) with 1 mM DTT, 1 mM NaF, 2 mM sodium orthovanadate and 1× protease inhibitor cocktail (Sigma-Aldrich) followed by scraping into tubes and centrifugation for 10 min at 16,100 g at 4°C. Protein concentration in the supernatants was determined using Bio-Rad Bradford solution. Proteins were mixed with 1× Laemmli sample buffer and equal amounts were size-fractionated on a SDS-PAGE gel. Proteins were then transferred onto an Immobilon-P PVDF membrane (Millipore) using the wet transfer method for 2 h at constant current (0.4 A). Following blocking for 1 h in 5% skimmed milk prepared in Tris-buffered saline (TBS) with 0.05% Tween 20, the membranes were incubated with appropriate primary antibodies overnight at 4°C. After washes, the blots were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature and visualized with Immobilon Western Chemiluminescent HRP Substrate (Millipore, WBKLS0500) on a Fusion Fx7 chemiluminescence reader.

siRNA and plasmid transfections

For RNAi experiments, cells were seeded in six-well plates and reverse transfected with 20 nM of siRNA against tenascin-C (Ambion silencer select, 4390771; siRNA ID: s75240) or a negative control (Ambion, 4390846) or 50 nM of custom designed siRNA against ET-20l and ET-20s using Lipofectamine RNAiMax (Invitrogen). siET-20l and siET-20s were custom designed by Thermo Fisher Scientific using On Target Plus technology. Sequences were: murine siET-20l, 5′-GAAAGAGGCUGGAAGCAAAUU-3′; murine siET-20s, 5′-UGAGAUACCAGGAAAGAAAUU-3′; human sihET-20s-1, 5′-AUAUGCUUGACUUGUAUUGUU-3′; human sihET-20s-2, 5′-UAUCUCAGGACACUUCUAUUU-3′. Transfections were repeated after 2 days and cells were harvested for subsequent analysis after total 4 days of transfection. In EMT experiments, TGFβ was added at the time of second transfection for 2 days.

For transient overexpression experiments, cells in 6 cm dishes were transfected with 1 µg of pcDNA-HA-empty vector or pcDNA-HA-Sox4 vector (Tiwari et al., 2013) using Lipofectamine 3000. Cells were harvested for RNA isolation and subsequent RT-qPCR analysis after 72 h. For overexpression of ET-20, 5 µg of pcDNA3.1 ET-20l or ET-20s plasmid was used. Cells were harvested after 72 h of transfection. pCDNA3.1 ET-20l and ET-20s were generated by Thermo Fisher Scientific.

Py2T cells stably expressing HA-tagged Sox4 were generated by transduction with pLenti-HA-Sox4 as previously described (Tiwari et al., 2013).

EMT kinetics and RNA sequencing

The EMT time course and RNA sequencing have previously been described (Meyer-Schaller et al., 2019). Briefly, NMuMG (E9) cells were treated with 2 ng/ml TGFβ for 2 h, 6 h, 12 h, 24 h, 36 h, 48 h, 72 h, 96 h, 7 days or 10 days. Untreated cells served as control. Biological duplicates were prepared for RNA sequencing. Total RNA was isolated using the miRNeasy kit (Qiagen, 217004). 5 µg of RNA was subjected to rRNA depletion (Ribo-Zero rRNA removal kit, Epicentre, MRZG12324) and concentrated (MinElute PCR purification kit, Qiagen, 74204) followed by library preparation with the Scriptseq v2 kit (Illumina). All libraries were barcoded and 4-6-plex were used for sequencing with the TruSeq SBS Kit v3 (Illumina) on a HiSeq 2000 sequencer.

RNA sequencing analysis

Single-end RNA sequencing reads were mapped to the mouse genome assembly version mm10 with RNA-STAR (Dobin et al., 2013) (default parameters except for allowing only unique hits to the genome (outFilterMultimapNmax=1), and filtering reads without evidence in the spliced junction table (outFilterType=‘BySJout’). Raw reads and mapping quality were assessed by the qQCreport function from the R/Bioconductor software package QuasR (Gaidatzis et al., 2015). Using RefSeq mRNA coordinates from UCSC (https://genome.ucsc.edu/, downloaded in December 2015) and the qCount function from QuasR package, gene expression was quantified as the number of reads that started within any annotated exon of a gene (exon-union model). All RNA sequencing count data were analyzed using the R package DESeq2 (Love et al., 2014). The sequencing files have previously been deposited under GEO database GSE112797 (Meyer-Schaller et al., 2019).

Bioinformatic analyses

Genome browsers used were: UCSC genome browser (http://genome.ucsc.edu) using human (GRCh38/hg38, Dec. 2013), mouse (GRCm38/mm10, Dec. 2011), and mouse (GRCm39/mm39, Jun. 2020) assemblies (Lee et al., 2020); Ensembl browser (https://www.ensembl.org) (Cunningham et al., 2019); and NCBI gene and genome data viewer at National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/) (Sayers et al., 2020). Sequence similarity searches were carried out using BLAT within the UCSC browser and blastn at NCBI (Johnson et al., 2008; Kent, 2002).

