L1CAM overexpression promotes tumor progression through recruitment of regulatory T cells in esophageal carcinoma

Abstract

Objective: L1 cell adhesion molecule (L1CAM) exhibits oncogenic activity in tumors. However, the link between L1CAM and the tumor microenvironment remains poorly understood in patients with esophageal squamous cell carcinoma (ESCC). In this study, we investigated how L1CAM expression in ESCC affects the oncogenic characteristics of tumor cells and the tumor microenvironment.

Methods: Human ESCC samples were collected, and the mRNA and protein levels of L1CAM were examined by real-time PCR and immunohistochemistry. Overexpression and knockdown gene expression assays were used for mechanistic studies. The cell proliferation and cell cycle were measured with CCK-8 assays and flow cytometry. Cell migration and invasion ability were measured with Transwell assays. Multiplex bead-based assays were performed to identity the factors downstream of L1CAM. Xenograft studies were performed in nude mice to evaluate the effects of L1CAM on tumor growth and regulatory T cell (Treg) recruitment.

Results: L1CAM expression was significantly elevated in ESCC tissues (P < 0.001) and correlated with poorer prognosis (P < 0.05). Ablation of L1CAM in ESCC cells inhibited tumor growth and migration, and increased tumor cell apoptosis (P < 0.05). In the tumor microenvironment, L1CAM expression correlated with Treg infiltration in ESCC by affecting CCL22 secretion. Mechanistically, L1CAM facilitated CCL22 expression by activating the PI3K/Akt/NF-κB signaling pathway. Furthermore, CCL22 promoted Treg recruitment to the tumor site; the Tregs then secreted TGF-β, which in turn promoted L1CAM expression via Smad2/3 in a positive feedback loop.

Conclusions: Our findings provide new insight into the mechanism of immune evasion mediated by L1CAM, suggesting that targeting L1CAM-CCL22-TGF-β crosstalk between tumor cells and Tregs may offer a unique means to improve treatment of patients with ESCC.

Keywords: CCL22; L1CAM; TGF-β; Tregs; esophageal squamous cell carcinoma.

Figure 1

Expression of L1CAM is elevated in ESCC tissues. (A) qRT-PCR analysis of L1CAM mRNA expression in paired fresh tissues from 106 patients with ESCC. GAPDH was used to normalize the data, which were analyzed with the 2^-ΔΔCt method. (B) Samples were divided according to their tumor differentiation, depth of invasion, and tumor stage, and the mRNA expression of L1CAM in ESCC tissues was analyzed. (C) IHC score analysis of L1CAM protein expression in 69 ESCC and paired adjacent normal tissues (***P < 0.001). (D) Representative images showing different intensities of L1CAM staining (200×). (E) Kaplan-Meier survival analysis of OS and PFS, on the basis of high (n = 54) and low (n = 15) L1CAM protein expression via IHC. *P < 0.05, ***P < 0.001.

Figure 2

L1CAM promotes ESCC tumor malignancy. (A) The mRNA and protein levels of L1CAM, examined with qRT-PCR and Western blot, in one immortalized esophageal cell line (Het-1α) and 4 human ESCC cell lines (KYSE450, KYSE70, TE1, and EC1). (B) L1CAM expression was detected with qRT-PCR and Western blot in shNC, shL1CAM-EC1 cells, scramble, and OE-L1CAM-KYSE450 cells. The effects of L1CAM on the proliferation of EC1 and KYSE450 cell lines were analyzed with CCK-8 (C), cell cycle (D), and apoptosis (E) assays. Summary bar chart (left) and representative images (right) of cell migration and invasion abilities in shNC, shL1CAM-EC1 cells, scramble, and OE-L1CAM-KYSE450 cells detected with Transwell (F) and Matrigel invasion (G) assays. Cells present in the substrate were stained with crystal violet and counted under a microscope (200×). The volumes (H) and weights (I) of the tumors grown in the xenograft mouse model are presented as the mean ± standard deviation (n = 5). (J) Photographs of the dissected tumors from different groups *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

