Sterile Alpha Motif Domain-Containing 5 Suppresses Malignant Phenotypes and Tumor Growth in Breast Cancer: Regulation of Polo-Like Kinase 1 and c-Myc Signaling in a Xenograft Model

Abstract

Background Breast cancer, particularly the triple-negative breast cancer (TNBC) subtype, remains a significant clinical challenge due to its resistance to standard chemotherapy and high recurrence rate. In this study, we explored the role of Sterile Alpha Motif Domain-Containing 5 (SAMD5) as a potential regulatory partner with the c-Myc oncogenic signaling pathway in breast cancer. Materials and methods Functional assays were conducted to investigate the effects of SAMD5 overexpression on cell viability, colony formation, and invasive behavior in TNBC cell lines. This study further assessed the expression levels of proliferation and invasion markers, including Ki67 (a marker for cell proliferation), Matrix Metalloproteinase-2 (MMP2), and Matrix Metalloproteinase-9 (MMP9). Mechanistic analyses identified a negative correlation between SAMD5 and Polo-like Kinase 1 (PLK1), a gene frequently overexpressed in breast cancer, particularly in TNBC. The effects of PLK1 knockdown on cell viability, colony formation, and invasion were observed, along with the impact of PLK1 overexpression on SAMD5's inhibitory activity. In vivo studies were performed using a xenograft tumor model in nude mice to evaluate the impact of SAMD5 overexpression on tumor weight and volume. Results SAMD5 overexpression significantly reduced cell viability, colony formation, and invasion in TNBC cells, and downregulated key proteins in the c-Myc signaling pathway, including c-Myc itself, β-catenin, Cyclin-Dependent Kinase 4 (CDK4), Cyclin-Dependent Kinase 6 (CDK6), and Cyclin D1. PLK1 overexpression was found to counteract SAMD5's inhibitory effects. In vivo experiments demonstrated that SAMD5 overexpression led to a marked reduction in tumor weight and volume, effects that were partially reversed by PLK1 overexpression. Conclusions SAMD5 acts as a tumor suppressor in breast cancer, particularly in TNBC, by inhibiting critical cellular processes and downregulating the c-Myc signaling pathway. This effect appears to be mediated, in part, through its negative association with PLK1. Targeting the SAMD5/PLK1 axis offers a promising therapeutic strategy for addressing aggressive breast cancers.

Keywords: breast cancer; plk1; samd5; the c-myc signaling; triple-negative breast cancer.

Figure 1. Differentially expressed genes between breast cancer and normal samples

(A) Microarray data were obtained from Xena (https://xenabrowser.net/datapages/) using the IlluminaHiSeq platform, which included 114 normal tissue samples and 1,104 breast cancer samples. Differential gene expression was analyzed using The Cancer Genome Atlas Breast Invasive Carcinoma (TCGA-BRCA) dataset with the Linear Models for Microarray and RNA-Seq Data (limma) package. (B)-(C) Differential gene expression was further analyzed in the Gene Expression Omnibus (GEO) datasets GSE57297 and GSE22820 using the limma package. (D) The Venn diagram shows the four common differentially expressed genes (DEGs) identified in the TCGA-BRCA, GSE57297, and GSE22820 datasets. SAMD5: Sterile Alpha Motif Domain-Containing 5; PIGR: Polymeric Immunoglobulin Receptor; SOX10: SRY-Box Transcription Factor 10; WIF1: WNT Inhibitory Factor 1; mRNA: messenger RNA

Figure 2. Screening and validation of Sterile Alpha Motif Domain-Containing 5 (SAMD5) gene expression in breast cancer

(A) The expression of Sterile Alpha Motif Domain Containing 5 (SAMD5) was analyzed in The Cancer Genome Atlas Breast Invasive Carcinoma (TCGA-BRCA) breast cancer dataset (normal = 114, tumor = 1,104) and validated using Gene Expression Profiling Interactive Analysis (GEPIA2) with integrated TCGA-BRCA/Genotype-Tissue Expression (GTEx) data (normal = 291, tumor = 1,085). The expression of SAMD5 was examined across multiple Gene Expression Omnibus (GEO) datasets, including GSE70947, GSE57297, and GSE38959 (B); GSE33447, GSE7904, and GSE22820 (C); and GSE10780, GSE21442, and GSE21444 (D). ***p < 0.001