RNA fluorescence in situ hybridization

FISH studies were performed using the Biosearch Stellaris protocol (see https://biosearchassets.blob.core.windows.net/assets/bti_stellaris_protocol_adherent_cell.pdf) for RNA-FISH on adherent cells. Briefly, cells grown on coverslips (#1, 15 mm round, Menzel-Glaser) were fixed with 4% formaldehyde in PBS for 15 min at room temperature, followed by permeabilization with 70% ethanol for 1 h. Hybridization with custom designed, CalFluor 610 conjugated FISH probes for ET-20l and ET-20s was carried out overnight at 37°C. Coverslips were then washed and counterstained with DAPI followed by mounting with Vectashield mounting medium (#H-1000, Vectalabs). The slides were imaged on a DMI8 inverted wide-field fluorescence microscope at 100X magnification. Z- stacks were taken and the maximum projection image was represented in the figures. CalFluor610-conjugated GAPDH mRNA probes were used as a positive control.

Co-localization analysis of ET-20 lncRNA and plakoglobin was performed by following the Biosearch Stellaris protocol for sequential immunofluorescence and FISH in adherent cells.

Immunofluorescence of cultured cells

Cells were grown on uncovered glass coverslips (#1, 12 mm round, Menzel-Glaser) and treated as indicated in the respective experiments. Cells were fixed with 4% paraformaldehyde for 20 min at room temperature, followed by permeabilization with 0.5% NP40 for 5 min and blocking with 3% BSA and 0.01% Triton X-100 in PBS for 30 min. Cells were then incubated with primary antibodies diluted in blocking solution at room temperature for 2 h followed by incubation with a fluorophore-coupled secondary antibody (Alexa Fluor, Invitrogen) for 1 h at room temperature in dark. Cell nuclei were counterstained with 5 ng/ml DAPI (Sigma-Aldrich, D9542). After staining, the coverslips were mounted in fluorescence mounting medium (Dako, S302380-2) on microscope slides and imaged using a fluorescence microscope (Leica DMI 4000).

Transwell migration and invasion assay

50,000 untreated or long-term (>20 days) TGFβ-treated cells were suspended in 500 μl of DMEM with 0.2% FBS and seeded into 24 transwell migration inserts (BD-Corning, 353097) or in Matrigel-covered invasion inserts (BD-Corning, 354483) in duplicates. The bottom chambers were filled with 700 μl of DMEM with 20% FBS to create a chemo-attractant gradient. The cells were incubated in a tissue culture incubator at 37°C with 5% CO2. After 18 h, inserts were fixed with 4% paraformaldehyde for 10 min. Cells that had not crossed the membrane were removed with a cotton swab, and cells on the bottom of the membrane were stained with DAPI. Images of five fields per insert were taken with a Leica DMI 4000 microscope. For quantification of the number of cells migrated or invaded, the following steps were performed on ImageJ: (i) Load .lif file in ImageJ with default settings; (ii) Image>Adjust>Threshold> set and apply; (iii) Process>Binary>Watershed; and (iv) Analyze>Analyze particles>Show outlines and summarize.

Chromatin immunoprecipitation

Chromatin immunoprecipitation (ChIP) experiments were performed as described previously (Cortázar et al., 2011). Briefly, crosslinked protein-bound DNA of Py2T-HA-Sox4 cells was sonicated (Bioruptor, Diagenode) to achieve chromatin fragments of size between 100 and 500 bps. For ChIP, at least 150 μg of chromatin was incubated with 10 μg of anti-HA-tag antibody (Cell Signaling Technology, 3724S) or normal rabbit IgG antibody (Cell Signaling Technology, 2729) and immunocomplexes were precipitated with 40 μl of pre-blocked Sepharose–Protein A beads (Affi-Prep Protein A Support, Bio-Rad; 1560006). Immunocomplexes were eluted from the beads, de-crosslinked and genomic DNA was purified by phenol/chloroform extraction and precipitated with sodium acetate. 1/40th of the ChIP sample and 1% of input DNA were used for quantitative RT-PCR. Fold enrichments for specific ET-20 promoter regions were calculated by determining the level of IP over input samples and normalized to isotype-specific IgG as negative control. Enrichment of HA–Sox4 at Tnc and Ezh2 promoters was used as a positive control. The SRY-binding sites for Sox4 on ET-20 promoter were identified by (i) performing a Matinspector analysis (Genomatix) on the promoter of ET-20 and (ii) by manually looking for the SRY motif (motif sequence was taken from JASPAR) along the promoter sequence. The promoter sequence of ET-20 was downloaded from UCSC Genome browser (mm10): 2500 bases upstream and 200 bases downstream of the transcription start site: >mm10_knownGene_uc008thj.1 range=chr4:63977354-64150124 5'pad=0 3'pad=0 strand=+ repeatMasking=none. Primers used in the ChIP experiments are listed in Table S3.

