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. 2016 Oct 25;7(43):69649-69665.
doi: 10.18632/oncotarget.11935.

CDCP1 Is a Novel Marker of the Most Aggressive Human Triple-Negative Breast Cancers

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Free PMC article

CDCP1 Is a Novel Marker of the Most Aggressive Human Triple-Negative Breast Cancers

Federica Turdo et al. Oncotarget. .
Free PMC article

Abstract

CDCP1, a transmembrane noncatalytic receptor, the expression of which has been associated with a poor prognosis in certain epithelial cancers, was found to be expressed in highly aggressive triple-negative breast cancer (TNBC) cell models, in which it promoted aggressive activities-ie, migration, invasion, anchorage-independent tumor growth, and the formation of vascular-like structures in vitro. By immunohistochemical (IHC) analysis of 100 human TNBC specimens, CDCP1 was overexpressed in 57% of samples, 38% of which exhibited a gain in CDCP1 copy number by fluorescence in situ hybridization (FISH). CDCP1 positivity was significantly associated between FISH and IHC. CDCP1 expression and gains in CDCP1 copy number synergized with nodal (N) status in determining disease-free and distant disease-free survival. The hazard ratios (HRs) of the synergies between CDCP1 positivity by IHC and FISH and lymph node positivity in predicting relapse did not differ significantly, indicating that CDCP1 overexpression in human primary TNBCs, regardless of being driven by gains in CDCP1, is for a critical factor in the progression of N-positive TNBCs. Thus, CDCP1 is a novel marker of the most aggressive N-positive TNBCs and a potential therapeutic target.

Keywords: CDCP1; CDCP1 copy number; metastasis; prognosis; triple-negative breast cancer.

Conflict of interest statement

CONFLICTS OF INTEREST

No potential conflicts of interest are disclosed by the authors.

Figures

Figure 1
Figure 1. Expression of gene pathways by treatment with WHFs
Enrichment Map of pathways (Gene Ontology Biological Processes) significantly enriched (negative enrichment, blue; positive enrichment, red) (FDR < 0.2) in WHF-treated compared to untreated TNBC cell lines by GSEA analysis.
Figure 2
Figure 2. Regulation of CDCP1 expression by WHF in TNBC cell lines
(A) The TNBC cell lines MDA-MB-231, BT-549, SUM149, SUM159, MDA-MB-157, HCC1937 and MDA-MB-468, considered basal-like (B) or mesenchymal-like (M) per Lehmann classification (4), were starved (0% FBS) for 24 h, stimulated for 48 h with a WHF pool, and processed for western blot analysis of CDCP1 (the full-length 135-kD and 70-kD forms) using polyclonal anti-CDCP1. Monoclonal anti-actin was used as a loading control. The results are representative of 3 independent experiments. (B) The graph shows the fold-increase ± standard error of the mean (SEM) in full-length CDCP1 on WHF stimulation for each cell line in 3 western blot experiments, evaluated by densitometry and normalized to actin levels.
Figure 3
Figure 3. Effect of silencing CDCP1 on signaling mediators
(A) MDA-MB-231 and BT-549 cells were transfected with a pool of 4 oligonucleotides (100 nM) that bind and degrade CDCP1 mRNA (see Materials and Methods for sequences) or with the appropriate negative control siRNAs. Cells were harvested at 48 h post-transfection, and CDCP1 expression was verified by western blot. Monoclonal anti-actin was used as a loading control. The knockdown efficiency was ≥ 60% for both CDCP1 forms. (B) CDCP1 siRNA-treated and control-treated MDA-MB-231 and BT-549 cells were analyzed for activation of CDCP1, Src, and PKCδ. Monoclonal anti-actin and anti-vinculin were used as loading controls.
Figure 4
Figure 4. Correlation between CDCP1 and migration and invasion in TNBC cell lines
CDCP1 siRNA-treated and control-treated MDA-MB-231 and BT-549 cells were plated into Boyden chambers for the migration assay (A, B) and in Transwells that were coated with Matrigel for the invasion assay (C, D). The area occupied by migrated cells in the Transwell assay (mean ± SEM in 3 independent experiments) was measured by digital image analysis (Image Pro-Plus 7.0 application, Media Cybernetics). *p < 0.05.
Figure 5
Figure 5. CDCP1 correlation with growth of TNBC cell lines
(A) Proliferation under anchorage-dependent conditions (2D) of CDCP1 siRNA- and control siRNA-treated cells was evaluated by SRB (mean ± SEM in 3 independent experiments). (B) Anchorage-independent tumor growth (3D) was performed as described in Materials and Methods on MDA-MB-231 and BT-549 cells expressing CDCP1 or in which CDCP1 was knocked down. Colony growth was determined by counting all colonies in each well 15 days after seeding at 40x magnification. (C) Tube formation assay was performed as described in Materials and Methods on MDA-MB-231 cells expressing CDCP1 or in which CDCP1 was knocked down. *p < 0.05; ns, not significant.
Figure 6
Figure 6. CDCP1 expression in human primary TNBCs
(A) FFPE TNBC sections were immunostained with polyclonal anti-CDCP1 (Thermo Fisher Scientific), and membrane staining intensity was scored as positive or negative as described in Materials and Methods. Scale bar: 50 μm (insets 2×). (B) Frequency distribution of human specimens by percentage of CDCP1-positive cells.
Figure 7
Figure 7. Genetic alterations in CDCP1 in human primary TNBCs
(A) Physical map of 3p21.31 region (spanning CDCP1), including genomic clones selected for FISH experiments covering the CDCP1 locus, their size in Kb, and the genes that they encompass. Maps are derived from University of California Santa Cruz Genome Browser (http://genome.ucsc.edu/), with adaptations. (B) Representative images of dual-color FISH using CDCP1/CEP3 probes on FFPE human primary TNBC specimens, showing tumor cells with: > 3 red signals for CEP3 and > 3 green signals for the CDCP1 locus (polysomy); < 3 signals for CEP3 and > 3 green signals for CDCP1 (amplification); < 3 signals for CEP3 and < 3 green signals for CDCP1 (disomy); and > 3 signals for CEP3 and < 3 green signals for the CDCP1 locus (deletion). (C) Correlation analysis between percentage of FISH-positive cells with percentage of IHC-positive cells (n = 75; p < 0.0001; Pearson r = 0.6121; 95% confidence interval 0.4472 to 0.7367).
Figure 8
Figure 8. Prognostic value of CDCP1 in TNBC primary tumors
Association between CDCP1 IHC status in N+ and N− patients with DFS (A) and DDFS (B) and between CDCP1 FISH status in N+ and N− patients with DFS (C) and DDFS (D). L.R. p = p-value of the log-rank test.

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