L1CAM promotes the recruitment of Tregs by upregulating CCL22. (A) Volcano plot of differentially expressed genes (DEGs), on the basis of the median value of the L1CAM mRNA expression level. (B) The upregulated DEGs were used to perform GO enrichment analysis. (C) The secretion of cytokines and chemokines in the supernatants of shNC and shL1CAM-EC1 cells (up) or scramble and OE-L1CAM-KYSE450 cells (down) cultured for 48 h was assessed with a human multiplex bead-based kit. The expression of CCL22 was detected by qRT-PCR (D) and ELISA (E) between shNC and shL1CAM-EC1 cells, or scramble and OE-L1CAM-KYSE450 cells. (F) Expression of L1CAM and CCL22 mRNAs in the different groups tumor samples derived from Figure 2H. (G) IHC of tumor tissues in different groups derived from Figure 2H. Representative images are shown (200×). (H) The IHC scores of L1CAM and CCL22 in different groups. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

L1CAM promotes the recruitment of Tregs through CCL22 in vivo. (A) The correlation between FOXP3 and CCL22 in the ESCC TCGA database, n = 94. (B) The purity of CD4⁺CD25⁺CD127^- Tregs sorted from the peripheral blood of patients with ESCC, according to FACS. (C) The numbers of migrating Tregs obtained from ESCC patient blood samples was calculated for the shNC, shL1CAM, shL1CAM + recombinant protein CCL22 (100 ng/mL), shL1CAM + PBS, scramble, OE-L1CAM, OE-L1CAM + CCL22 antibody (500 ng/mL), and OE-L1CAM + IgG groups. (D) Flow chart of the in vivo experiments. (E) mRNA expression of FOXP3 and CCR4 in the indicated groups. (F) Percentage of CD4⁺FOXP3⁺ Tregs and FOXP3⁺CCR4⁺ Tregs recruited to tumor sites, as analyzed by flow cytometry. (G) Expression of FOXP3 in tumor tissues of different groups, determined by IHC (200×). (H) The expression of p-Akt, Akt, p-NF-κB, NF-κB, and CCL22 was detected by Western blot between shL1CAM and OE-L1CAM cells treated with or without p-Akt inhibitor (RF-04691502) and p-NF-κB inhibitor (QNZ). β-actin expression was used as a loading control. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

Tregs secrete TGF-β, which in turn induces L1CAM expression by activating Smad2/Smad3. (A) L1CAM expression in KYSE450 and EC1 cells with or without co-culturing with Tregs (1:1), as determined by qRT-PCR. (B) A multiple-cytokine kit was used to detect cytokines in the supernatants from the EC1/Treg co-culture system and from the EC1 and Tregs cultured alone. (C) The TGF-β protein level was validated using ELISA. (D) TGF-β expression in the tumor cells and Tregs obtained from the co-culture system, and in the tumor cells and Tregs cultured alone. (E) EC1 and KYSE450 cells pretreated with or without TGF-β recombinant protein (20 ng/mL) or TGF-β inhibitor (SB431542) (10 μM). The expression of L1CAM, p-Smad2, Smad2, p-Smad3, and Smad3 was examined with Western blot analysis. β-actin expression was used as a loading control. (F) L1CAM expression was detected in tumor cells with or without Tregs, rhTGF-β, or SB431542. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

High expression of CCL22 predicts poor survival in patients with ESCC. (A–B) qRT-PCR analysis of CCL22 expression in paired fresh tissues from 47 patients with ESCC. GAPDH was used to normalize the data, which were analyzed with the 2^-ΔΔCt method. (C) Kaplan–Meier survival analysis of overall survival on the basis of high (n = 24) and low (n = 23) CCL22 expression by qRT-PCR. (D) The percentage of CD4⁺FOXP3⁺ Tregs in tumor tissues obtained from patients with ESCC. Correlations between CD4⁺FOXP3⁺ Tregs and L1CAM (E), CD4⁺FOXP3⁺ Tregs, and CCL22 (F), L1CAM, and CCL22 in the tumor tissues of patients with ESCC (G) were evaluated with linear regression analysis. (H) Schematic diagram of the proposed molecular mechanism: L1CAM promotes the recruitment of Tregs into the tumor site through PI3K/Akt/NF-κB-CCL22-CCR4. Tregs produce TGF-β, which further induces L1CAM upregulation in ESCC tumor cells. **P < 0.01.