Chromatin isolation by RNA purification-mass spectrometry

The ChIRP experiment was performed as described previously with few modifications (Chu et al., 2012, 2015). Briefly, cells were seeded in 15 cm dishes and treated with TGFβ for 4 days; ∼20 million cells per sample were used in a ChIRP experiment. Cells were crosslinked with 1% formaldehyde for 30 min followed by harvesting and snap freezing. After cell lysis, sonication was carried out using a Bioruptor sonicator in 15 ml falcon tubes for 90 cycles (30 s ON, 30 s OFF). After preclearing of lysates, hybridization with custom designed biotinylated DNA tiling probes (LGC Biosearch Technology) against ET-20l was carried out overnight at 37°C. The probes were divided into odd and even pools and both were used in the pull-down experiment. Probes used for ChIRP experiment are listed in Table S4. RNase A-treated samples and incubation with probes against lacZ mRNA served as negative controls. Post hybridization, the samples were incubated for 1 h at 37°C with C-1 magnetic beads (Invitrogen, 65002). A tenth of the beads were taken for RNA elution to check for efficiency of RNA pulldown. The rest of the beads were taken for elution of proteins and subsequent mass spectrometric analysis. Protein elution was performed using biotin elution buffer (12.5 mM biotin, 7.5 mM HEPES pH 7.5, 75 mM NaCl, 1.5 mM EDTA, 0.15% SDS, 0.075% sarkosyl and 0.02% sodium deoxycholate). For the non-quantitative mass spectrometric runs, proteins were precipitated with 25% trichloroacetic acid. Sample preparation for quantitative mass spectrometry was performed essentially as described previously (Sharma et al., 2015). After protein elution from magnetic beads using biotin elution buffer, the eluent was subjected to methanol/chloroform precipitation (Wessel and Flügge, 1984). Proteins were then alkylated with 40 mM 2-chloroacetamide, diluted tenfold with digestion buffer (20 mM Tris-HCl, pH 8.5 and 10% acetonitrile) and digested with 1:50 (w/w) Lys-C (Wako) and 1:50 (w/w) trypsin (Promega) at 37°C overnight. The resulting peptide mixture was acidified by addition of 1% TFA and desalted on StageTips with three layers of SDB-RPS. Peptides were separated on 50-cm columns of ReproSil-Pur C18-AQ 1.9 μm resin (Dr. Maisch GmbH) packed in-house. Liquid chromatography was performed on an EASY-nLC 1000 ultrahigh-pressure system coupled through a nanoelectrospray source to a Q-Exactive mass spectrometer (all from Thermo Fisher Scientific). Peptides were loaded in buffer A (0.1% formic acid) and separated by applying a nonlinear gradient of 5–60% buffer B (0.1% formic acid, 80% acetonitrile) at a flow rate of 250 nl min−1 over 120 min. Data acquisition switched between a full scan and five data-dependent MS/MS scans. Multiple sequencing of peptides was minimized by excluding the selected peptide candidates for 15 s. Raw mass spectrometry data were analyzed with the MaxQuant workflow. Peak lists were searched against the Uniprot FASTA database combined with 262 common contaminants by the integrated Andromeda search engine. The false discovery rate (FDR) was set to 1% for both peptides (minimum length of 7 amino acids) and proteins. ‘Match between runs’ (MBR) with a maximum time difference of 0.7 min was enabled. Relative protein amounts were determined by the MaxLFQ algorithm, with a minimum ratio count of two peptides.

RNA immunoprecipitation

RIP was performed using the EZ-Magna RNA-Binding Protein Immunoprecipitation Kit (Catalog No. 17-701). PyMT-1099 cells treated with TGFβ for 2 days were used for the experiment. Pull down of U1 snRNA using the SNRNP70 antibody served as a positive control. Fold enrichment of ET-20 l pull-down with plakoglobin was calculated by determining the level of IP over input samples and normalized to isotype-specific IgG as negative control.

We thank P. Lorentz and the DBM microscopy facility (DBM, University of Basel) for support with microscopy. We are grateful to P. Jenoe and S. Moes for mass spectroscopy analysis, and to C. Beisel, K. Eschbach and the Genomics Facility Basel for library preparation and next-generation RNA Sequencing. We thank Prof. Nicola Aceto for providing us the CTC cell lines Brx16, Brx50 and also the MDA-MB-231 LM2 cells.

Author contributions

Conceptualization: M.S., G.C.; Methodology: M.S., M.H., M.N., M.D., R.I., K.S., R.K.R.K., T.R.B., S.R., G.C.; Software: M.S., R.I., R.K.R.K., T.R.B.; Validation: M.S., M.H., G.C.; Formal analysis: M.S., M.H., M.N., M.D., R.I., K.S., R.K.R.K., T.R.B., S.R., G.C.; Investigation: M.S., M.H., M.N., M.D., R.I., K.S., R.K.R.K., T.R.B., S.R., G.C.; Resources: G.C.; Data curation: M.S., M.H., M.N., M.D., R.I., K.S., R.K.R.K., T.R.B., S.R.; Writing - original draft: M.S.; Writing - review & editing: M.S., R.I., T.R.B., G.C.; Visualization: M.S., M.H., M.N., M.D., R.I., K.S., R.K.R.K., T.R.B., S.R., G.C.; Supervision: M.S., G.C.; Project administration: M.S., G.C.; Funding acquisition: G.C.

Funding

This work has been supported by the SystemsX.ch MTD project MetastasiX (2014/268), a project grant by the Swiss National Science Foundation (Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung) (310030B_163471), the Swiss Cancer Research Foundation (KFS-3479-08-2014), the Krebsliga Beider Basel (KlbB-4469-03-2018), and the Research Funds of the University of Basel.

The peer review history is available online at https://journals.biologists.com/jcs/article-lookup/doi/10.1242/jcs.258418.

