Article Text

Original article
TBL1XR1 promotes lymphangiogenesis and lymphatic metastasis in esophageal squamous cell carcinoma
  1. Liping Liu1,2,3,
  2. Chuyong Lin1,
  3. Weijiang Liang4,
  4. Shu Wu1,
  5. Aibin Liu5,
  6. Jueheng Wu6,
  7. Xin Zhang1,
  8. Pengli Ren5,
  9. Mengfeng Li6,
  10. Libing Song1
  1. 1State Key Laboratory of Oncology in Southern China, Department of Experimental Research, Cancer Center, Sun Yat-sen University, Guangzhou, Guangdong, China
  2. 2Department of Cardiothoracic Surgery, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China
  3. 3Guangzhou Research Institute of Respiratory Disease & China State Key Laboratory of Respiratory Disease, Guangzhou, Guangdong, China
  4. 4Department of Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China
  5. 5Department of Biochemistry, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China
  6. 6Department of Microbiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China
  1. Correspondence to Professor Libing Song, State Key Laboratory of Oncology in Southern China and Department of Experimental Research, Sun Yat-sen University Cancer Center, Guangzhou 510060, China; lb.song1{at}gmail.com

Abstract

Objective Transducin (β)-like 1 X-linked receptor 1 (TBL1XR1) plays an important role in controlling the precisely regulated switch between gene repression and gene activation in transcriptional regulation. We investigated its biological function and clinical significance in esophageal squamous cell carcinoma (ESCC).

Design Immunoblotting and immunochemistry were used to determine TBL1XR1 expression in ESCC cell lines, ESCC clinical tissues and 230 clinicopathologically characterised ESCC specimens. The role of TBL1XR1 in lymphangiogenesis and lymphatic metastasis was examined by tube formation, cell invasion and wound-healing assays in vitro, and by a popliteal lymph node metastasis model in vivo. The molecular mechanism by which TBL1XR1 upregulates vascular endothelial growth factor C (VEGF-C) expression was explored using real-time PCR, ELISA, luciferase reporter assay and chromatin immunoprecipitation.

Results TBL1XR1 expression was significantly upregulated in ESCC, positively correlated with disease stage and patient survival, and identified as an independent prognostic factor for patient outcome. We found that TBL1XR1 overexpression promoted lymphangiogenesis and lymphatic metastasis in ESCC in vitro and in vivo, whereas TBL1XR1 silencing had the converse effect. We demonstrated that TBL1XR1 induced VEGF-C expression by binding to the VEGF-C promoter. We confirmed the correlation between TBL1XR1 and VEGF-C expression in a large cohort of clinical ESCC samples and through analysis of published datasets in gastric, colorectal and breast cancer.

Conclusions Our results demonstrated that TBL1XR1 induced lymphangiogenesis and lymphatic metastasis in ESCC via upregulation of VEGF-C, and may represent a novel prognostic biomarker and therapeutic target for patients with ESCC.

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Significance of this study

What is already known about this subject?

  • Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive and lethal human malignancies. The poor outcome of patients with ESCC is attributed to the high rates of regional lymph node metastasis and distant metastasis.

  • Vascular endothelial growth factor C (VEGF-C) is a putative lymphangiogenic growth factor that has been identified as an independent prognostic factor in ESCC.

  • Although studies have demonstrated that TBL1XR1 controls the precisely regulated switch from gene repression to gene activation, its clinical significance and biological role in the progression of ESCC remain largely unknown.

What are the new findings?

  • TBL1XR1 was upregulated in ESCC and positively correlated with ESCC progression.

  • Patients with higher TBL1XR1 expression had shorter overall survival time.

  • Overexpression of TBL1XR1 promoted lymphangiogenesis and lymphatic metastasis in ESCC in vitro and in vivo, whereas downregulation of TBL1XR1 suppressed these effects.

  • TBL1XR1 induced VEGF-C expression by binding to the VEGF-C promoter.

How might it impact on clinical practice in the foreseeable future?

  • By highlighting an important oncogenic role for TBL1XR1 through promotion of lymphangiogenesis and lymphatic metastasis in ESCC, our results suggest that TBL1XR1 is a potential novel prognostic marker and therapeutic target for the treatment of patients with ESCC.

