LNMAT1 promotes lymphatic metastasis of bladder cancer via CCL2 dependent macrophage recruitment

Abstract

Tumor-associated macrophages (TAMs) are the most abundant inflammatory infiltrates in the tumor microenvironment and contribute to lymph node (LN) metastasis. However, the precise mechanisms of TAMs-induced LN metastasis remain largely unknown. Herein, we identify a long noncoding RNA, termed Lymph Node Metastasis Associated Transcript 1 (LNMAT1), which is upregulated in LN-positive bladder cancer and associated with LN metastasis and prognosis. Through gain and loss of function approaches, we find that LNMAT1 promotes bladder cancer-associated lymphangiogenesis and lymphatic metastasis. Mechanistically, LNMAT1 epigenetically activates CCL2 expression by recruiting hnRNPL to CCL2 promoter, which leads to increased H3K4 tri-methylation that ensures hnRNPL binding and enhances transcription. Furthermore, LNMAT1-induced upregulation of CCL2 recruits macrophages into the tumor, which promotes lymphatic metastasis via VEGF-C excretion. These findings provide a plausible mechanism for LNMAT1-modulated tumor microenvironment in lymphatic metastasis and suggest that LNMAT1 may represent a potential therapeutic target for clinical intervention in LN-metastatic bladder cancer.

Fig. 1

LNMAT1 overexpression is associated with poor prognosis for bladder cancer. a, b Unsupervised hierarchical clustering of the lncRNAs differentially expressed in MIBC tissues and paired normal adjacent tissues NAT (a) and in LN-positive and LN-negative bladder cancer tissues (b). The pseudocolor represents the intensity scale of MIBC tissues vs. NAT or the LN-positive vs. LN-negative bladder cancer tissues, generated by a log₂ transformation (fold changes > 5.0, p < 0.01). c Schematic representation of LNMAT1 upregulation in MIBC tissues and LN-positive tissues. d qRT-PCR analysis of LNMAT1 expression in a 266-case cohort of freshly collected human bladder cancer samples and NATs. The nonparametric Mann–Whitney U-test was used. e, f Correlation of LNMAT1 expression in bladder cancer tissues (n = 266) assessed by qRT-PCR with pathological grade (e) and LN status (f). The nonparametric Mann–Whitney U-test was used. g Comparison of LNMAT1 expression in primary human bladder cancer samples and paired metastatic LNs. The nonparametric Mann–Whitney U-test was used. h, i Kaplan–Meier curves for OS (h) and DFS (i) of bladder cancer patients with low vs. high expression of LNMAT1. The median LNMAT1 expression was used as the cutoff value. j‚ k Representative images (j) and percentages (k) of the ISH of LNMAT1 expression (blue) in the paraffin-embedded NAT and tumor sections of bladder cancer with or without LN metastasis (n = 266). Samples were counterstained with nuclear fast red. The scramble probe was used as a negative control. Statistical significance was assessed by χ² test. Scale bars: 50 μm. The error bars represent standard deviations of three independent experiments. *p < 0.05 and **p < 0.01

Fig. 2

LNMAT1 overexpression promotes LN metastasis in vivo. a Representative images of the nude mouse model of popliteal LN metastasis. The indicated UM-UC-3 cells were injected into the footpads of the nude mice, and the popliteal LNs were enucleated and analyzed. b, c qRT-PCR analysis of LNMAT1 expression in LNMAT1-transduced, LNMAT1-silenced and control cell as indicated. Statistical significance was assessed using one-way analyses of variance (ANOVA) followed by Dunnett’s tests for multiple comparison and two-tailed t-tests. d, e Representative images of bioluminescence (popliteal LNs) and histogram analysis of popliteal LN metastasis in the indicated cells (n = 16 per group). Statistical significance was assessed using one-way analyses of variance (ANOVA) followed by Dunnett’s tests and two-tailed t-tests. f Representative images of enucleated popliteal LNs and histogram analysis of the LN volume in the indicated cells. Statistical significance was assessed using one-way analyses of variance (ANOVA) followed by Dunnett’s tests and two-tailed t-tests. (n = 16, Scale bar: 5 mm). g Kaplan–Meier (Mantal-Cox) test of the mice (n = 16) that were inoculated in the indicated cells. h, i Representative images and percentages of mice tissues with high or low levels of F4/80-positive cells and LYVE-1-positive cells in the intratumoral tissues with different LNMAT1 expression levels. LNMAT1 expression levels were quantified by ISH, macrophage density was quantified by IHC using anti-F4/80 antibody and microlymphatic vessel density was quantified by IHC using the anti-LYVE-1 antibody. Two representative cases are shown. Statistical significance was assessed by χ² test. Scale bars: 100 μm. The error bars represent standard deviations of three independent experiments. *p < 0.05 and**p < 0.01