Aiello
,
N. M.
,
Brabletz
,
T.
,
Kang
,
Y.
,
Nieto
,
M. A.
,
Weinberg
,
R. A.
and
Stanger
,
B. Z.
(
2017
).
Upholding a role for EMT in pancreatic cancer metastasis
.
Nature
547
,
E7
-
E8
.
Aigner
,
K.
,
Descovich
,
L.
,
Mikula
,
M.
,
Sultan
,
A.
,
Dampier
,
B.
,
Bonné
,
S.
,
van Roy
,
F.
,
Mikulits
,
W.
,
Schreiber
,
M.
,
Brabletz
,
T.
et al. 
(
2007
).
The transcription factor ZEB1 (δEF1) represses Plakophilin 3 during human cancer progression
.
FEBS Lett.
581
,
1617
-
1624
.
Aishima
,
S.
,
Taguchi
,
K.
,
Terashi
,
T.
,
Matsuura
,
S.
,
Shimada
,
M.
and
Tsuneyoshi
,
M.
(
2003
).
Tenascin expression at the invasive front is associated with poor prognosis in intrahepatic cholangiocarcinoma
.
Mod. Pathol.
16
,
1019
-
1027
.
Aslakson
,
C. J.
and
Miller
,
F. R.
(
1992
).
Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor
.
Cancer Res.
52
,
1399
-
1405
.
Batista
,
P. J.
and
Chang
,
H. Y.
(
2013
).
Long noncoding RNAs: cellular address codes in development and disease
.
Cell
152
,
1298
-
1307
.
Beaudry
,
V. G.
,
Jiang
,
D.
,
Dusek
,
R. L.
,
Park
,
E. J.
,
Knezevich
,
S.
,
Ridd
,
K.
,
Vogel
,
H.
,
Bastian
,
B. C.
and
Attardi
,
L. D.
(
2010
).
Loss of the p53/p63 regulated desmosomal protein Perp promotes tumorigenesis
.
PLoS Genet.
6
,
e1001168
.
Binder
,
A. K.
,
Rodriguez
,
K. F.
,
Hamilton
,
K. J.
,
Stockton
,
P. S.
,
Reed
,
C. E.
and
Korach
,
K. S.
(
2013
).
The absence of ER-β results in altered gene expression in ovarian granulosa cells isolated from in vivo preovulatory follicles
.
Endocrinology
154
,
2174
-
2187
.
Brabletz
,
T.
,
Kalluri
,
R.
,
Nieto
,
M. A.
and
Weinberg
,
R. A.
(
2018
).
EMT in cancer
.
Nat. Rev. Cancer
18
,
128
-
134
.
Brellier
,
F.
and
Chiquet-Ehrismann
,
R.
(
2012
).
How do tenascins influence the birth and life of a malignant cell?
J. Cell. Mol. Med.
16
,
32
-
40
.
Cai
,
J.
,
Du
,
S.
,
Wang
,
H.
,
Xin
,
B.
,
Wang
,
J.
,
Shen
,
W.
,
Wei
,
W.
,
Guo
,
Z.
and
Shen
,
X.
(
2017
).
Tenascin-C induces migration and invasion through JNK/c-Jun signalling in pancreatic cancer
.
Oncotarget
8
,
74406
-
74422
.
Chaffer
,
C. L.
and
Weinberg
,
R. A.
(
2011
).
A perspective on cancer cell metastasis
.
Science
331
,
1559
-
1564
.
Chu
,
C.
,
Quinn
,
J.
and
Chang
,
H. Y.
(
2012
).
Chromatin isolation by RNA purification (ChIRP)
.
J. Vis. Exp.
61
,
e3912
.
Chu
,
C.
,
Zhang
,
Q. C.
,
da Rocha
,
S. T.
,
Flynn
,
R. A.
,
Bharadwaj
,
M.
,
Calabrese
,
J. M.
,
Magnuson
,
T.
,
Heard
,
E.
and
Chang
,
H. Y.
(
2015
).
Systematic discovery of Xist RNA binding proteins
.
Cell
161
,
404
-
416
.
Chun
,
M. G. H.
and
Hanahan
,
D.
(
2010
).
Genetic deletion of the desmosomal component desmoplakin promotes tumor microinvasion in a mouse model of pancreatic neuroendocrine carcinogenesis
.
PLoS Genet.
6
,
e1001120
.
Cortázar
,
D.
,
Kunz
,
C.
,
Selfridge
,
J.
,
Lettieri
,
T.
,
Saito
,
Y.
,
MacDougall
,
E.
,
Wirz
,
A.
,
Schuermann
,
D.
,
Jacobs
,
A. L.
,
Siegrist
,
F.
et al. 
(
2011
).
Embryonic lethal phenotype reveals a function of TDG in maintaining epigenetic stability
.
Nature
470
,
419
-
423
.
Cunningham
,
F.
,
Achuthan
,
P.
,
Akanni
,
W.
,
Allen
,
J.
,
Amode
,
M. R.
,
Armean
,
I. M.
,
Bennett
,
R.
,
Bhai
,
J.
,
Billis
,
K.
,
Boddu
,
S.
et al. 
(
2019
).
Ensembl 2019
.
Nucleic Acids Res.
47
,
D745
-
D751
.
De Craene
,
B.
and
Berx
,
G.
(
2013
).
Regulatory networks defining EMT during cancer initiation and progression
.
Nat. Rev. Cancer
13
,
97
-
110
.
Dobin
,
A.
,
Davis
,
C. A.
,
Schlesinger
,
F.
,
Drenkow
,
J.
,
Zaleski
,
C.
,
Jha
,
S.
,
Batut
,
P.
,
Chaisson
,