Introduction

Human esophageal squamous cell carcinoma (ESCC) is one of the most aggressive and lethal malignancies.1 Although the diagnosis, staging and treatment of ESCC have improved over the past three decades, the prognosis remains poor. The overall 5-year survival rate in patients with stage III disease is 10–15% and the median survival time in patients with stage IV disease is less than 1 year.1–3 The poor clinical outcome of patients with ESCC is attributed to the high rates of local invasion and regional lymph node metastasis. The early occurrence of lymphatic metastasis is considered a crucial step in the development of distant metastasis in ESCC.4 ,5

Lymphangiogenesis is the growth of new lymphatic vessels and plays an important role in various physiological and pathological processes, including metastasis.6 ,7 Tumour-induced lymphangiogenesis has been reported to enhance the development of lymphatic vessels within and near tumours, thereby promoting the spread of tumour cells to regional lymph nodes.8 Tumours provoke the growth of lymphatic vessels by secreting lymphangiogenic growth factors, such as those in the vascular endothelial growth factor (VEGF) receptor family.9 ,10 As the first identified putative lymphangiogenic growth factor, VEGF-C stimulates proliferation and migration of lymphatic endothelial cells and induces vascular permeability by binding to and activating VEGFR-3.8 ,11 Studies have consistently demonstrated that VEGF-C is overexpressed and positively correlated with lymphangiogenesis and lymph node metastasis in a variety of malignancies, including ESCC, gastric cancer, colorectal cancer, lung cancer and breast cancer.12–18 Importantly, blocking the VEGF-C/VEGFR-3 lymphangiogenic axis has been shown to reduce the rate of lymph node metastasis by 60–70% in a mouse model.19 ,20 These reports indicate that VEGF-C plays a central role in lymphangiogenesis and lymph node metastasis, and may therefore be an effective therapeutic target for anticancer strategies.

Transducin (β)-like 1 X-linked receptor 1 (TBL1XR1) was initially identified as a component of the SMRT/N-CoR corepressor complex and demonstrated to control the precisely regulated switch from gene repression to gene activation in response to nuclear receptors and other transcriptional regulators.21–26 TBL1XR1 is an F-box/WD-40 domain-containing protein reported to act as an E3-ligase in the recruitment of the ubiquitin conjugating/19S proteasome complex, thereby leading to proteasomal degradation of the SMRT/N-CoR complex to promote corepressor/coactivator exchange and subsequent transcriptional activation.21 ,23 ,27 Although overexpression of TBL1XR1 has been implicated in cancer progression, and has been found to be upregulated in squamous cell lung carcinoma and breast cancer,28 ,29 the biological role and clinical significance of TBL1XR1 in ESCC remain largely unknown.

We found that TBL1XR1 was markedly upregulated in ESCC and that its overexpression was significantly correlated with the clinicopathological characteristics and survival of patients with ESCC. Overexpression of TBL1XR1 provoked, whereas silencing TBL1XR1 abrogated, lymphangiogenesis and lymphatic metastasis in ESCC in vitro and in vivo. We further demonstrated that TBL1XR1 transcriptionally upregulated VEGF-C by binding to the VEGF-C promoter. Our findings suggest that TBL1XR1 plays a key role in lymphangiogenesis and lymphatic metastasis in ESCC, and might be a novel prognostic biomarker and potential therapeutic target in the treatment of patients with this malignancy.

Materials and methods

Cell lines and cell culture

Primary cultures of normal esophageal epithelial cells (NEECs) were established from fresh specimens of adjacent non-cancerous oesophageal tissue >5 cm from the cancerous tissue, as previously described.30 Human lymphatic endothelial cells (HLECs) were purchased from ScienCell Research Laboratories (Carlsbad, California , USA) and cultured according to the manufacturer's instructions. The ESCC cell lines EC18, Eca109 and HKESC1 were kindly provided by Professors S.W. Tsao and G. Srivastava (University of Hong Kong). The ESCC cell lines KYSE-30, KYSE-140, KYSE-180, KYSE-410, KYSE-510 and KYSE-520 were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), the German Resource Centre for Biological Material.31 The cells were grown in Dulbecco's modified Eagle medium (Invitrogen, Carlsbad, California , USA) supplemented with 10% fetal bovine serum (HyClone, Logan, Utah, USA).

Tissue specimens and clinicopathological characteristics

The total 230 paraffin-embedded, archived ESCC samples used in this study were histopathologically and clinically diagnosed at the Sun Yat-sen University Cancer Center between 2003 and 2009. Written informed consent was obtained from all patients prior to the study. The use of the clinical specimens for research purposes was approved by the Institutional Research Ethics Committee. The clinicopathological characteristics of the samples are summarised in online supplementary table S1. Eight freshly collected ESCC tissues and matched adjacent non-tumour oesophageal tissues from the same patient were frozen and stored in liquid nitrogen until required.