Figure 3. Clinical analysis of Sterile Alpha Motif Domain-Containing 5 (SAMD5) in breast cancer

(A) The prognostic value of Sterile Alpha Motif Domain-Containing 5 (SAMD5) expression was evaluated using the Kaplan-Meier Plotter (KMplot) online tool, which analyzed both gene chip data and RNA sequencing (RNAseq) data to determine its impact on overall survival (OS) in breast cancer patients. (B) The relationship between SAMD5 expression and clinical features was examined using data from The Cancer Genome Atlas Breast Invasive Carcinoma (TCGA-BRCA) dataset, including the analysis of its expression across different pathological stages and Tumor-Node-Metastasis (TNM) classifications. (C) The association of SAMD5 expression with tumor purity and immune cell infiltration was investigated using the Tumor Immune Estimation Resource (TIMER) (http://timer.comp-genomics.org/), which analyzed the correlation between SAMD5 expression and the infiltration of cytotoxic natural killer (NK) cells. ***p < 0.001

Figure 4. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of Sterile Alpha Motif Domain-Containing 5 (SAMD5) expression related genes in The Cancer Genome Atlas Breast Invasive Carcinoma (TCGA-BRCA)

Pearson's correlation analysis between Sterile Alpha Motif Domain-Containing 5 (SAMD5) expression and other genes was performed using data from The Cancer Genome Atlas Breast Invasive Carcinoma (TCGA-BRCA) dataset (n = 1,217), covering 20,530 gene expressions. The correlated genes were subsequently subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis.

Figure 5. Expression of Sterile Alpha Motif Domain-Containing 5 (SAMD5) in breast cancer clinical tissues and cell lines

(A) Breast cancer tissues and adjacent normal breast tissues were collected. Hematoxylin and eosin (H&E) staining was performed to confirm pathological status. (B) The messenger RNA (mRNA) expression levels of Sterile Alpha Motif Domain-Containing 5 (SAMD5), Polymeric Immunoglobulin Receptor (PIGR), SRY-Box Transcription Factor 10 (SOX10), and Wnt Inhibitory Factor 1 (WIF1) were determined in tissue samples using Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) (n = 12). (C) The protein abundance of SAMD5 in tissues was assessed using immunohistochemistry (IHC). (D) The mRNA levels of SAMD5 in normal breast epithelial cells (MCF-10A and MCF-12A) and breast cancer cell lines (MDA-MB-231, HCC1937, and BT-483) were determined using RT-qPCR. (E) The protein expression levels of SAMD5 were examined in the cell lines using immunoblotting. *p < 0.05; **p < 0.01

Figure 6. In vitro effects of Sterile Alpha Motif Domain-Containing 5 (SAMD5) on breast cancer cell phenotypes

(A) and (C) Sterile Alpha Motif Domain-Containing 5 (SAMD5) overexpression was achieved in two triple-negative breast cancer cell lines (MDA-MB-231 and HCC1937) by introducing SAMD5 overexpression (OE), which was validated using Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) and immunoblotting. (B) MDA-MB-231 and HCC1937 cells were transfected with SAMD5 OE and examined for cell viability using the Cell Counting Kit-8 (CCK-8) assay. (D) Colony formation was assessed in the transfected cells. (E) Cell invasion was evaluated using the Transwell assay. (F) The protein levels of the proliferation marker Ki67 and the invasion markers Matrix Metalloproteinase 2 (MMP2) and Matrix Metalloproteinase 9 (MMP9) were determined using immunoblotting. *p < 0.05; **p < 0.01

Figure 7. Polo-like Kinase 1 (PLK1) is negatively correlated with Sterile Alpha Motif Domain-Containing 5 (SAMD5)

