PRAME promotes epithelial-to-mesenchymal transition in triple negative breast cancer

Abstract

Background: The triple negative breast cancer (TNBC) paradox marks a major challenge in the treatment-decision making process. TNBC patients generally respond better to neoadjuvant chemotherapy compared to other breast cancer patients; however, they have a substantial higher risk of disease recurrence. We evaluated the expression of the tumor-associated antigen PReferentially Antigen expressed in MElanoma (PRAME) as a prognostic biomarker in breast cancer and explored its role in cell migration and invasion, key hallmarks of progressive and metastatic disease.

Methods: TCGA and GTeX datasets were interrogated to assess the expression of PRAME in relation to overall and disease-free survival. The role of PRAME in cell migration and invasion was investigated using gain- and loss-of-function TNBC cell line models.

Results: We show that PRAME promotes migration and invasion of TNBC cells through changes in expression of E-cadherin, N-cadherin, vimentin and ZEB1, core markers of an epithelial-to-mesenchymal transition. Mechanistic analysis of PRAME-overexpressing cells showed an upregulation of 11 genes (SNAI1, TCF4, TWIST1, FOXC2, IL1RN, MMP2, SOX10, WNT11, MMP3, PDGFRB, and JAG1) and downregulation of 2 genes (BMP7 and TSPAN13). Gene ontology analyses revealed enrichment of genes that are dysregulated in ovarian and esophageal cancer and are involved in transcription and apoptosis. In line with this, interrogation of TCGA and GTEx data demonstrated an increased PRAME expression in ovarian and esophageal tumor tissues in addition to breast tumors where it is associated with worse survival.

Conclusions: Our findings indicate that PRAME plays a tumor-promoting role in triple negative breast cancer by increasing cancer cell motility through EMT-gene reprogramming. Therefore, PRAME could serve as a prognostic biomarker and/or therapeutic target in TNBC.

Keywords: Epithelial-to-mesenchymal transition; Invasion; Migration; PRAME; PReferentially Antigen expressed in Melanoma; Triple negative breast cancer.

Fig. 1

PRAME expression in common malignancies and association with survival in breast cancer. a PRAME mRNA expression across the most common human malignancies comparing expression in tumor (T, red) and normal (N, grey) tissue. Boxplot represents Median and IQR. *p ≤ 0.05, One-way ANOVA with log₂FC cutoff ≥ 1.5 or ≤ − 1.5. SKCM, Skin Cutaneous Melanoma; BRCA, breast invasive carcinoma; COAD, colon adenocarcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; LAML, acute myeloid leukemia. b Kaplan–Meier curves for overall and disease-free survival of PRAME expression in breast cancer. Patients were classified into subgroups with low (n = 531) or high (n = 534) expression defined as lower or as higher than median PRAME mRNA expression. p-value obtained by log-rank test

Fig. 2

PRAME mRNA and protein expression in silenced and overexpressing TNBC cell line models. a Relative PRAME mRNA expression, normalized to the housekeeping gene RPLPO. b Representative picture and densitometric quantification of PRAME protein expression, as determined by western blot. *p ≤ 0.05. c Representative immunofluorescence pictures of PRAME localization. Magnification ×40. DAPI, blue; PRAME, red. Insert at 130% zoom

Fig. 3

PRAME alters migratory potential of triple negative breast cancer cells. Migratory ability of a TNBC cells treated with siCTR or siPRAME for 72 h or b TNBC cells stably transduced with control vector or a vector encoding full length PRAME. Migration potential was measured by both the wound closure assay and the QCM™ 24-well colorimetric cell migration assay (Boyden chamber principle). **p < 0.01, *p < 0.05, n = 3 biological replicates

Fig. 4

PRAME overexpression mediates invasion through 3D Matrigel. a Inverted Matrigel invasion of TNBC cells stably transduced with control vector or full-length PRAME. Live cells were visualized using Calcein-AM and serial optical sections were acquired at 15 μm intervals, with increasing depth from left to right. Rate of invasion beyond 30 μm, indicated by red line, was quantified in 3 fields of view/biological replicate (n = 3). **p < 0.01. b Matrigel Boyden Chamber invasion assay of TNBC cells stably transduced with control vector or full-length PRAME, performed in QCM ECMatrix Cell Invasion Assay. *p < 0.05, n = 3 biological replicates

Fig. 5

PRAME induces an epithelial-to-mesenchymal transition towards a more mesenchymal phenotype. a Protein expression and densitometric quantification of epithelial (E-Cadherin) and mesenchymal (Vimentin, N-Cadherin) markers in PRAME TNBC cell line models. N-Cadherin expression could not be detected in the MDA-MB-468 cell line. *p ≤ 0.05; **p ≤ 0.01. b Representative immunofluorescence image of E-cadherin, demonstrating subcellular redistribution in PRAME overexpressing cells. Magnification ×40. DAPI, blue; E-Cadherin, green. Insert at 130% zoom

Fig. 6

PRAME plays a role in gene expression reprogramming during epithelial-to-mesenchymal transition. a PRAME overexpression results in upregulation of 11 and downregulation of 2 EMT-related genes as determined by the EMT RT2 Profiler qPCR assay. b Gene ontology analyses of PRAME-associated upregulated genes according to enrichment for biological processes and disease-inference. c TCGA and GTEx data analysis of PRAME mRNA expression in esophageal and ovarian cancer using the GEPIA web server. T, tumor (red); N, normal (gray). Boxplot represents median and IQR. *p < 0.05, One-way ANOVA with log₂FC cutoff ≥ 1.5 or ≤ − 1.5

Fig. 7

PRAME induces the expression of several epithelial-to-mesenchymal transition transcription factors. a PRAME overexpression results in upregulation of 4 EMT-transcription factors as determined by the EMT RT2 Profiler qPCR assay. b Protein expression of TWIST1 and ZEB1 in PRAME TNBC cell line models