Western blotting analysis

Western blotting (WB) was performed as previously described,32 using anti-TBL1XR1 antibody (Abcam, Cambridge, Massachusetts, USA), anti-phospho-AKT (Ser 473), anti-AKT, anti-p-ERK1/2 (T202/Y204) and anti-ERK antibodies (Cell Signaling, Danvers, Massachusetts, USA). Following the initial western blot assay, the membranes were stripped and re-probed with anti-α-tubulin antibody (Sigma, Saint Louis, Missouri, USA) as a protein loading control.

Immunohistochemistry

Immunohistochemistry (IHC) analysis was performed on the 230 paraffin-embedded ESCC tissue sections as previously described.32 The degree of immunostaining of formalin-fixed, paraffin-embedded sections was reviewed and scored separately by two independent pathologists. The scores were determined by combining the proportion of positively stained tumour cells and the intensity of staining. Tumour cell proportions were scored as follows: 0, no positive tumour cells; 1, <10% positive tumour cells; 2, 10–35% positive tumour cells; 3, 35–75% positive tumour cells; 4, >75% positive tumour cells. Staining intensity was graded according to the following standard: 1, no staining; 2, weak staining (light yellow); 3, moderate staining (yellow brown); 4, strong staining (brown). The staining index (SI) was calculated as the product of the staining intensity score and the proportion of positive tumour cells. Using this method of assessment, we evaluated protein expression in benign oesophageal epithelia and malignant lesions by determining the SI, with possible scores of 0, 2, 3, 4, 6, 8, 9, 12 and 16. Samples with a SI ≥ 8 were determined as high expression and samples with a SI<8 were determined as low expression. Cutoff values were determined on the basis of a measure of heterogeneity using the log-rank test with respect to overall survival.

Plasmids, virus constructs and retroviral infection of target cells

Human TBL1XR1 was amplified by PCR and cloned into the pSin-EF2 vector. Knockout of endogenous TBL1XR1 was performed by cloning two short hairpin RNA (shRNA) oligonucleotides into the pSuper-retro-puro vector to generate pSuper-retro-TBL1XR1-RNAi(s). Fragments of human VEGF-C promoter, generated by PCR amplification, were cloned into the pGL3 luciferase reporter plasmid (Promega, Madison, Wisconsin, USA). Transfection of siRNAs or plasmids was performed using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, California, USA) according to the manufacturer's instructions. Stable cell lines expressing TBL1XR1 and TBL1XR1-shRNA were generated by retroviral infection using HEK293T cells, as previously described, and selected with 0.5 μg/mL puromycin for 10 days.32

Human lymphatic endothelial cell tube formation assay

The human lymphatic endothelial cells (HLECs) tube formation assay was performed by first pipetting 200 μL Matrigel (BD Biosciences, Bedford, Massachusetts, USA) into each well of a 24-well plate, which was then polymerised for 30 min at 37°C. HLECs (2×104) in 200 μL of conditioned medium were added to each well and incubated at 37°C, 5% CO2 for 12 h. Images were taken using a bright-field microscope at ×100 magnification. The capillary tubes were quantified by measuring the total length of completed tubule structures.

Popliteal lymph node metastasis model

BALB/c-nu mice (5–6 weeks old, 18–20 g) were purchased from the Experimental Animal Center, Guangzhou University of Chinese Medicine (Guangzhou, China). All experimental procedures were approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University. The BALB/c nude mice were randomly divided into four groups (n=6/group). The TBL1XR1, TBL1XR1-RNAi or vector-transduced Eca109 cells (5×105), which stably express firefly luciferase, were inoculated into the foot-pads of the mice on day 0. The mice were sacrificed on day 28, and the primary tumours and popliteal lymph nodes were enucleated and paraffin embedded. Serial 4.0 µm sections were taken and analysed by IHC with anti-lyve-1 and anti-luciferase antibodies (Abcam). The images were captured using an AxioVision Rel.4.6 computerised image analysis system (Carl Zeiss, Jena, Germany).

Chromatin immunoprecipitation assay

Cells (2×106) plated in a 100 mm culture dish were treated with 1% formaldehyde to cross-link proteins to DNA. The cell lysates were sonicated to shear the DNA into 300–1000 bp lengths. Aliquots containing equal amounts of chromatin supernatants were incubated on a rocking bed at 4°C overnight with either 1 μg anti-TBL1XR1 antibody, or 1 μg anti-H3K14Ac antibody, or HDAC3 or anti-polymerase II antibodies (Abcam), or 1 μg anti-IgG antibody as a negative control. Following reverse cross-linking of protein–DNA complexes to free the DNA, real-time PCR was carried out. The primers used in this study are listed in the online supplementary materials and methods.