(A) The 258 genes of the Hallmark MYC Targets Version 1/Version 2 (HALLMARK_MYC_TARGETS_V1/V2) pathway were analyzed for expression correlation with Sterile Alpha Motif Domain-Containing 5 (SAMD5). (B)-(F) Correlation analysis of Polo-like Kinase 1 (PLK1) and SAMD5 was conducted using data from the Gene Expression Omnibus (GEO) datasets GSE70947, GSE57297, GSE33447, GSE7904, and The Cancer Genome Atlas Breast Invasive Carcinoma (TCGA-BRCA). (G)-(H) Immunohistochemistry (IHC) staining and Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR), PLK1 expression was higher in breast cancer tissues compared to adjacent non-tumor tissues. *p < 0.05; **p < 0.01

Figure 8. In vitro effects of Polo-like Kinase 1 (PLK1) knockdown on breast cancer cell phenotypes

(A) Polo-like Kinase 1 (PLK1) knockdown was achieved in two triple-negative breast cancer cell lines (MDA-MB-231 and HCC1937) by introducing small interfering RNA against PLK1 (siRNA-PLK1-1/2/3), which was validated using immunoblotting. si-PLK1-2 was selected for further investigations due to its higher efficiency. (B) MDA-MB-231 and HCC1937 cells were transfected with si-PLK1 and examined for cell viability using the Cell Counting Kit-8 (CCK-8) assay. (C) Colony formation was assessed in the transfected cells. (D) Cell invasion was evaluated using the Transwell assay. (E) The protein levels of proliferation marker Ki67 and invasion markers Matrix Metalloproteinase 2 (MMP2) and Matrix Metalloproteinase 9 (MMP9) were determined using immunoblotting. *p < 0.05; **p < 0.01

Figure 9. Polo-like Kinase 1 (PLK1) overexpression partially attenuates Sterile Alpha Motif Domain-Containing 5 (SAMD5) overexpression effects on breast cancer cells

(A) Polo-like Kinase 1 (PLK1) overexpression was achieved in two triple-negative breast cancer cell lines (MDA-MB-231 and HCC1937) by introducing PLK1 overexpression (OE), which was validated using immunoblotting. (B) MDA-MB-231 and HCC1937 cells were then co-transfected with Sterile Alpha Motif Domain-Containing 5 (SAMD5) overexpression (OE) and PLK1 OE, and the protein levels of SAMD5 were analyzed using immunoblotting. (C) Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay. (D) Colony formation was evaluated in the transfected cells. (E) Cell invasion was measured using the Transwell assay. (F)-(G) The protein levels of proliferation marker Ki67 and invasion markers Matrix Metalloproteinase 2 (MMP2) and Matrix Metalloproteinase 9 (MMP9) were determined using immunoblotting. **p < 0.01 vs. vector group; #p < 0.05; ##p < 0.01 vs. SAMD5 group

Figure 10. Sterile Alpha Motif Domain-Containing 5 (SAMD5) interacts with Polo-like Kinase 1 (PLK1) to modulate the c-Myc signaling in breast cancer cells

(A)-(B) MDA-MB-231 and HCC1937 cells were co-transfected with Sterile Alpha Motif Domain-Containing 5 (SAMD5) overexpression (OE) and Polo-like Kinase 1 (PLK1) OE and examined for the protein levels of MYC Proto-Oncogene (c-Myc), β-catenin, Cyclin-Dependent Kinase 4 (CDK4), Cyclin-Dependent Kinase 6 (CDK6), and Cyclin D1 using immunoblotting. **p < 0.01 vs. vector group; ##p < 0.01 vs. SAMD5 group

Figure 11. Dynamic effects of Sterile Alpha Motif Domain-Containing 5 (SAMD5) and Polo-like Kinase 1 (PLK1) on tumor growth in a mouse model

(A) A xenograft tumor model was established in nude mice, and lentivirus-mediated overexpression of Sterile Alpha Motif Domain-Containing 5 (SAMD5) and/or Polo-like Kinase 1 (PLK1) was introduced into the tumors. (B)-(C) Tumor weight and volume were measured. (D) The protein levels of MYC Proto-Oncogene (c-Myc), β-catenin, Cyclin-Dependent Kinase 4 (CDK4), Cyclin-Dependent Kinase 6 (CDK6), and Cyclin D1 in tumor tissues were determined using immunoblotting. **p < 0.01 vs. vector group; #p < 0.05; ##p < 0.01 vs. SAMD5 group