Fig. 3

LNMAT1 overexpression promotes bladder cancer cells invasion. a, b Representative images of Transwell assay using UM-UC-3 and 5637 cells showing cell motility after knockdown (a) or overexpression (b) of LNMAT1 and a histogram analysis of migrated cell counts are shown. Scale bars: 100 μm. Statistical significance was assessed using two-tailed t-tests and one-way analyses of variance (ANOVA) followed by Dunnett’s tests for multiple comparison. c, d Representative images of wound healing assay using UM-UC-3 and 5637 cells (left panels) showing cell motility after knockdown (c) or overexpression (d) of LNMAT1 and a histogram analysis of cell migration distance are shown (right panels). Statistical significance was assessed using two-tailed t-tests and one-way analyses of variance (ANOVA) followed by Dunnett’s tests for multiple comparison. e–g Representative images of lung colonization by UM-UC-3 cells injected into the tail veins of nude mice, histogram analysis of luminescence and the number of metastatic foci representing lung metastasis measured on day 60 (n = 16). Scale bars: black, 200 µm; red, 50 µm. Statistical significance was assessed using two-tailed t-tests and one-way analyses of variance (ANOVA) followed by Dunnett’s tests for multiple comparison. The error bars represent standard deviations of three independent experiments. *p < 0.05 and **p < 0.01

Fig. 4

CCL2 is required for LNMAT1-induced LN metastasis. a, b Heatmap representing unsupervised hierarchical clustering of genes regulated by LNMAT1 based on next-generation sequencing (NGS) analysis. Rows represent probe sets, and columns represent samples treated as indicated. Green, downregulation; red, upregulation. CCL2 is indicated by black arrows. c, d Bio-Plex multiplex immunoassay analysis of protein expression of 40 cytokines and chemokines regulated by LNMAT1. Two-tailed t-tests was used. e, f qRT-PCR of CCL2 expression in LNMAT1-transduced, LNMAT1-silenced and control cell as indicated. Statistical significance was assessed using one-way ANOVA followed by Dunnett’s tests for multiple comparison and two-tailed t-tests. g, h ELISA of CCL2 expression in the indicated cells. Statistical significance was assessed using one-way ANOVA followed by Dunnett’s tests for multiple comparison and two-tailed t-tests. i Representative images of popliteal LNs, enucleated LNs and IHC staining with anti-luciferase antibody in the indicated mice group (n = 16 per group). Scale bars: black, 500 µm; red, 50 µm. j, k The ratios of the metastatic to volume quantification (j) and Kaplan–Meier survival analysis (k) for the indicated group (n = 16 per group). Two-tailed t-tests was used. l Representative images of HE and IHC staining confirming the LN status (n = 16). Scale bars: black, 500 µm; red, 100 µm. m Volume quantification of popliteal LN metastasis after shRNA-mediated depletion of CCL2. One-way ANOVA followed by Dunnett’s tests for multiple comparison was used. The error bars represent standard deviations of three independent experiments. *p < 0.05 and **p < 0.01

Fig. 5

LNMAT1 forms a triplex with the CCL2 promoter and interacts with hnRNPL. a FISH analysis of the subcellular distribution of LNMAT1 in UM-UC-3 cells. Scale bar: 10 μm. b Nuclear fractionation analyses and qRT-PCR analyses of LNMAT1 expression in the nucleus and cytoplasm. c Transcriptional activity of the CCL2 promoter was evaluated using sequential deletions and by examining the CCL2 promoter linked to Renilla luciferase activity. Two-tailed t-tests was used. d Schematic presentation of the potential LNMAT1 binding sites in the CCL2 promoter. e, f ChIRP analysis of LNMAT1-associated chromatin in UM-UC-3 cells. Retrieved chromatin was quantified by qRT-PCR. Statistical significance was assessed using one-way ANOVA followed by Dunnett’s tests for multiple comparison. g–i Fluorescence resonance energy transfer (FRET) of a 5:1 mixture of TFO in LNMAT1 with TTS in the CCL2 promoter sequences is shown in red. The individual TFO is blue and TTS is shown in black (g, h). FENDRR/PITX2 was used as the positive control (i). j–l Circular dichroism (CD) spectroscopy of a 1:1 mixture of TFO in LNMAT1 with TTS in the CCL2 promoter sequences is shown in blue. The sum of individual TFO and TTS is shown in red (j, k). FENDRR/PITX2 was used as the positive control (l). m An RNA pull-down assay was performed using LNMAT1 sense and antisense RNAs in UM-UC-3 cells, followed by silver staining. A red arrow indicates hnRNPL. n, o The interaction between LNMAT1 and hnRNPL was confirmed by RNA pull-down and western blotting with nuclear extract or purified recombinant hnRNPL. p RNA immunoprecipitation (RIP) analysis using the anti-hnRNPL antibody revealed that LNMAT1 interacted with endogenous hnRNPL in UM-UC-3. U1 was used as the negative control. Statistical significance was assessed using Two-tailed t-tests was used. The error bars represent standard deviations of three independent experiments. *p < 0.05 and **p < 0.01