Statistical analysis

All statistical analyses were carried out using SPSS V.13.0 statistical software. The χ2 test was used to analyse the relationship between TBL1XR1 expression and clinicopathological characteristics. Survival curves were plotted using the Kaplan–Meier method and compared by log-rank test. Survival data were evaluated by univariate and multivariate Cox regression analyses. p<0.05 was considered statistically significant.

Results

Upregulation of TBL1XR1 correlates with poor prognosis in human ESCC

To investigate the biological role of TBL1XR1 in human ESCC progression, we examined expression of TBL1XR1 in ESCC cell lines and human ESCC tissue samples. As shown in figure 1A, TBL1XR1 was markedly overexpressed in the ESCC cell lines, at protein and mRNA levels, compared with NEECs. Consistently, comparative analyses showed that TBL1XR1 was upregulated in the eight ESCC samples compared with their matched adjacent non-tumour tissues (figure 1B). These results suggest that TBL1XR1 is upregulated in human ESCC.

Figure 1

Upregulation of transducin (β)-like 1 X-linked receptor 1 (TBL1XR1) correlates with poor prognosis in human esophageal squamous cell carcinoma (ESCC). (A, B) Western blot and real-time PCR analyses of TBL1XR1 expression in (A) cultured ESCC cell lines and normal human esophageal epithelial cells (NEECs) and (B) in primary ESCC tissues (T) and matched adjacent non-tumour tissues. The transcript levels were normalised to GAPDH; α-tubulin was used as a protein loading control. The error bars represent the mean±SD from three independent experiments. (C) Immunohistochemical staining shows that TBL1XR1 expression is upregulated in human ESCC tissues compared with normal oesophageal tissues. Scale bars: 50 μm; insets: 10 μm. (D) Kaplan–Meier curves compare overall survival between patients with ESCC with low and high levels of TBL1XR1 expression (n=230; p<0.001, log-rank test). (E) Representative micrographs and the percentages of tissue specimens with high and low levels Lyve-1-positive microvessels in ESCC with low or high expression of TBL1XR1. Left panel, intratumoural; right panel, peritumoural; scale bars: 50 μm.

To determine the clinical relevance of TBL1XR1 in ESCC, TBL1XR1 expression was examined in 230 paraffin-embedded, archived ESCC tissues using IHC. As shown in figure 1C, TBL1XR1 was markedly upregulated in the ESCC tissues, but was only marginally detectable in normal oesophageal tissues. Furthermore, statistical analyses revealed that expression of TBL1XR1 was significantly correlated with clinical stage (p<0.001), patient survival (p<0.001) and tumour-node-metastasis stage (T: p<0.001; N: p<0.001; M: p=0.001; see online supplementary table S2). Additionally, Kaplan–Meier survival analysis and log-rank test showed that TBL1XR1 overexpression was correlated with shorter overall survival (p<0.001; figure 1D). Univariate and multivariate analyses indicated that TBL1XR1 expression was an independent prognostic factor for outcome in ESCC (p=0.001; see online supplementary table S3). Taken together, these data suggest a potential link between TBL1XR1 overexpression and ESCC progression.

TBL1XR1 correlates with microlymphatic vessel density in ESCC

Since TBL1XR1 levels were significantly correlated with lymphatic metastasis (p<0.001; see online supplementary figure S1 and table S2), we examined the correlation between TBL1XR1 expression and microlymphatic vessel density (MLD) in ESCC samples. As shown in figure 1E, expression of TBL1XR1 was significantly correlated with MLD in the intratumour and peritumour regions (p<0.001; p<0.001, respectively), as indicated by Lyve-1-positive microvessels, suggesting that TBL1XR1 might contribute to ESCC lymphangiogenesis.

TBL1XR1 promotes ESCC lymphangiogesis in vitro

To further investigate the role of TBL1XR1 in the promotion of lymphangiogenesis in ESCC, we established Eca109 and KYSE-410 ESCC cells stably expressing TBL1XR1 cDNA and TBL1XR1 RNAi(s) (figure 2A). As shown in figure 2B,C, tube formation and migration assays revealed that overexpressing TBL1XR1 strongly provoked, whereas silencing TBL1XR1 abrogated, the ability of ESCC cells to induce tube formation and migration in human lymphatic endothelial cells (HLECs), suggesting that TBL1XR1 promotes ESCC lymphangiogenesis in vitro.

Figure 2

Transducin (β)-like 1 X-linked receptor 1 (TBL1XR1) promotes esophageal squamous cell carcinoma (ESCC) lymphangiogenesis in vitro. (A) Western blot analysis of TBL1XR1 expression in Eca109 and KYSE-410 cells. α-Tubulin was used as a loading control. (B, C) Representative images of human lymphatic endothelial cells (HLECs) cultured with conditioned medium derived from TBL1XR1 overexpressing cells, TBL1XR1 silenced cells and control cells. (B) Matrigel tube formation assay. (C) Transwell migration assays. Scale bars: 100 μm; error bars represent the mean±SD from three independent experiments, *p<0.05.

