Unveiling transcriptional mechanisms of B7-H3 in breast cancer stem cells through proteomic approaches

Abstract

B7-H3, an immune checkpoint molecule, is prominently overexpressed in various solid tumors, correlating with poor clinical outcomes. Despite its critical role in promoting tumorigenesis, metastasis, and immune evasion, the regulatory mechanisms governing B7-H3 expression, particularly in cancer stem cells (CSCs), remain elusive. In this comprehensive study, we focused on breast CSCs to uncover the transcriptional regulators driving B7-H3 overexpression. Utilizing DNA affinity purification-mass spectrometry (DAP-MS) to analyze B7-H3 promoter regions, we identified a novel set of transcription factors, including DDB1, XRCC5, PARP1, RPA1, and RPA3, as key modulators of B7-H3 expression. Functional assays revealed that targeting DDB1 with nitazoxanide significantly downregulated B7-H3 expression, subsequently impairing tumor sphere formation and cell migration in breast CSCs. These findings not only elucidate the complex transcriptional network controlling B7-H3 expression but also open new avenues for developing targeted immunotherapies aimed at disrupting CSC-driven cancer progression.

Keywords: cancer; cell biology; molecular biology; omics.

Graphical abstract

Figure 1

B7-H3 expression in CSCs populations (A) Western blot analysis of B7-H3 in breast cancer CSCs, NCSCs, and parental MDA-MB453 cells. GAPDH was used as a loading control. (B) Flow cytometry analysis of B7-H3 expression in breast cancer CSCs, NCSCs, and MDA-MB453 (parental cells). Histograms show B7-H3 staining (blue line) versus isotype control IgG1κ (black line). (C) Quantitative RT-PCR analysis of B7-H3 in breast cancer CSCs, NCSCs, and parental MDA-MB453 cells. β-actin mRNA was used for normalization. (D) B7-H3 expression levels in CSCs population (CD44^highCD24^low), NCSCs population (CD44^lowCD24^high), and their respective parental cells in HCT116 and PANC-1 cell lines. Histograms show B7-H3 staining (blue line) versus secondary Ab only (black line). B7-H3 expression was assessed by (E) western blot and (F) quantitative RT-PCR in CSC-eniched populations (CSC-HCT116 and CSC-PANC-1) and their parental cell lines (HCT116, PANC-1). Representative results from three independent experiments are shown. All values are expressed as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Figure 2

Proteomic analysis of B7-H3 promoter-binding proteins in CSCs and NCSCs (A) Dual luciferase reporter assays using truncated B7-H3 promoter constructs in CSCs and NCSCs. Promoter fragments of human B7-H3 were cloned into the pGL3 Basic vector, and relative luciferase activity was measured following transfection into CSCs and NCSCs. The pGL3 Basic vector without an insert served as a negative control. Luciferase activity was normalized as the ratio of Firefly luciferase to Renilla luciferase. Representative results from three independent experiments are shown. Data are presented as mean ± SD. Statistical significance was determined as follows: ∗p < 0.05 and ∗∗p < 0.01. (B) Volcano plots of protein abundance between CSCs and NCSCs using 500 and 1,000 bp B7-H3 promoter DNA fragments against a 500 bp scrambled DNA control. Each dot represents a protein, black cutoff lines indicate q value <0.05. Red dots represent upregulated proteins, while gray dots represent downregulated proteins or non-significant proteins. Comparisions include: (1) 1,000 bp promoter vs. scrambled DNA in CSCs, (2) 500 bp promoter vs. scrambled DNA in CSCs, (3) CSCs vs. NCSCs using 1,000 bp promoter DNA, and (4) CSCs vs. NCSCs using 500 bp promoter DNA. (C) Venn diagram showing the overlap of significantly upregulated proteins across the four comparision groups. (D) The heatmap displays the Z scores of the 47 proteins across the four comparision groups. (E) Gene Ontology (GO) analysis of the 47 upregulated proteins. Only the top five significant enriched terms from each GO catagory are shown. Point size indicates the number of proteins associated with each term, and point color represents the fold enrichment score. The x axis displays the statistical significance of enrichment as Log10(FDR).

Figure 3

Regulation of B7-H3 expression by TFs in CSCs (A) mRNA expression levels of B7-H3 in CSCs following transfection with siRNA targeting candidate proteins (siXRCC5, siPARP1, siDDB1, siDDB2, siRPA1, and siRPA3), with siControl as a negative control and siB7-H3 as a positive control. Representative results from three independent experiments are shown. All values are expressed as mean ± SD. ∗p < 0.05 and ∗∗p < 0.01. (B) Protein expression levels of B7-H3 following siRNA-mediated knockdown of candidate TFs were assessed by Western blot, with GAPDH used as a loading control. (C) Flow cytometry analysis of B7-H3 expression following siRNA-mediated knockdown of candidate proteins. Histograms show B7-H3 staining (red line) compared to isotype control IgG1κ (black line).

Figure 4

Verification of TFs binding to the B7-H3 promoter using a DNA pull-down assay Biotinylated B7-H3 promoter DNA fragments [-1,000 bp, -900 bp, -700 bp, -500 bp, -300 bp, and -100 bp relative to the TSS (+1)] were pulled down with streptavidin beads. A 500 bp scrambled DNA fragment was used as a negative control. (A) Western blot analysis confirming the binding of TFs to the B7-H3 promoter DNA in CSCs and NCSCs. Bands correpond to the endogenous levels of the five candidate TFs at thier predicted molecular weights. Total cell lysates (input) from each group were used as positive controls, and GAPDH was included as a loading control. (B) Straptavidin pull-down assay reveals the binding of TFs to the B7-H3 promoter DNA. Biotinylated promoter DNA fragments were incubated with lysates from HEK293 cells overexpressing FLAG-tagged TFs, and bound proteins were detected using an anti-FLAG antibody. Total cell lysates (input) were used as a positive control to confrim TF overexpression. GAPDH was included as a loading control.

Figure 5

Characterization of TFs regulating CSC properties (A) Tumor sphere formation in CSCs following siRNA-mediated knockdown of DDB1, PARP1, XRCC5, RPA1, RPA3, and B7-H3, compared to cells treated with non-silencing control siRNA (siControl). (B) Wound-healing assay and (C) transwell migration assay performed in CSCs following siRNA-mediated knockdown of DDB1, PARP1, XRCC5, RPA1, RPA3, and B7-H3. Representative results from three independent experiments are shown. Data are presented as means ± SD. Statistical significance was determined as follows: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Figure 6

Effect of NTZ on B7-H3 expression and stemness properties in CSCs (A) B7-H3 protein and (B) mRNA expression levels in CSCs followingNTZ treatment. Western blot analysis was performed using GAPDH as a loading control, and quantitative RT-PCR was conducted with β-actin mRNA as the normalization control. (C) Cell viability assay of CSCs and NCSCs following NTZ treatment. (D) Comparison of NTZ-induced cytotoxicity between CSCs and B7-H3 knockout CSCs. (E) Tumor-sphere formation in CSCs and NCSCs following NTZ treatment. Cell migration assessment using (F) wound healing assay and (G) transwell migration assay to access cell migration in CSCs treated with NTZ. Representative results from three independent experiments are shown. Data are presented as mean ± SD. Statistical significance was determined as follows: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.