Copy number aberrations of genes regulating normal thymus development in thymic epithelial tumors

Abstract

Purposes: To determine whether the deregulation of genes relevant for normal thymus development can contribute to the biology of thymic epithelial tumors (TET).

Experimental design: Using array comparative genomic hybridization, we evaluated the copy number aberrations of genes regulating thymus development. The expression of genes most commonly involved in copy number aberrations was evaluated by immunohistochemistry and correlated with patients' outcome. Correlation between FOXC1 copy number loss and gene expression was determined in a confirmation cohort. Cell lines were used to test the role of FOXC1 in tumors.

Results: Among 31 thymus development-related genes, PBX1 copy number gain and FOXC1 copy number loss were presented in 43.0% and 39.5% of the tumors, respectively. Immunohistochemistry on a series of 132 TETs, including those evaluated by comparative genomic hybridization, revealed a correlation between protein expression and copy number status only for FOXC1 but not for PBX1. Patients with FOXC1-negative tumors had a shorter time to progression and a trend for a shorter disease-related survival. The correlation between FOXC1 copy number loss and mRNA expression was confirmed in a separate cohort of 27 TETs. Ectopic FOXC1 expression attenuated anchorage-independent cell growth and cell migration in vitro.

Conclusion: Our data support a tumor suppressor role of FOXC1 in TETs.

Trial registration: ClinicalTrials.gov NCT00965627.

Figures

Figure 1. Copy number aberrations of genes regulating normal thymus development

Genomic CN aberrations of 59 FFPE samples of thymic epithelial tumors were evaluated using array CGH. The frequency of CN loss (red) and gain (blue) of genes implicated in normal thymus development are reported. 35.9% of the tumors presented CN gain of PBX1 locus and 33.9% CN loss of FOXC1.

Figure 2. CN loss of FOXC1 and CN gain of PBX1 in thymic epithelial tumors and prognostic value of immunohistochemistry results

(A) An example of a focal CN loss of chromosome 6p affecting FOXC1 locus. The map of chromosome 6 is reported horizontally, the red highlighted area indicates the region of CN loss. (B) Frequency of FOXC1 CN loss according to WHO histotypes. (C) A representative PBX1 CN gain along with CN gain of approximately the entire long arm of chromosome 1. (D) Frequency of PBX1 CN gain according to WHO histotypes. Examples of (E) FOXC1 positive G3 and (F) negative G0 staining. Kaplan-Maier curves for (G) time to progression and (H) disease related survival of patients with FOXC1 positive and negative tumors.

Figure 3. FOXC1 and TBX1 gene expression in the confirmation cohort of thymic epithelial tumors

(A) FOXC1 relative gene expression (expressed as 2−ΔCt of FOXC1 and the housekeeping gene RPLO) according to FOXC1 CN status in thymic epithelial tumors (thymomas and thymic carcinomas). (B) Relative FOXC1 expression vs FOXC1 CN status in thymomas. (C) Relative TBX1 expression (expressed as 2−Ct of TBX1 and RPLO) vs FOXC1 CN status in thymic epithelial tumors. (D) No correlation between FOXC1 and TBX1 expression. (E) Relative FOXC1 expression vs WHO histotypes. (F) RelativeTBX1 expression vs WHO histotypes.

Figure 4. FOXC1, PBX1 and TBX1 expression in cell lines

(A) Relative gene expression of FOXC1 (expressed as 2−Ct of FOXC1 and the housekeeping gene RPLO) and TBX1 (expressed as 2−Ct of TBX1 and RPLO) in thymic epithelial tumors cell lines. For FOXC1 gene expression, H82 and H69 small cell lung cancer cell lines were used as negative controls and H23, H460 and H1355 non-small cell lung cancer cell lines as positive controls, according to the cancer cell line encyclopedia(26). Results are the average of 4 independent replicate experiments with the relative standard deviation. (B) Western blots evaluating protein expression of FOXC1, PBX1 isoform-α, PBX1 isoform-β and TBX1 compared to α-Tubulin in TET cell lines. HEK-293T cells were used as a positive control for TBX1 expression. (C) Ectopic expression of FOXC1 in U2OS stable clones. (D) Soft agar (top panel) and migration (bottom panel) assays of parental, vector- and FOXC1-transfected U2OS cells. (E) Quantification of colony formation in soft agar assay shown in panel D. * denotes (p-value <0.05). (F) Ectopic expression of FOXC1 in NIH3T3. Note that FOXC1 stable clones stably expressing FOXC1 could not be established from NIH3T3 cells. * Control was NIH-3T3 cells transiently transfected with FOXC1.