TBL1XR1 increases the motility and invasiveness of ESCC cells in vitro

Wound migration assays showed that upregulation of TBL1XR1 dramatically increased, whereas downregulation of TBL1XR1 decreased, the migratory speed of Eca109 and KYSE-410 cells (figure 3A,B). Furthermore, the transwell matrix penetration assay showed that TBL1XR1-transduced ESCC cells exhibited a higher invasive capacity than vector control cells, whereas TBL1XR1-inhibited cells had a lower invasive capacity (figure 3C,D). These results suggest that TBL1XR1 promotes motility and invasiveness in ESCC cells.

Figure 3

Transducin (β)-like 1 X-linked receptor 1 (TBL1XR1) promotes motility and invasiveness in esophageal squamous cell carcinoma (ESCC) cells. (A, B) Representative micrographs of the wound healing assay in ESCC cells showing the motilities of TBL1XR1-overexpressing cells and TBL1XR1-silenced cells at 0 h and 22 h compared with vector controls. (C, D) Representative micrographs of the transwell matrix penetration assay showing the invasiveness of TBL1XR1-overexpressing cells and TBL1XR1-silenced cells compared with vector controls. Scale bars: 100 μm; error bars represent the mean±SD from three independent experiments, *p<0.05.

TBL1XR1 promotes lymph node metastasis in vivo

The effect of TBL1XR1 on lymphangiogesis and lymphatic metastasis in ESCC was investigated in vivo using a popliteal lymph node metastasis model. The TBL1XR1, TBL1XR1-RNAi or vector-transduced Eca109 cells, which stably expressing firefly luciferase, were inoculated into the foot-pads of nude mice (n=6/group; figure 4A). The resulting foot-pad tumours and popliteal lymph nodes were enucleated and analysed after 4 weeks. As shown in figure 4B, the tumours formed by Eca109/TBL1XR1 cells displayed increased levels of MLD compared with the control tumours, within the tumours and their adjacent non-tumour tissue, as indicated by the Lyve-1-positive microvessels. Conversely, MLD was markedly reduced in the tumours formed by Eca109/TBL1XR1-RNAi cells (figure 4B). Meanwhile, we found that the lymph nodes in tumours formed from TBL1XR1-transduced cells had larger volumes and displayed higher numbers of luciferase-positive tumour cells than tumours formed from vector-control cells (figure 4C,D). Conversely, the lymph nodes formed from TBL1XR1-silenced cells had smaller volumes than vector-control tumours (figure 4C). Strikingly, the ratios of metastatic to total dissected popliteal lymph nodes were markedly higher in the Eca109-TBL1XR1 group (100% (6/6)) than in the vector-control groups (33.3% (2/6)) (figure 4E); in contrast, no metastatic lymph nodes were found in the TBL1XR1-silenced groups (figure 4D,E). Taken together, these results indicated that TBL1XR1 promotes lymphangiogenesis and lymph node metastasis in ESCC in vivo.

Figure 4

Transducin (β)-like 1 X-linked receptor 1 (TBL1XR1) promotes lymph node metastasis in vivo. A popliteal lymph node metastasis model was established in nude mice by inoculating the foot pads with the indicated cells stably expressing firefly luciferase. (A) The foot-pad tumours and popliteal lymph nodes were enucleated and analysed 4 weeks after inoculation. (B) Representative micrographs of tumour sections immunostained with anti-Lyve-l antibody (left panel), and quantification (right panel), indicates the microlymphatic vessel density. Scale bars: 50 μm. (C) Representative micrographs (left panel) and the volumes of popliteal lymph nodes (right panel). (D) Representative micrographs of the popliteal lymph nodes immunostained with anti-luciferase antibody. Scale bars: upper panel, 200 μm; lower panel, 20 μm. (E) The ratios of metastatic to total dissected popliteal lymph nodes from mice inoculated with the indicated cells. Error bars represent the mean±SD of three independent experiments, *p<0.05.