Fig. 6

LNMAT1/hnRNPL promotes H3K4me3 of the CCL2 promoter. a Serial deletions of LNMAT1 were used in the RNA pull-down assays to identify the core regions of LNMAT1 for the physical interaction with hnRNPL. b Silver staining image of proteins pulled down by the 5′-terminus of the truncated LNMAT1 (350–550 nt). c The interaction between the truncated LNMAT1 and hnRNPL was confirmed by RNA pull-down and western blotting with nuclear extract or purified recombinant hnRNPL. d–f Endogenous LNMAT1 was depleted with a shRNA and the efficiency of the expression of the truncated LNMAT1 was examined by qRT-PCR and ELISA analyses. Two-tailed t-tests was used. g, h qRT-PCR and ELISA analyses of CCL2 expression in the hnRNPL-silenced and control cell as indicated. Statistical significance was assessed using two-tailed t-tests and one-way analyses of variance (ANOVA) followed by Dunnett’s tests for multiple comparison. i, j CCL2 wild-type (−2000 ~+121) or with LNMAT1 binding site mutated promoter were subjected to luciferase reporter assays in LNMAT1 or hnRNPL overexpressing cells. Two-tailed t-tests and one-way analyses of variance (ANOVA) followed by Dunnett’s tests for multiple comparison. k–n ChIP-qPCR analysis of hnRNPL occupancy (k, m) and H3K4 methylation status (l, n) in the CCL2 promoter after overexpression/knock down of LNMAT1 in UM-UC-3 cells as indicated. Two-tailed t-tests and one-way analyses of variance (ANOVA) followed by Dunnett’s tests for multiple comparison. o–r qRT-PCR and ELISA analyses of CCL2 expression in the hnRNPL knockdown (o, p) or overexpression (q, r) or on LNMAT1-induced CCL2 expression in UM-UC-3 cells. Two-tailed t-tests and one-way analyses of variance (ANOVA) followed by Dunnett’s tests for multiple comparison. The error bars represent standard deviations of three independent experiments. *p < 0.05 and **p < 0.01

Fig. 7

LNMAT1-upregulated CCL2 activates TAMs. a, b Representative images and percentages of bladder cancer tissues with high or low levels of CD68-positive cells and LYVE-1-positive cells in the intratumoral tissues with different LNMAT1 expression levels. LNMAT1 expression levels were quantified by ISH, macrophage density was quantified by IHC using anti-CD68 antibody and microlymphatic vessel density was quantified by IHC using the anti-LYVE-1 antibody. Two representative cases are shown. Statistical significance was assessed by χ² test. Scale bars: 100 μm. c Flow cytometric analysis and quantification of the expressions of CD206/HLA-DR in macrophages treated with medium collected from the indicated cells for 24 h. CCL2-neutralizing treatment was carried out for an additional 18 h. Statistical significance was assessed by two-tailed t-tests. d, e Representative images (d) IHC staining evaluating macrophages with anti-F4/80 as indicating with red arrows and histogram analysis (e) in primary tumor from the footpads of nude mice. Scale bars: 100 μm. Statistical significance was assessed by two-tailed t-tests. The error bars represent standard deviations of three independent experiments. *p < 0.05, and **p < 0.01

Fig. 8

LNMAT1-upregulated CCL2 induces LN metastasis of bladder cancer. a, b ELISA (a) and qRT-PCR (b) analyses of VEGF-C expression in TAM-like macrophages induced by conditioned medium collected from the indicated cells. One-way ANOVA followed by Dunnett’s tests for multiple comparison. c–e Representative images (c) and quantifications (d, e) of tube formation and Transwell migration by HLECs treated with conditioned medium collected from macrophages induced by LNMAT1 from UM-UC-3 cells. HLECs were cultured with conditioned medium derived from TAM-like macrophages induced by the indicated cancer cells. Scale bars: 100 µm. Two-tailed t-tests and one-way analyses of variance (ANOVA) followed by Dunnett’s tests for multiple comparison. f Representative images of HE and IHC staining confirming the LN status (n = 16). Scale bars: black, 500 µm; red, 100 µm. g Volume quantification of popliteal LN metastasis after inhibition of VEGF-C with neutralizing antibody. Two-tailed t-tests. The error bars represent standard deviations of three independent experiments. *p < 0.05 and **p < 0.01

Fig. 9

Clinical relevance of LNMAT1/CCL2/lymphangiogenesis in bladder cancer. a Correlation of CCL2 expression in bladder cancer tissues (n = 266) assessed by qRT-PCR with LN status. The nonparametric Mann–Whitney U-test was used. b, c Kaplan–Meier analysis for OS and DFS of bladder cancer patients with low vs. high expression of CCL2. The median CCL2 expression was used as the cutoff value. d–f TCGA data showed that LNMAT1 overexpression correlated with LN metastasis (d), high grade (e), and poor prognosis (f). g–h The LNMAT1 levels were positively correlated with CCL2 expression in the bladder cancer specimens (n = 266; p < 0.001). Scale bars: 100 μm. Statistical significance was assessed by χ² test. i Correlation analysis of LNMAT1 and CCL2 mRNA expression by qRT-PCR. j Illustrative model showing the proposed mechanism by which LNMAT1 promotes lymphatic metastasis in bladder cancer via CCL2-dependent TAMs recruitment. The error bars represent standard deviations of three independent experiments. *p < 0.05 and **p < 0.01