The prognostic value of JUNB-positive CTCs in metastatic breast cancer: from bioinformatics to phenotypic characterization

Abstract

Background: Circulating tumor cells (CTCs) are important for metastatic dissemination of cancer. They can provide useful information, regarding biological features and tumor heterogeneity; however, their detection and characterization are difficult due to their limited number in the bloodstream and their mesenchymal characteristics. Therefore, new biomarkers are needed to address these questions.

Methods: Bioinformatics functional enrichment analysis revealed a subgroup of 24 genes, potentially overexpressed in CTCs. Among these genes, the chemokine receptor CXCR4 plays a central role. After prioritization according to the CXCR4 corresponding pathways, five molecules (JUNB, YWHAB, TYROBP, NFYA, and PRDX1) were selected for further analysis in biological samples. The SKBR3, MDA-MB231, and MCF7 cell lines, as well as PBMCs from normal (n = 10) blood donors, were used as controls to define the expression pattern of all the examined molecules. Consequently, 100 previously untreated metastatic breast cancer (mBC) patients (n = 100) were analyzed using the following combinations of antibodies: CK (cytokeratin)/CXCR4/JUNB, CK/NFYA/ΥWHΑΒ (14-3-3), and CK/TYROBP/PRDX1. A threshold value for every molecule was considered the mean expression in normal PBMCs.

Results: Quantification of CXCR4 revealed overexpression of the receptor in SKBR3 and in CTCs, following the subsequent scale (SKBR3>CTCs>Hela>MCF7>MDA-MB231). JUNB was also overexpressed in CTCs (SKBR3>CTCs>MCF7>MDA-MB231>Hela). According to the defined threshold for each molecule, CXCR4-positive CTCs were identified in 90% of the patients with detectable tumor cells in their blood. In addition, 65%, 75%, 14.3%, and 12.5% of the patients harbored JUNB-, TYROBP-, NFYA-, and PRDX-positive CTCs, respectively. Conversely, none of the patients revealed YWHAB-positive CTCs. Interestingly, JUNB expression in CTCs was phenotypically and statistically enhanced compared to patients' blood cells (p = 0.002) providing a possible new biomarker for CTCs. Furthermore, the detection of JUNB-positive CTCs in patients was associated with poorer PFS (p = 0.015) and OS (p = 0.002). Moreover, JUNB staining of 11 primary and 4 metastatic tumors from the same cohort of patients revealed a dramatic increase of JUNB expression in metastasis.

Conclusions: CXCR4, JUNB, and TYROBP were overexpressed in CTCs, but only the expression of JUNB was associated with poor prognosis, providing a new biomarker and a potential therapeutic target for the elimination of CTCs.

Keywords: Bioinformatics; Breast cancer; CTCs; CXCR4; JUNB.

Fig. 1

Bioinformatics and functional enrichment analysis for biomarker discovery. Twenty-four genes were obtained from a data-driven computational procedure (data integration, cross-site comparisons) and were further explored by using functional enrichment analysis. CXCR4 plays a central role in this analysis. Based on four databases (G2SBC, WebGestalt, GSEA, and KEGG), significant biological information was validated and five genes of the 24-signature were prioritized according to their direct or indirect association with the CXCR4 pathway. These six molecules (CXCR4, JUNB, YWHAB, TYROBP, NFYA, and PRDX1) were experimentally evaluated in biological samples

Fig. 2

Expression of cytokeratin, CXCR4, and JUNB in CTCs isolated from breast cancer patients. a Quantification of CXCR4 expression (intensity per pixel) in breast cancer cell lines, Hela cells, PBMCs, and patients’ CTCs, using ARIOL system automate software. b Quantification of JUNB expression (intensity per pixel) in breast cancer cell lines, in Hela cells, PBMCs, and patients’ CTCs, using ARIOL system automate software. c Cytospins obtained from metastatic breast cancer patients were triple stained with CK (green), CXCR4 (orange), and JUNB (red) antibodies plus DAPI (blue) and analyzed with ARIOL system (magnification X40). d Representative images from confocal laser scanning microscopy. Patients’ samples were triple stained with CK (green), CXCR4 (red), and JUNB (blue) antibodies plus DAPI (not shown due to the absence of corresponding laser) magnification (× 60)

Fig. 3

Expression of cytokeratin, TYROBP, and PRDX1 in CTCs isolated from breast cancer patients. a Quantification of TYROBP expression (intensity per pixel) in breast cancer cell lines, Hela cells, PBMCs, and patients’ CTCs, using ARIOL system automate software. b Quantification of PRDX1 expression (intensity per pixel) in breast cancer cell lines, in Hela cells, in PBMCs, and in patients’ CTCs, using ARIOL system automate software. c Cytospins obtained from metastatic breast cancer patients were triple stained with CK (green), PRDX1(orange), and TYROBP (red) antibodies plus DAPI (blue) and analyzed with ARIOL system (magnification × 40). d Representative images from confocal laser scanning microscopy. Patients’ samples were triple stained with CK (green), TYROBP (blue), and PRDX1 (red) antibodies plus DAPI (not shown due to the absence of corresponding laser) and analyzed with confocal laser scanning microscopy (magnification × 60)

Fig. 4

Expression of cytokeratin, NFYA, and YWHAB in CTCs isolated from breast cancer patients. a Quantification of NFYA expression (intensity per pixel) in breast cancer cell lines, Hela cells, PBMCs, and patients’ CTCs, using ARIOL system automate software. b Quantification of YWHAB expression (intensity per pixel) in breast cancer cell lines, in Hela cells, in PBMCs, and in patients’ CTCs, using ARIOL system automate software. c Cytospins obtained from metastatic breast cancer patients were triple stained with CK (green), YWHAB (orange), and NFYA (red) antibodies plus DAPI (blue) and analyzed with ARIOL system (magnification × 40). d Representative images from confocal laser scanning microscopy. Patients’ samples were triple stained with CK (green), NFYA (blue), and YWHAB (red) antibodies plus DAPI (not shown due to the absence of the corresponding laser) and analyzed with confocal laser scanning microscopy (magnification × 60)

Fig. 5

I (A) Overall survival in patients with JUNB-positive CTCs compared to JUNB-negative patients (p = 0.02, Kaplan-Meier analysis). In the group of patients harboring JUNB-positive CTCs, 90% died during follow-up period vs 75% of deaths in the cohort of patients without JUNB expression. I (B) Kaplan-Meier analysis revealed that the progression-free survival in patients harboring JUNB-positive CTCs was statistically lower compared to patients without JUNB expression (p = 0.015). Patients without JUNB-positive CTCs experienced longer PFS vs JUNB-negative patients [3.5 (range 0–30) vs 9 (range 0–72) months]. II (A) JUNB staining in primary tumor obtained from BC patient. JUNB-positive tumor cells (brown) were very rare in the sample. Inner frame is shown control sample from a squamous cell carcinoma (magnification × 40). II (B) JUNB staining in metastatic tumor obtained from mBC patient. The majority of the tumor cells were positive for JUNB (magnification × 40)