TBL1XR1 upregulates VEGF-C in ESCC cells

Previous reports have suggested that the expression of VEGF-C is biologically and clinically linked to the lymphangiogenic and lymphatic metastatic capacities of tumour cells.13 This prompted us to investigate whether VEGF-C was also involved in TBL1XR1-induced lymphangiogenesis and lymphatic metastasis in ESCC. As shown in figure 5A,B, VEGF-C expression was upregulated in TBL1XR1-transduced ESCC cells, but downregulated in TBL1XR1-silenced ESCC cells, at the mRNA and protein levels, compared with control cells, suggesting that TBL1XR1 transcriptionally upregulates VEGF-C in ESCC cells. Furthermore, luciferase reporter assay showed that the luciferase activity driven by the VEGF-C promoter was increased in TBL1XR1-transduced cells but decreased in TBL1XR1-silenced cells (figure 5C and see online supplementary figure S2). Meanwhile, chromatin immunoprecipitation (ChIP) assay revealed that TBL1XR1 bound to region 6 (nucleotides −1162 to −1019) and region 11 (nucleotides +186 to +347) within the VEGF-C promoter region (figure 5D), indicating that TBL1XR1 upregulated VEGF-C expression by targeting the VEGF-C promoter. In addition, we found that the recruitment of RNA polymerase II (Pol II) and H3K14Ac to the VEGF-C promoter was increased in TBL1XR1-transduced cells but decreased in TBL1XR1-silenced cells; and that overexpressing TBL1XR1 reduced, whereas silencing TBL1XR1 promoted, binding of HDAC3 to the VEGF-C promoter (see online supplementary figure S3A–C). In combination, these results provide strong evidence that TBL1XR1 transcriptionally upregulates VEGF-C expression. Importantly, we found that silencing VEGF-C or treatment with a neutralising anti-VEGF-C antibody could reduce the ability of TBL1XR1-transduced ESCC cells to induce migration and tube formation in HLECs (figure 5E,F), indicating that VEGF-C plays a central role in TBL1XR1-induced lymphangiogenesis in ESCC.

Figure 5

Transducin (β)-like 1 X-linked receptor 1 (TBL1XR1) upregulates vascular endothelial growth factor C (VEGF-C) expression in esophageal squamous cell carcinoma (ESCC) cells. (A) Real-time PCR analysis of VEGF-C mRNA expression in the indicated cells. Expression levels were normalised to GAPDH. (B) ELISA of VEGF-C protein levels in the supernatants of the indicated cells. (C) Luciferase activity assays in Eca109 and KYSE-410 cells show transactivation of the VEGF-C promoter by TBL1XR1 overexpression and repression of the VEGF-C promoter by TBL1XR1 silencing. (D) Chromatin immunoprecipitation (ChIP) assay showing the nucleotide regions of the VEGF-C promoter that are physically associated with TBL1XR1. Left panel: schematic illustration of PCR-amplified fragments of the VEGF-C promoter; right panel: ChIP assays were performed using TBL1XR1 antibody to screen TBL1XR1-bound VEGF-C promoter regions. IgG was used as a negative control. (E, F) Representative images and quantifications of Matrigel tube formation and transwell migration assays, respectively, of human lymphatic endothelial cells (HLECs) cultured with conditioned medium derived from the indicated cells. Scale bars: 100 μm; error bars represent the mean±SD of three independent experiments, *p<0.05.

As AKT and ERK are key downstream effectors of the VEGF-C signalling pathway,33–35 the effect of TBL1XR1-induced VEGF-C expression on AKT and ERK activation was examined in HLECs. As shown in online supplementary figure S4A, phospho-AKT (Ser 473) and phospho-ERK1/2 (T202/Y204) were dramatically upregulated in HLECs treated with conditioned media from TBL1XR1-transduced Eca109 or KYSE-410 cells, and downregulated in HLECs treated with conditioned media from TBL1XR1-silenced Eca109 or KYSE-410 cells, compared with vector-control cells. Furthermore, silencing VEGF-C, or treatment with a neutralising anti-VEGF-C antibody, abrogated the ability of TBL1XR1-transduced ESCC cells to induce AKT and ERK activation in HLECs, suggesting that VEGF-C is a functionally relevant effector of TBL1XR1-induced AKT and ERK activation (see online supplementary figure 4B).

VEGF-C is required for TBL1XR1-induced lymphatic metastasis in vivo

The in vivo results showed that silencing VEGF-C significantly decreased the intratumour and peritumour MLD in tumours formed by Eca109/TBL1XR1 (figure 6A). Meanwhile, we found that downregulation of VEGF-C dramatically reduced the volume and number of luciferase-positive tumour cells in the lymph nodes from transfected mice (figure 6B,C). In addition, the ratios of metastatic to total dissected popliteal lymph nodes were also markedly reduced from 100% (6/6) to 16.7% (1/6) in the Eca109/TBL1XR1 and Eca109/TBL1XR1/VEGF-C RNAi groups, respectively (figure 6D). Taken together, these results demonstrated that VEGF-C is a key mediator of TBL1XR1-induced ESCC lymphangiogenesis and lymphatic metastasis in vivo.

