PLIN2 promotes colorectal cancer progression through CD36-mediated epithelial-mesenchymal transition

Abstract

Colorectal cancer (CRC) is one of the most common malignant tumors with high incidence and mortality. The challenge remains to construct reliable prognostic prediction models and to further elucidate the key molecular mechanisms of tumor progression. To address this, we performed WGCNA based on 120 immune cell expression profiles from GEO sources to obtain a collection of monocytes/macrophages-related genes. The prognostic model was constructed by univariate survival analysis and LASSO regression analysis. Then, the prognostic model was validated by Multivariate Cox regression, Kaplan-Meier survival analysis and ROC analysis. In this prognostic model, we identified that PLIN2 has a potential value for CRC prognosis. PLIN2 expression in monocytes/macrophages was verified by scRNA-seq datasets and spatial transcriptome datasets, and PLIN2 was found to promote macrophage transformation to M2 subtype. Clinical specimens and tissue microarrays confirmed the differential expression and prognostic value of PLIN2 in CRC patients. Functional experiments demonstrated that PLIN2 gene overexpression promoted the proliferation, migration and invasion of CRC cells and significantly facilitated tumor growth in vivo. Mechanistically, we revealed that CD36 is a potential downstream target gene of PLIN2. The CD36 inhibitor Sulfo-N-succinimidyl Oleate significantly reversed PLIN2-induced proliferation, migration, invasion, and EMT activity of CRC cells in vitro and in vivo. Immunoprecipitation and immunofluorescence experiments confirmed that PLIN2 could interact with CD36. PLIN2 stabilized CD36 protein expression by inhibiting the proteasomal degradation pathway, thereby promoting CD36-mediated EMT activity. Overall, our study highlights that the PLIN2/CD36 axis regulates EMT activity and CRC progression, suggesting that interventions in this signaling pathway may offer a promising therapeutic approach to CRC progression. Schematic diagram elucidating the role of PLIN2 in CRC by Figdraw. FA is transported into the cell via CD36-mediated endocytosis. In CRC cells, PLIN2 promotes stability of CD36 and interacts with CD36 to activate the EMT process. However, the CD36 inhibitor SSO inhibits the binding of FAs to CD36 and attenuates its endocytosis, thereby reversing the PLIN2-mediated EMT process. Ultimately, the PLIN2-induced enhancement of CRC cell proliferation, migration, and invasion is attenuated by the CD36 inhibitor SSO.

Schematic diagram elucidating the role of PLIN2 in CRC by Figdraw. FA is transported into the cell via CD36-mediated endocytosis. In CRC cells, PLIN2 promotes stability of CD36 and interacts with CD36 to activate the EMT process. However, the CD36 inhibitor SSO inhibits the binding of FAs to CD36 and attenuates its endocytosis, thereby reversing the PLIN2-mediated EMT process. Ultimately, the PLIN2-induced enhancement of CRC cell proliferation, migration, and invasion is attenuated by the CD36 inhibitor SSO.

Fig. 1. Construction and verification of a prognostic risk model based on monocytes/macrophages-related genes.

A Correlation results between the 16 modules and each clinical phenotype. B Forest diagram of the intersected monocytes/macrophages-related genes significantly associated with the prognosis of CRC in TCGA-CRC cohorts. C Confidence intervals under each lambda. D Kaplan–Meier survival curve distribution of the 6-gene signature in TCGA-CRC database. E Time-dependent ROC analysis of 1-, 3-, and 5-year survival for CRC patients. F–I Survival analysis of our constructed model combined with clinical factors (age, gender, lymph node metastasis and stage).

Fig. 2. Distribution of PLIN2 in the spatial transcriptome and its regulation of M2 polarization in THP-1-derived macrophages.

A Spatial distribution of PLIN2 expression across the tissue section. Color intensity indicates the relative expression level of PLIN2. B Unsupervised clustering of spatial transcriptomic spots. Each color denotes a distinct cluster identified based on global gene expression patterns. C Bar plot showing the expression of PLIN2 across the identified transcriptomic clusters. The y-axis indicates the normalized expression level, and the x-axis lists each cluster. D UMAP projection illustrating PLIN2 expression at the single-cell level. E UMAP projection annotated by cell type inferred from canonical marker genes. F Bar plot depicting PLIN2 expression levels across the major annotated cell types. The y-axis indicates normalized expression, and the x-axis lists each cell type. G Schematic diagram of the experimental procedure for induction of macrophage polarisation from THP-1 cells. H–I qRT-PCR analysis revealed that PLIN2 overexpression significantly downregulated M1 macrophage markers while upregulating M2-associated genes in THP-1 cells. J–L PLIN2 promotes the transformation of THP-1-derived macrophages to the M2 subtype as detected by flow cytometry. Data are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 3. PLIN2 gene is highly expressed in CRC and correlates with poor prognosis.

