Syndecan-1 as an immunogene in Triple-negative breast cancer: regulation tumor-infiltrating lymphocyte in the tumor microenviroment and EMT by TGFb1/Smad pathway

Abstract

Background: Immune checkpoint inhibitors are the most studied forms of immunotherapy for triple-negative breast cancer (TNBC). The Cancer Genome Map (TCGA) and METABRIC project provide large-scale cancer samples that can be used for comprehensive and reliable immunity-related gene research.

Methods: We analyzed data from TCGA and METABRIC and established an immunity-related gene prognosis model for breast cancer. The SDC1 expression in tumor and cancer associated fibroblasts (CAFs) was then observed in 282 TNBC patients by immunohistochemistry. The effects of SDC1 on MDA-MB-231 proliferation, migration and invasion were evaluated. Qualitative real-time PCR and western blotting were performed to identify mRNA and protein expression, respectively.

Results: SDC1, as a key immunity-related gene, was significantly correlated with survival in the TCGA and METABRIC databases, while SDC1 was found to be highly expressed in TNBC in the METABRIC database. In the TNBC cohort, patients with high SDC1 expression in tumor cells and low expression in CAFs had significantly lower disease-free survival (DFS) and fewer tumor-infiltrating lymphocytes (TILs). The downregulation of SDC1 decreased the proliferation of MDA-MB-231, while promoting the migration of MDA-MB-231 cells by reducing the gene expression of E-cadherin and TGFb1 and activating p-Smad2 and p-Smad3 expression.

Conclusion: SDC1 is a key immunity-related gene that is highly expressed TNBC patients. Patients with high SDC1 expression in tumors and low expression in CAFs had poor prognoses and low TILs. Our findings also suggest that SDC1 regulates the migration of MDA-MB-231 breast cancer cells through a TGFb1-Smad and E-cadherin-dependent mechanism.

Keywords: Prognosis; SDC1; TGFb1-Smad; Triple negative breast cancer; Tumor-infiltrating lymphocyte.

Fig. 1

A Heatmap and C volcano plot showing differentially expressed genes between breast cancer tissue and normal tissue in the TCGA dataset. Red dots represent differentially up-regulated genes, green dots represent differentially downregulated genes and black dots represent no differentially expressed genes. B Heatmap and D volcano plot showed different immune-related genes between breast cancer tissue and normal tissue in the TCGA dataset. Red dots represent differentially up-regulated genes, green dots represent differentially downregulated genes and black dots represent no differentially expressed genes. E The most significant genomes pathways by gene functional enrichment in immune-related genes. F Sixteen immunity-related differentially expressed genes significantly associated with overall survival in the TCGA datasets

Fig. 2

A The survival outcome and B the overall survival between high risk and low-risk patients distinguished by immunity-related gene model. C The prognostic value of the model by receiver operating characteristic (ROC) curve. Univariate analysis (D) and multivariate analysis (E) of immunity-related gene prognosis model and distant metastasis in the METABRIC datasets. The relationships between B cell infiltration (F), CD4⁺ T cell infiltration (G), CD8⁺ T cell infiltration (H), dendritic infiltration (I), macrophage infiltration (J), neutrophil infiltration (K) and the risk value by the immunity-related gene prognosis model

Fig. 3

A The overall survival and B disease-free survival in METABRIC datasets between patients with high expression and low expression of SDC1. C SDC1 expression in patients with or without lymph node metastasis in the TCGA dataset. In the METABRIC dataset, D SDC1 expression in patients with TNBC and in other patients, E SDC1 expression in HER2-positive patients and HER2-negative patients, F SDC1 expression in patients aged ≤ 60 years old and > 60 years old

Fig. 4

A Hematoxylin and eosin (HE) staining and immunohistochemistry (IHC) of SDC1 expression in TNBC patients. SDC1-negative expression on tumor cell by (a) HE staining and b IHC (original magnification, × 200). SDC1 1 + expression on tumor cells by (c) HE staining and d IHC (original magnification, × 200). SDC1 2 + expression on tumor cells by (e) HE staining and f IHC (original magnification, × 200). SDC1 3 + expression on tumor cells by (g) HE staining and h IHC (original magnification, × 200). SDC1-negative expression in cancer-associated fibroblasts (CAFs) by (i) HE staining and j IHC (original magnification, × 200). SDC1-positive expression on CAF by (k) HE staining and l IHC (original magnification, × 200). B The percentage of TNBC patients with the expression of SDC1 in tumor cells and in paracancerous normal mammary duct cells (PNMDCs). Disease-free survival between SDC1-positive patients and SDC1-negative patients in tumor cells C and in CAFs D in the TNBC cohort

Fig. 5

A SDC1 expression in tumor cells associated with TILs in the TNBC cohort. a, b Patients with SDC1-positive expression and a low percentage of CD3⁺ TILs. c, d Patients with SDC1-positive expression and a low percentage of CD4⁺ TILs. e, f Patients with SDC1-positive expression and a low percentage of CD8⁺ TILs. g, h Patients with SDC1-positive expression and a low percentage of CD19⁺ TILs. B Frequencies of TILs between patients with SDC1-negative expression and SDC1-positive expression in tumor cells in the TNBC cohort. a Frequencies of TILs, b CD3⁺ TILs, c CD4⁺ TILs, d CD8⁺ TILs, e CD19⁺ TILs associated with SDC1 expression in tumor cells. C SDC1 expression in CAFs associated with TILs in the TNBC cohort. a, b Patients with SDC1-positive expression and a low percentage of CD3⁺ TILs. c, d Patients with SDC1-positive expression and a high percentage of CD4⁺ TILs. e, f Patients with SDC1-positive expression and a low percentage of CD8⁺ TILs. g, h The patients with SDC1-positive expression and a high percentage of CD19⁺ TILs. D Frequencies of TILs between patients with SDC1-negative expression and SDC1-positive expression in CAF in the TNBC cohort. a Frequencies of TILs, b CD3⁺ TILs, c CD4⁺ TILs, d CD8⁺ TILs, e CD19⁺ TILs associated with SDC1 expression in CAFs

Fig. 6

The effect of SDC1 on breast cancer cell proliferation and migration. A Relative SDC1 mRNA expression after knockdown and overexpression. B Relative SDC1 protein expression after knockdown and overexpression. A CCK-8 assay evaluated the effects of SDC1 on cell proliferation after the C knockdown and D overexpression of SDC1. E Transwell assay determined that SDC1 knockdown promoted the invasion of MDA-MB-231 cells. F The protein levels of EMT-related proteins. G Protein expression in the TGF-β1/Smad signaling pathway. Results with P < 0.05 were considered statistically significant. An unpaired t-test was used for data analysis *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001