Figure 6

Vascular endothelial growth factor C (VEGF-C) is required for transducin (β)-like 1 X-linked receptor 1 (TBL1XR1)-induced lymphatic metastasis in vivo. Tumours and popliteal lymph nodes from the foot pads of nude mice inoculated with TBL1XR1-transduced esophageal squamous cell carcinoma (ESCC) cells infected with VEGF-C shRNA or vector controls. (A) Representative micrographs of tumour sections immunostained with anti-Lyve-l antibody (left panel) and quantification (right panel) indicting microlymphatic vessel density. Scale bars: 50 μm. (B) Representative micrographs and the volumes of popliteal lymph nodes. (C) Representative micrographs of popliteal lymph nodes immunostained with anti-luciferase antibody from mice inoculated with the indicated cells. Scale bars: upper panel, 200 μm; lower panel, 20 μm. (D) The ratios of metastatic to total dissected popliteal lymph nodes from mice inoculated with the indicated cells. Error bars represent the mean±SD of three independent experiments, *p<0.05.

Clinical relevance of TBL1XR1-induced upregulation of VEGF-C in human ESCC

Finally, we examined whether the TBL1XR1/VEGF-C axis identified in our ESCC cell models could also be evidenced in clinical ESCC samples. As shown in figure 7A, IHC analysis revealed that TBL1XR1 levels in 230 ESCC specimens were significantly correlated with VEGF-C expression (p<0.001). Furthermore, we found that in nine freshly collected clinical ESCC samples, TBL1XR1 expression was also strongly associated with the mRNA levels of VEGF-C (r=0.817, p=0.007) (figure 7B). Importantly, analysis of published microarray datasets showed that TBL1XR1 levels were significantly correlated with VEGF-C expression in gastric cancer (NCBI/GEO/GSE37023; n=112, r=0.475, p<0.001), colorectal cancer (NCBI/GEO/GSE14095; n=189, r=0.429, p<0.001) and breast cancer (GSE6532; n=327, r=0.607, p<0.001) (see online supplementary figure S5A). Concordantly, silencing TBL1XR1 in gastric cancer, colorectal cancer and breast cancer cells resulted in a significant decrease in VEGF-C expression (see online supplementary figure S5B–5D). Taken together, these results further support the notion that overexpression of TBL1XR1 upregulated VEGF-C, thereby promoting lymphangiogenesis and lymphatic metastasis, and leading to poor prognosis in patients with ESCC (figure 7C).

Figure 7

Clinical relevance of transducin (β)-like 1 X-linked receptor 1 (TBL1XR1)-induced upregulation of vascular endothelial growth factor C (VEGF-C) in human esophageal squamous cell carcinoma (ESCC). (A) TBL1XR1 levels are positively correlated with VEGF-C expression in primary human ESCC specimens (n=230; p<0.001). Left panel: micrographs of two representative cases. Scale bars: 50 μm. Right panel: percentages of ESCC specimens with low or high expression of TBL1XR1 relative to VEGF-C expression. (B) Analyses showing the expression (left) of TBL1XR1 protein and its correlation (right) with expression of VEGF-C mRNA in nine freshly collected human ESCC samples. Error bars represent the mean±SD of three independent experiments, *p<0.05. (C) Illustrative model showing the proposed mechanism by which TBL1XR1 promotes lymphangiogenesis and lymphatic metastasis in ESCC via upregulation of VEGF-C expression.

Discussion

The role of lymphangiogenesis and lymphatic metastasis in aggressive malignancies has been demonstrated in multiple studies,5 ,7 ,36–39 and the occurrence of regional lymph node metastasis at an early stage is thought to be a crucial step in cancer progression. Although the detection of lymphangiogenesis and tumour cells in the regional lymph nodes has been used as a prognostic factor in the diagnosis and staging of patients with ESCC,4 ,5 the mechanisms that control lymph node metastasis are unclear. In this study, we found that TBL1XR1 was significantly upregulated in ESCC. Furthermore, our results revealed a potential molecular mechanism by which TBL1XR1 may promote lymphangiogenesis and lymphatic metastasis in ESCC, in vitro and in vivo, via upregulation of VEGF-C. Therefore, our results uncover the novel molecular mechanism for lymphangiogenesis and lymphatic metastasis of ESCC.