M.
and
Gingeras
,
T. R.
(
2013
)
STAR: ultrafast universal RNA-seq aligner
.
Bioinformatics
29
,
15
-
21
.
Dongre
,
A.
and
Weinberg
,
R. A.
(
2019
).
New insights into the mechanisms of epithelial-mesenchymal transition and implications for cancer
.
Nat. Rev. Mol. Cell Biol.
20
,
69
-
84
.
Dusek
,
R. L.
and
Attardi
,
L. D.
(
2011
).
Desmosomes: new perpetrators in tumour suppression
.
Nat. Rev. Cancer
11
,
317
-
323
.
Evans
,
J. R.
,
Feng
,
F. Y.
and
Chinnaiyan
,
A. M.
(
2016
).
The bright side of dark matter: lncRNAs in cancer
.
J. Clin. Invest.
126
,
2775
-
2782
.
Fatica
,
A.
and
Bozzoni
,
I.
(
2014
).
Long non-coding RNAs: new players in cell differentiation and development
.
Nat. Rev. Genet.
15
,
7
-
21
.
Fischer
,
K. R.
,
Durrans
,
A.
,
Lee
,
S.
,
Sheng
,
J.
,
Li
,
F.
,
Wong
,
S. T. C.
,
Choi
,
H.
,
El Rayes
,
T.
,
Ryu
,
S.
,
Troeger
,
J.
et al. 
(
2015
).
Epithelial-to-mesenchymal transition is not required for lung metastasis but contributes to chemoresistance
.
Nature
527
,
472
-
476
.
Gaidatzis
,
D.
,
Lerch
,
A.
,
Hahne
,
F.
and
Stadler
,
M. B.
(
2015
).
QuasR: quantification and annotation of short reads in R
.
Bioinformatics
31
,
1130
-
1132
.
Gil
,
N.
and
Ulitsky
,
I.
(
2020
).
Regulation of gene expression by cis-acting long non-coding RNAs
.
Nat. Rev. Genet.
21
,
102
-
117
.
Grelet
,
S.
,
Link
,
L. A.
,
Howley
,
B.
,
Obellianne
,
C.
,
Palanisamy
,
V.
,
Gangaraju
,
V. K.
,
Diehl
,
J. A.
and
Howe
,
P. H.
(
2017
).
A regulated PNUTS mRNA to lncRNA splice switch mediates EMT and tumour progression
.
Nat. Cell Biol.
19
,
1105
-
1115
.
Gutschner
,
T.
and
Diederichs
,
S.
(
2012
).
The hallmarks of cancer: a long non-coding RNA point of view
.
RNA Biol.
9
,
703
-
719
.
Hao
,
Y.
,
Baker
,
D.
and
Ten Dijke
,
P.
(
2019
).
TGF-beta-mediated epithelial-mesenchymal transition and cancer metastasis
.
Int. J. Mol. Sci.
20
,
2767
.
Huang
,
R. Y.-J.
,
Guilford
,
P.
and
Thiery
,
J. P.
(
2012
).
Early events in cell adhesion and polarity during epithelial-mesenchymal transition
.
J. Cell Sci.
125
,
4417
-
4422
.
Iyer
,
M. K.
,
Niknafs
,
Y. S.
,
Malik
,
R.
,
Singhal
,
U.
,
Sahu
,
A.
,
Hosono
,
Y.
,
Barrette
,
T. R.
,
Prensner
,
J. R.
,
Evans
,
J. R.
,
Zhao
,
S.
et al. 
(
2015
).
The landscape of long noncoding RNAs in the human transcriptome
.
Nat. Genet.
47
,
199
-
208
.
Jahkola
,
T.
,
Toivonen
,
T.
,
von Smitten
,
K.
,
Blomqvist
,
C.
and
Virtanen
,
I.
(
1996
).
Expression of tenascin in invasion border of early breast cancer correlates with higher risk of distant metastasis
.
Int. J. Cancer
69
,
445
-
447
.
Jahkola
,
T.
,
Toivonen
,
T.
,
Nordling
,
S.
,
von Smitten
,
K.
and
Virtanen
,
I.
(
1998a
).
Expression of tenascin-C in intraductal carcinoma of human breast: relationship to invasion
.
Eur. J. Cancer
34
,
1687
-
1692
.
Jahkola
,
T.
,
Toivonen
,
T.
,
Virtanen
,
I.
,
von Smitten
,
K.
,
Nordling
,
S.
,
von Boguslawski
,
K.
,
Haglund
,
C.
,
Nevanlinna
,
H.
and
Blomqvist
,
C.
(
1998b
).
Tenascin-C expression in invasion border of early breast cancer: a predictor of local and distant recurrence
.
Br. J. Cancer
78
,
1507
-
1513
.
Johnson
,
M.
,
Zaretskaya
,
I.
,
Raytselis
,
Y.
,
Merezhuk
,
Y.
,
McGinnis
,
S.
and
Madden
,
T. L.
(
2008
).
NCBI BLAST: a better web interface
.
Nucleic Acids Res.
36
,
W5
-
W9
.
Johnson
,
J. L.
,
Najor
,
N. A.
and
Green
,
K. J.
(
2014
).
Desmosomes: regulators of cellular signaling and adhesion in epidermal health and disease
.
Cold Spring Harb. Perspect. Med.
4
,
a015297
.
Kaarteenaho-Wiik
,
R.
,
Soini
,
Y.
,
Pöllänen
,
R.
,
Pääkkö