A The expression PLIN2 in CRC tissues (n = 942) and normal tissues (n = 389) analyzed in the TCGA-CRC and GTEx databases. B, C The Kaplan - Meier method for comparing the survival of patients with high and low PLIN2 expression in the TCGA-CRC dataset (n = 596) and GSE39582 dataset (n = 577). D, E PLIN2 expression in CRC tissues at different stages, from I to IV based on AJCC Cancer Staging Manual, were measured using IHC staining (D). PLIN2 expression scores were shown in (E). F, G PLIN2 expression in normal colon tissue, adenoma and metastatic adenocarcinoma was measured using IHC staining (F). PLIN2 expression scores were shown in (G). Data are shown as mean ± SD. ***p < 0.001, ****p < 0.0001.

Fig. 4. PLIN2 overexpression promotes the proliferation, migration and invasion and EMT of CRC cells in vitro.

A Western blot analysis of the expression of PLIN2 after treatment with Lv-Control and Lv-PLIN2 in RKO and SW480 cells. B CCK8 assay was used to assess cell proliferation. C, D Wound-healing assay was used to assay cell migration. E, F Cell migration was determined by using transwell migration assays. G, H Cell invasion was determined by using transwell invasion assays. Cells invading through uncoated inserts and Matrigel-coated inserts were stained. I, J Western blot analysis showed that PLIN2 decreased the expression levels of the epithelial cell markers E-cadherin and occludin and increased the mesenchymal markers N-cadherin and vimentin in RKO and SW480 cells. Data are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 5. The interaction between PLIN2 and CD36.

A Western blot analysis showed that PLIN2 increased the expression level of CD36 protein in RKO and SW480 cells. B Quantification of the protein expression of CD36 represented in (C). C qRT‒PCR showed that PLIN2 increased the expression level of CD36 protein in RKO and SW480 cells. D Molecular docking simulation of PLIN2 (blue) and CD36 (pink) (left). Amino acid sites and reciprocal hydrogen bonds (yellow) and salt bridges (purple) of PLIN2 and CD36 interaction (right). E Immunofluorescence assays were performed to detect co-localization of PLIN2 with CD36. F Co-IP showed a direct interaction between CD36 and PLIN2 in RKO and SW480 cells. Data are shown as mean ± SD. **p < 0.01, ***p < 0.001.

Fig. 6. CD36 inhibition suppresses the proliferation in CRC cells induced by PLIN2 in vitro.

A, B CCK8 assay was used to assess cell proliferation. C, D Wound-healing assay was used to assay cell migration. E, F Cell migration was determined by using transwell migration assays. G, H Cell invasion was determined by using transwell invasion assays. Cells invading through uncoated inserts and Matrigel-coated inserts were stained. Data are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Lv-control vs Lv-PLIN2); ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001 (Lv-PLIN2 vs Lv-PLIN2 + SSO).

Fig. 7. CD36 inhibition suppresses EMT in CRC cells.

A Western blot analysis showed that CD36 inhibition decreased the expression levels of the epithelial cell markers E-cadherin and occludin and increased the mesenchymal markers N-cadherin and vimentin in RKO and SW480 cells compared with the Lv-PLIN2 group. B Quantification of the protein expression of E-cadherin, N-cadherin, Vimentin and Occludin represented in (A). C, D Immunofluorescence of E-cadherin and Vimentin in RKO and SW480 cells. Data are shown as mean ± SD. **p < 0.01, ***p < 0.001, ****p < 0.0001 (Lv-control vs Lv-PLIN2); ^#p < 0.05, ^##p < 0.01, ^####p < 0.0001 (Lv-PLIN2 vs Lv-PLIN2 + SSO).

Fig. 8. PLIN2 promotes CRC progression via CD36 in vivo.

A, B Schematic diagram of the subcutaneous xenograft model (A) and CRC orthotopic model (B) by Figdraw. C–E Tumor tissue images (C), tumor volume statistical curves (D) and tumor weights (E) of Lv control, Lv-PLIN2, and Lv-PLIN2 + SSO groups of tumor-bearing mice. F Representative IHC staining images of E-cadherin, N-cadherin, occludin, and vimentin and Ki-67 in subcutaneous xenograft model tumor tissue sections of each group. G The statistical analysis of IHC staining in (F). H, I Tumor tissue images (I) and tumor weights (H) of Lv-control, Lv-PLIN2, and Lv-PLIN2 + SSO groups of CRC orthotopic model mice. J Representative HE staining images of CRC orthotopic model tumor. K Representative IHC staining images of E-cadherin, N-cadherin, occludin, vimentin and Ki-67 in CRC orthotopic model tumor tissue sections of each group. L The statistical analysis of IHC staining in (K). Data are shown as mean ± SD. **p < 0.01, ***p < 0.001, ****p < 0.0001 (Lv-control vs Lv-PLIN2); ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001 (Lv-PLIN2 vs Lv-PLIN2 + SSO).