Previous independent studies have demonstrated that inhibiting tumour-induced lymphangiogenesis can significantly reduce metastatic spread of tumour cells and improve survival in patients with breast cancer, gastric cancer, colorectal cancer, lung cancer and ESCC.8 ,40–42 Overexpression of VEGF-C has been implicated in a variety of human cancers and is associated with increased lymphangiogenesis and enhanced lymphatic metastasis.18 ,43 ,44 Consistent with these reports, we found that TBL1XR1 was significantly upregulated in ESCC tissues compared with their matched adjacent normal oesophageal tissues, and was strongly correlated with N and M staging in clinical samples of ESCC (p<0.001; p=0.001, respectively). In addition, we demonstrated that overexpressing TBL1XR1 significantly increased, and silencing TBL1XR1 decreased, the lymphangiogenic and lymphatic metastatic capacity of ESCC cells.

Previous studies have demonstrated that VEGF-C signalling induces lymphangiogenesis via activation of ERK1/2 or PI3 K/AKT pathways.33 ,34 ,35 In agreement with these studies, we found that overexpresing TBL1XR1 markedly increased, whereas silencing TBL1XR1 decreased, the expression of p-AKT and p-ERK1/2 in HLECs, and that inhibition of VEGF-C abrogated the ability of TBL1XR1-transduced ESCC cells to induce activation of AKT and ERK in HLECs, suggesting a potential mechanism for TBL1XR1-induced lymphangiogenesis and lymphatic metastasis in ESCC via activation of VEGF-C signalling.

The precisely regulated switch between gene repression and gene activation represents a critical step in transcriptional regulation. TBL1XR1 and its homologue TBL1 are intrinsic components of the SMRT/N-CoR corepressor complex, which is required for transcriptional corepressor/coactivator exchange in response to nuclear receptors and activating signals in many physiological processes, such as embryonic development and immunity response.21 ,24 ,27–29 ,45 ,46 For instance, TBL1XR1 and TBL1 were demonstrated to be required for the effective commitment of embryonic stem cells to adipocytes through its decisional role in peroxisome proliferator-activated receptor γ induced gene activation.16 Recently, Li and colleagues found that silencing TBL1XR1 in HT29 colorectal cancer cells significantly increased the spontaneous apoptosis and reduced cell invasion and tumour growth in a nude mice model.24 Kadota et al reported that knockdown TBL1XR1 expression using a shRNA in breast cancer cells resulted in reduction of cell migration/invasion and suppression of tumourigenesis in mouse xenografts.25 In our study, overexpression of TBL1XR1 drastically increased, whereas downregulation of TBL1XR1 abrogated, the lymphangiogenesis and lymphatic metastatic ability of ESCC cells. We found that recruitment of RNA polymerase II (Pol II) to the VEGF-C promoter was enhanced in TBL1XR1-transduced cells, and that upregulation of TBL1XR1 increased, while silencing TBL1XR1 decreased, expression of H3K14Ac at the VEGF-C promoter. It has previously been reported that TBL1XR1 plays roles in transcription activation through dismissal of the transcriptional corepressors, such as NCoR/SMRT/HDAC3, Groucho/TLE1/HDAC1 and BCL-3/CtBP/HDAC3, to the downstream gene promoter upon ligand binding or other signal-dependent transcription factors.21 ,23 ,24 ,25 The results of our ChIP assay were consistent with these reports by showing that HDAC3 binding to the VEGF-C promoter was reduced by TBL1XR1 overexpression and increased by TBL1XR1 silencing. Taken together, our results indicated that TBL1XR1 may promote VEGF-C transcription through chromatin-remodelling events.

Considerable research has been carried out to determine the mechanism of lymph node metastasis in ESCC. In addition, understanding the mechanisms underlying lymphangiogenesis and lymphatic metastasis could provide new therapeutic targets for cancer therapies. We found that expression of TBL1XR1 was correlated with expression of VEGF-C in multiple cancer types, including ESCC. In combination with reports from other studies, our results have provided strong evidence that dysregulation of TBL1XR1 plays important roles in multiple pathological processes. Therefore, further investigations into the mechanisms by which TBL1XR1 is overexpressed in human cancers are necessary to increase our knowledge of the biological basis of cancer progression.

References

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Footnotes

  • Contributors LL, CL and WL carried out most of the experimental work. SW and AL conducted animal experiments. XZ and PR conducted luciferase reporter assay. JW conducted real-time PCR analysis and data analysis. ML and LS supervised the project and wrote the manuscript. LL, CL and WL contributed equally.

  • Funding This work was supported by the Ministry of Science and Technology of China grant (No. 973-2014CB910604); The Natural Science Foundation of China (No. 81272198, 81301998, U1201121); The Science and Technology Department of Guangdong Province (No. S2011020002757, S2012040007113); Ministry of Education of China (20130171110085).

  • Competing interests None.

  • Patient consent Obtained.

  • Ethics approval Samples used in this study were approved by the committees for ethical review of research at the Sun Yat-sen University.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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