,
P.
and
Kinnula
,
V. L.
(
2003
).
Over-expression of tenascin-C in malignant pleural mesothelioma
.
Histopathology
42
,
280
-
291
.
Kallen
,
A. N.
,
Zhou
,
X.-B.
,
Xu
,
J.
,
Qiao
,
C.
,
Ma
,
J.
,
Yan
,
L.
,
Lu
,
L.
,
Liu
,
C.
,
Yi
,
J.-S.
,
Zhang
,
H.
et al. 
(
2013
).
The imprinted H19 lncRNA antagonizes let-7 microRNAs
.
Mol. Cell
52
,
101
-
112
.
Katoh
,
D.
,
Nagaharu
,
K.
,
Shimojo
,
N.
,
Hanamura
,
N.
,
Yamashita
,
M.
,
Kozuka
,
Y.
,
Imanaka-Yoshida
,
K.
and
Yoshida
,
T.
(
2013
).
Binding of alphavbeta1 and alphavbeta6 integrins to tenascin-C induces epithelial-mesenchymal transition-like change of breast cancer cells
.
Oncogenesis
2
,
e65
.
Kent
,
W. J.
(
2002
).
BLAT--the BLAST-like alignment tool
.
Genome Res.
12
,
656
-
664
.
Kong
,
L.
,
Zhang
,
Y.
,
Ye
,
Z.-Q.
,
Liu
,
X.-Q.
,
Zhao
,
S.-Q.
,
Wei
,
L.
and
Gao
,
G.
(
2007
).
CPC: assess the protein-coding potential of transcripts using sequence features and support vector machine
.
Nucleic Acids Res.
35
,
W345
-
W349
.
Kopp
,
F.
and
Mendell
,
J. T.
(
2018
).
Functional classification and experimental dissection of long noncoding RNAs
.
Cell
172
,
393
-
407
.
Krebs
,
A. M.
,
Mitschke
,
J.
,
Lasierra Losada
,
M.
,
Schmalhofer
,
O.
,
Boerries
,
M.
,
Busch
,
H.
,
Boettcher
,
M.
,
Mougiakakos
,
D.
,
Reichardt
,
W.
,
Bronsert
,
P.
et al. 
(
2017
).
The EMT-activator Zeb1 is a key factor for cell plasticity and promotes metastasis in pancreatic cancer
.
Nat. Cell Biol.
19
,
518
-
529
.
Lamouille
,
S.
,
Xu
,
J.
and
Derynck
,
R.
(
2014
).
Molecular mechanisms of epithelial-mesenchymal transition
.
Nat. Rev. Mol. Cell Biol.
15
,
178
-
196
.
Lee
,
C. M.
,
Barber
,
G. P.
,
Casper
,
J.
,
Clawson
,
H.
,
Diekhans
,
M.
,
Gonzalez
,
J. N.
,
Hinrichs
,
A. S.
,
Lee
,
B. T.
,
Nassar
,
L. R.
,
Powell
,
C. C.
et al. 
(
2020
).
UCSC genome browser enters 20th year
.
Nucleic Acids Res.
48
,
D756
-
D761
.
Lizio
,
M.
,
Harshbarger
,
J.
,
Shimoji
,
H.
,
Severin
,
J.
,
Kasukawa
,
T.
,
Sahin
,
S.
,
Abugessaisa
,
I.
,
Fukuda
,
S.
,
Hori
,
F.
,
Ishikawa-Kato
,
S.
et al. 
(
2015
).
Gateways to the FANTOM5 promoter level mammalian expression atlas
.
Genome Biol.
16
,
22
.
Lizio
,
M.
,
Abugessaisa
,
I.
,
Noguchi
,
S.
,
Kondo
,
A.
,
Hasegawa
,
A.
,
Hon
,
C. C.
,
de Hoon
,
M.
,
Severin
,
J.
,
Oki
,
S.
,
Hayashizaki
,
Y.
et al. 
(
2019
).
Update of the FANTOM web resource: expansion to provide additional transcriptome atlases
.
Nucleic Acids Res.
47
,
D752
-
D758
.
Love
,
M. I.
,
Huber
,
W.
and
Anders
,
S.
(
2014
).
Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2
.
Genome Biol.
15
,
550
.
Lowy
,
C. M.
and
Oskarsson
,
T.
(
2015
).
Tenascin C in metastasis: a view from the invasive front
.
Cell Adh. Migr.
9
,
112
-
124
.
Lu
,
W.
and
Kang
,
Y.
(
2019
).
Epithelial-mesenchymal plasticity in cancer progression and metastasis
.
Dev. Cell
49
,
361
-
374
.
Maeda
,
M.
,
Johnson
,
K. R.
and
Wheelock
,
M. J.
(
2005
).
Cadherin switching: essential for behavioral but not morphological changes during an epithelium-to-mesenchyme transition
.
J. Cell Sci.
118
,
873
-
887
.
Mercer
,
T. R.
,
Dinger
,
M. E.
and
Mattick
,
J. S.
(
2009
).
Long non-coding RNAs: insights into functions
.
Nat. Rev. Genet.
10
,
155
-
159
.
Meyer-Schaller
,
N.
,
Cardner
,
M.
,
Diepenbruck
,
M.
,
Saxena
,
M.
,
Tiede
,
S.
,
Luond
,
F.
,
Ivanek
,
R.
,
Beerenwinkel
,
N.
and
Christofori
,
G.
(
2019
).
A hierarchical regulatory landscape during the multiple stages of EMT
.
Dev. Cell
48
,
539
-
553.e6
.
Morris
,
K. V.
and
Mattick
,
J. S.
(
2014
).
The rise of regulatory RNA
