Lenalidomide regulates the CCL21/CCR7/ERK1/2 axis to inhibit migration and proliferation in diffuse large B-cell lymphoma

Abstract

Background: The prognostic significance of the chemokine receptor CCR7 in diffuse large B-cell lymphoma (DLBCL) has been reported previously. However, the detailed mechanisms of CCR7 in DLBCL, particularly regarding its interaction with lenalidomide treatment, are not fully understood.

Methods: Our study utilized bioinformatics approaches to identify hub genes in SU-DHL-2 cell lines treated with lenalidomide compared to control groups. Immunohistochemical data and clinical information from 122 patients with DLBCL were analyzed to assess the correlation of CCR7 and p-ERK1/2 expression with the prognosis of DLBCL. Furthermore, in vitro and in vivo experiments were conducted to clarify the role of CCR7 in the response of DLBCL to lenalidomide treatment.

Results: Our bioinformatics analysis pinpointed CCR7 as a hub gene in the context of lenalidomide treatment in DLBCL. Notably, 31.14% and 36.0% (44/122) of DLBCL cases showed positive expression for CCR7 and ERK1/2 respectively, establishing them as independent prognostic factors for adverse outcomes in DLBCL via multivariate Cox regression analysis. Additionally, our studies demonstrated that the external application of the protein CCL21 promoted proliferation, migration, invasion, and activation of the ERK1/2 pathway in SU-DHL-2 and OCI-LY3 cell lines with high levels of CCR7 expression. This effect was mitigated by CCR7 silencing through siRNA, application of ERK inhibitors, or lenalidomide treatment. In vivo experiments reinforced the efficacy of lenalidomide, significantly reducing tumor growth rate, tumor mass, serum total LDH levels, and expression of CCR7 and p-ERK1/2 in a SU-DHL-2 xenograft model in nude mice (p < 0.05).

Conclusion: Our study clarifies the potential role of the CCL21/CCR7/ERK1/2 axis in the therapeutic effects of lenalidomide in DLBCL treatment.

Keywords: CCL21; CCR7; Diffuse large B-cell lymphoma (DLBCL); ERK1/2; Lenalidomide.

Figure 1. Analysis of DEGs and pathways in DLBCL cell lines treated with lenalidomide. (A) Volcano plot illustrating the distribution of DEGs. (B) Top 10 GO terms identified in BP, CC, and MF. (C) Top 20 pathways highlighted by KEGG enrichment analysis. (D) Protein interaction network from DEGs in the cytokine-cytokine receptor interaction pathway. (E) Ranking of the top 15 hub genes based on MCC scores. (F) Validation of RNA-seq findings through qRT-PCR analysis.

Figure 2. Impact of lenalidomide concentration on CCR7 expression in DLBCL cell lines. (A) Dose-response curves for lenalidomide on SU-DHL-2 and OCI-LY3 cells over 72 h. (B) IHC analysis showing reduced CCR7 expression in cells treated with lenalidomide. (C) Bar chart indicating the IHC scores for CCR7 expression across different lenalidomide concentrations. (D) WB analysis demonstrating a dose-dependent decrease in CCR7 expression with increasing lenalidomide concentrations. (E) Graphical representation of CCR7’s relative expression levels (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 3. The impact of CCR7 and p-ERK1/2 expression on prognosis in DLBCL patients. (A) Representative i IHC images of positive and negative expressions of CCR7 and p-ERK1/2 in DLBCL tissue samples (×200). (B) Multicolor immunofluorescence staining illustrating the co-localization of CD20, p-ERK1/2, and CCR7 in DLBCL (×200). (C) Scatter plot demonstrating the correlation between CCR7 and p-ERK1/2 expression levels. (D) Kaplan-Meier survival curves comparing the outcomes of patients with positive vs. negative CCR7 expression. (E) Forest plots from univariate and multivariate Cox regression analyses assessing the prognostic risk associated with DLBCL patients.

Figure 4. Activation of the ERK1/2 pathway by CCL21 through CCR7 enhances proliferation, migration, and invasion in DLBCL cells. (A–C) Analysis of CCR7 expression in OCI-LY3, SU-DHL-2, and U2932 DLBCL cell lines using IHC (×200) (A) and WB (B and C). (D–F) The impact of CCL21 on cell proliferation was assessed via EdU staining (D–E) and CCK-8 assays (F) across the three cell lines. (G and H) Transwell experiments demonstrated the effect of CCL21 on cell migration (G) and invasion capabilities (H) in the cell lines. (I–J) Analysis of the activation of the ERK1/2 signaling pathway by CCL21 (I) and its quantitative assessment (J) (^nsp > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001).

Figure 5. Lenalidomide modulates the CCL21/CCR7/ERK axis to suppress malignant biological behaviors in DLBCL cells. (A–C) Impact of lenalidomide and CCL21 on ERK1/2 phosphorylation and CCR7 expression. (D) CCK-8 assay evaluates the effect of lenalidomide and CCL21 on DLBCL cell viability. (E and F) Transwell migration (E) and invasion (F) assays demonstrate the regulation of cell behavior by lenalidomide and CCL21. (G–I) Influence of U0126 and CCL21 on ERK1/2 phosphorylation and CCR7 expression. (J) CCK-8 determination of the impact of U0126 and CCL21 on cell viability. (K and L) Effects of U0126 and CCL21 in transwell migration (K) and invasion (L) assays. (M and N) Analysis of ERK1/2 activity changes following CCR7 knockdown with CCR7-siRNA. (O–Q) Assessment of CCL21’s impact on cell viability (O), migration (P), and invasion (Q) after siRNA treatment (^nsp > 0.05, *p < 0.05, **p < 0.01).

Figure 6. In vivo antitumor effects and mechanism of lenalidomide. Representative images of euthanized mice bearing tumors (A) and excised tumors from both groups (B). (C) Tumor growth curves following treatment with Lenalidomide and DMSO. (D) Tumor weights of mice after euthanasia. (E) Serum LDH levels in both groups. (F) HE and IHC staining showing CCR7 and p-ERK expression in tumor tissues from both groups (×200). (G and H) Protein expression of CCR7 and p-ERK in both groups (*p < 0.05, **p < 0.01).