Increased cathepsin D protein expression is a biomarker for osteosarcomas, pulmonary metastases and other bone malignancies

Abstract

Cancer proteomics provide a powerful approach to identify biomarkers for personalized medicine. Particularly, biomarkers for early detection, prognosis and therapeutic intervention of bone cancers, especially osteosarcomas, are missing. Initially, we compared two-dimensional gel electrophoresis (2-DE)-based protein expression pattern between cell lines of fetal osteoblasts, osteosarcoma and pulmonary metastasis derived from osteosarcoma. Two independent statistical analyses by means of PDQuest® and SameSpot® software revealed a common set of 34 differentially expressed protein spots (p < 0.05). 17 Proteins were identified by mass spectrometry and subjected to Ingenuity Pathway Analysis resulting in one high-ranked network associated with Gene Expression, Cell Death and Cell-To-Cell Signaling and Interaction. Ran/TC4-binding protein (RANBP1) and Cathepsin D (CTSD) were further validated by Western Blot in cell lines while the latter one showed higher expression differences also in cytospins and in clinical samples using tissue microarrays comprising osteosarcomas, metastases, other bone malignancies, and control tissues. The results show that protein expression patterns distinguish fetal osteoblasts from osteosarcomas, pulmonary metastases, and other bone diseases with relevant sensitivities between 55.56% and 100% at ≥87.50% specificity. Particularly, CTSD was validated in clinical material and could thus serve as a new biomarker for bone malignancies and potentially guide individualized treatment regimes.

Keywords: CTSD; bone malignancies; mass spectrometry; osteosarcoma; two-dimensional gel electrophoresis.

Figure 1. Workflow of the study design

* target reached significance in individual validation steps.

Figure 2. PCA map of the protein expression data

Included are 34 significant (a) and 17 identified (b) proteins that were able to group fetal osteoblasts (black), osteosarcomas (red) and pulmonary metastases (green). X and y-axis show first and second principal components. Cell lines were run in triplicates A., B., C.

Figure 3. Representative images and data of the CTSD validation on cytospins of all cell lines (A) and tissue microarray validations (B)

CTSD showed an increased staining from fetal osteoblasts, osteosarcomas to pulmonary metastasis (A, left) that reached significance between three groups (A, right). Customized (B, left) and commercially (B, right) tissue microarray-based evaluation of CTSD showed a strong overexpression in osteosarcomas, pulmonary metastasis and distinct bone diseases (except Ewing's sarcoma) compared to normal bone tissue. Based on the lack of representative fetal osteoblasts in the normal bone tissue, we chose isolated osteocytes – known to be descended from matured osteoblasts – as reference control (***: 0.0001 < p < 0.001; **: 0.001 < p < 0.01; * 0.01 < p < 0.05).