.
Nat. Rev. Genet.
15
,
423
-
437
.
Nagaharu
,
K.
,
Zhang
,
X.
,
Yoshida
,
T.
,
Katoh
,
D.
,
Hanamura
,
N.
,
Kozuka
,
Y.
,
Ogawa
,
T.
,
Shiraishi
,
T.
and
Imanaka-Yoshida
,
K.
(
2011
).
Tenascin C induces epithelial-mesenchymal transition-like change accompanied by SRC activation and focal adhesion kinase phosphorylation in human breast cancer cells
.
Am. J. Pathol.
178
,
754
-
763
.
Nieto
,
M. A.
,
Huang
,
R. Y.-J.
,
Jackson
,
R. A.
and
Thiery
,
J. P.
(
2016
).
Emt: 2016
.
Cell
166
,
21
-
45
.
Oskarsson
,
T.
,
Acharyya
,
S.
,
Zhang
,
X. H.-F.
,
Vanharanta
,
S.
,
Tavazoie
,
S. F.
,
Morris
,
P. G.
,
Downey
,
R. J.
,
Manova-Todorova
,
K.
,
Brogi
,
E.
and
Massagué
,
J.
(
2011
).
Breast cancer cells produce tenascin C as a metastatic niche component to colonize the lungs
.
Nat. Med.
17
,
867
-
874
.
Padmanaban
,
V.
,
Krol
,
I.
,
Suhail
,
Y.
,
Szczerba
,
B. M.
,
Aceto
,
N.
,
Bader
,
J. S.
and
Ewald
,
A. J.
(
2019
).
E-cadherin is required for metastasis in multiple models of breast cancer
.
Nature
573
,
439
-
444
.
Quinn
,
J. J.
and
Chang
,
H. Y.
(
2016
).
Unique features of long non-coding RNA biogenesis and function
.
Nat. Rev. Genet.
17
,
47
-
62
.
Ransohoff
,
J. D.
,
Wei
,
Y.
and
Khavari
,
P. A.
(
2018
).
The functions and unique features of long intergenic non-coding RNA
.
Nat. Rev. Mol. Cell Biol.
19
,
143
-
157
.
Richards
,
E. J.
,
Zhang
,
G.
,
Li
,
Z.-P.
,
Permuth-Wey
,
J.
,
Challa
,
S.
,
Li
,
Y.
,
Kong
,
W.
,
Dan
,
S.
,
Bui
,
M. M.
,
Coppola
,
D.
et al. 
(
2015
).
Long non-coding RNAs (LncRNA) regulated by transforming growth factor (TGF) beta: LncRNA-hit-mediated TGFbeta-induced epithelial to mesenchymal transition in mammary epithelia
.
J. Biol. Chem.
290
,
6857
-
6867
.
Rinn
,
J. L.
and
Chang
,
H. Y.
(
2012
).
Genome regulation by long noncoding RNAs
.
Annu. Rev. Biochem.
81
,
145
-
166
.
Savagner
,
P.
,
Kusewitt
,
D. F.
,
Carver
,
E. A.
,
Magnino
,
F.
,
Choi
,
C.
,
Gridley
,
T.
and
Hudson
,
L. G.
(
2005
).
Developmental transcription factor slug is required for effective re-epithelialization by adult keratinocytes
.
J. Cell. Physiol.
202
,
858
-
866
.
Saxena
,
M.
,
Kalathur
,
R. K. R.
,
Neutzner
,
M.
and
Christofori
,
G.
(
2018
).
PyMT-1099, a versatile murine cell model for EMT in breast cancer
.
Sci. Rep.
8
,
12123
.
Sayers
,
E. W.
,
Beck
,
J.
,
Brister
,
J. R.
,
Bolton
,
E. E.
,
Canese
,
K.
,
Comeau
,
D. C.
,
Funk
,
K.
,
Ketter
,
A.
,
Kim
,
S.
,
Kimchi
,
A.
et al. 
(
2020
).
Database resources of the National Center for Biotechnology Information
.
Nucleic Acids Res.
48
,
D9
-
D16
.
Scharer
,
C. D.
,
McCabe
,
C. D.
,
Ali-Seyed
,
M.
,
Berger
,
M. F.
,
Bulyk
,
M. L.
and
Moreno
,
C. S.
(
2009
).
Genome-wide promoter analysis of the SOX4 transcriptional network in prostate cancer cells
.
Cancer Res.
69
,
709
-
717
.
Schmitt
,
A. M.
and
Chang
,
H. Y.
(
2016
).
Long noncoding RNAs in cancer pathways
.
Cancer Cell
29
,
452
-
463
.
Sharma
,
K.
,
Schmitt
,
S.
,
Bergner
,
C. G.
,
Tyanova
,
S.
,
Kannaiyan
,
N.
,
Manrique-Hoyos
,
N.
,
Kongi
,
K.
,
Cantuti
,
L.
,
Hanisch
,
U.-K.
,
Philips
,
M.-A.
et al. 
(
2015
).
Cell type- and brain region-resolved mouse brain proteome
.
Nat. Neurosci.
18
,
1819
-
1831
.
Slack
,
F. J.
and
Chinnaiyan
,
A. M.
(
2019
).
The role of non-coding RNAs in oncology
.
Cell
179
,
1033
-
1055
.
Soule
,
H. D.
,
Maloney
,
T. M.
,
Wolman
,
S. R.
,
Peterson
,
W. D.
,
Jr., Brenz
,
R.
,
McGrath
,
C. M.
,
Russo
,
J.
,
Pauley
,
R. J.
,
Jones
,
R. F.
and
Brooks
,
S. C.
(
1990
).
Isolation and characterization of a spontaneously immortalized human breast epithelial cell line, MCF-10
