Screening of the novel immune-suppressive biomarkers of TMED family and whether knockdown of TMED2/3/4/9 inhibits cell migration and invasion in breast cancer

BACKGROUND: Transmembrane p24 trafficking protein (TMED) family members are implicated in several solid tumors, but their clinical relevance for breast cancer (BC) remains unclear. This study aimed to probe their prognostic values and relations with tumor immunity in BC. METHODS: TMED family mRNA expression was assessed in five microarray datasets (GSE65212, GSE42568, GSE5364, GSE22820 and GSE45827) from Gene Expression Omnibus (GEO) database and invasive breast cancer (BRCA) cohort from The Cancer Genome Atlas (TCGA). Receiver operating characteristic (ROC) curve was performed to determine the predictive values of filtered members of the TMED family. The protein expressions of screen genes were validated by Clinical Proteomic Tumor Analysis Consortium (CPTAC) data from University of ALabama at Birmingham CANcer data analysis portal (UALCAN) and detected in the clinical specimens by western blot assay. Clinicopathologic variables were analyzed with bc-GenExMiner, and patient prognostic data were obtained with Kaplan-Meier Plotter. In vitro wound healing and invasion assays were performed on siRNA-transfected BC cell lines. TIMER 2.0, SangerBox, and ImmPort were used to evaluate tumor immune infiltration, immune checkpoints, and other immune-related genes. CbioPortal, Metascape, Expression2kinases, and LinkedOmics were used to explore gene regulatory network. RESULTS: BC tissues expressed TMED2/3/4/9 at a higher level than normal tissues, providing diagnostic potential. All the areas under the ROC curve for TMED2/3/4/9 were more than 0.7. TMED2/3/4/9 correlated with numerous clinical variables, including lymph node status, Scarff-Bloom-Richardson score (SBR), Nottingham Prognostic Index (NPI), estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2), and triple-negative breast cancer (TNBC) status, and their high expression predicted the poor prognosis of BC patients. TMED2/3/4/9 knockdown drastically inhibited the migratory and invasive capacities of MDA-MB-231 and HCC1937 cells. TMED2/3/4/9 expressions correlated negatively with the infiltration of tumor-suppressive immune cells such as CD8(+) T cells, dendritic cells, and natural killer cells, and was inversely related to a variety of immune checkpoint genes, including programmed cell death 1 (PD-1) and cytotoxic T-lymphocyte associated protein 4 (CTLA4). A set of kinases, transcription factors, and microRNAs (miRNAs) may regulate TMED2/3/4/9 abnormalities at the genome level. CONCLUSIONS: TMED2/3/4/9 may serve as diagnostic, prognostic, and immune-suppressive biomarkers in BC.