.
Cancer Res.
50
,
6075
-
6086
.
Sun
,
Z.
,
Schwenzer
,
A.
,
Rupp
,
T.
,
Murdamoothoo
,
D.
,
Vegliante
,
R.
,
Lefebvre
,
O.
,
Klein
,
A.
,
Hussenet
,
T.
and
Orend
,
G.
(
2018
).
Tenascin-C promotes tumor cell migration and metastasis through integrin alpha9beta1-mediated YAP inhibition
.
Cancer Res.
78
,
950
-
961
.
Takahashi
,
Y.
,
Sawada
,
G.
,
Kurashige
,
J.
,
Matsumura
,
T.
,
Uchi
,
R.
,
Ueo
,
H.
,
Ishibashi
,
M.
,
Takano
,
Y.
,
Akiyoshi
,
S.
,
Iwaya
,
T.
et al. 
(
2013
).
Tumor-derived tenascin-C promotes the epithelial-mesenchymal transition in colorectal cancer cells
.
Anticancer Res.
33
,
1927
-
1934
.
Thiery
,
J. P.
,
Acloque
,
H.
,
Huang
,
R. Y. J.
and
Nieto
,
M. A.
(
2009
).
Epithelial-mesenchymal transitions in development and disease
.
Cell
139
,
871
-
890
.
Tiwari
,
N.
,
Gheldof
,
A.
,
Tatari
,
M.
and
Christofori
,
G.
(
2012
).
EMT as the ultimate survival mechanism of cancer cells
.
Semin. Cancer Biol.
22
,
194
-
207
.
Tiwari
,
N.
,
Tiwari
,
V. K.
,
Waldmeier
,
L.
,
Balwierz
,
P. J.
,
Arnold
,
P.
,
Pachkov
,
M.
,
Meyer-Schaller
,
N.
,
Schubeler
,
D.
,
van Nimwegen
,
E.
and
Christofori
,
G.
(
2013
).
Sox4 is a master regulator of epithelial-mesenchymal transition by controlling Ezh2 expression and epigenetic reprogramming
.
Cancer Cell
23
,
768
-
783
.
Ulitsky
,
I.
(
2016
).
Evolution to the rescue: using comparative genomics to understand long non-coding RNAs
.
Nat. Rev. Genet.
17
,
601
-
614
.
Ulitsky
,
I.
and
Bartel
,
D. P.
(
2013
).
lincRNAs: genomics, evolution, and mechanisms
.
Cell
154
,
26
-
46
.
Vandewalle
,
C.
,
Comijn
,
J.
,
De Craene
,
B.
,
Vermassen
,
P.
,
Bruyneel
,
E.
,
Andersen
,
H.
,
Tulchinsky
,
E.
,
Van Roy
,
F.
and
Berx
,
G.
(
2005
).
SIP1/ZEB2 induces EMT by repressing genes of different epithelial cell-cell junctions
.
Nucleic Acids Res.
33
,
6566
-
6578
.
Waldmeier
,
L.
,
Meyer-Schaller
,
N.
,
Diepenbruck
,
M.
and
Christofori
,
G.
(
2012
).
Py2T murine breast cancer cells, a versatile model of TGFbeta-induced EMT in vitro and in vivo
.
PLoS One
7
,
e48651
.
Wessel
,
D.
and
Flügge
,
U. I.
(
1984
).
A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids
.
Anal. Biochem.
138
,
141
-
143
.
Yang
,
J.
,
Antin
,
P.
,
Berx
,
G.
,
Blanpain
,
C.
,
Brabletz
,
T.
,
Bronner
,
M.
,
Campbell
,
K.
,
Cano
,
A.
,
Casanova
,
J.
,
Christofori
,
G.
et al. 
(
2020
).
Guidelines and definitions for research on epithelial-mesenchymal transition
.
Nat. Rev. Mol. Cell Biol.
21
,
341
-
352
.
Yao
,
R.-W.
,
Wang
,
Y.
and
Chen
,
L.-L.
(
2019
).
Cellular functions of long noncoding RNAs
.
Nat. Cell Biol.
21
,
542
-
551
.
Ye
,
X.
,
Brabletz
,
T.
,
Kang
,
Y.
,
Longmore
,
G. D.
,
Nieto
,
M. A.
,
Stanger
,
B. Z.
,
Yang
,
J.
and
Weinberg
,
R. A.
(
2017
).
Upholding a role for EMT in breast cancer metastasis
.
Nature
547
,
E1
-
E3
.
Yuan
,
J.-H.
,
Yang
,
F.
,
Wang
,
F.
,
Ma
,
J.-Z.
,
Guo
,
Y.-J.
,
Tao
,
Q.-F.
,
Liu
,
F.
,
Pan
,
W.
,
Wang
,
T.-T.
,
Zhou
,
C.-C.
et al. 
(
2014
).
A long noncoding RNA activated by TGF-beta promotes the invasion-metastasis cascade in hepatocellular carcinoma
.
Cancer Cell
25
,
666
-
681
.
Zheng
,
X.
,
Carstens
,
J. L.
,
Kim
,
J.
,
Scheible
,
M.
,
Kaye
,
J.
,
Sugimoto
,
H.
,
Wu
,
C.-C.
,
LeBleu
,
V. S.
and
Kalluri
,
R.
(
2015
).
Epithelial-to-mesenchymal transition is dispensable for metastasis but induces chemoresistance in pancreatic cancer
.
Nature
527
,
525
-
530
.

Competing interests

The authors declare no competing or financial interests.

Supplementary information