Cell culture,
proteomics,
bioinformatics,
clinical biochemistry,
ELISA,
Mass Spectrometry
biomarker discovery,
antibody production
Early detection of
prostate cancer is problematic due to the lack of a marker that has high diagnostic
sensitivity and specificity. The
prostate specific antigen test, in combination with
digital rectal examination, is the
gold standard for
prostate cancer diagnosis. However, this modality
suffers from low specificity. Therefore, specific markers for clinically relevant
prostate cancer are needed. Our objective was to proteomically characterize the conditioned media from human
prostate cancer cell lines to identify
secreted proteins that could serve as novel
prostate cancer biomarkers.
An initial proof of principle study of the
PC3 prostate cancer cell line was conducted. From this study over 200 proteins were identified in the conditioned media. Through
gene ontology analysis and literature searches Mac-2
binding protein was
selected as a candidate biomarker for validation in the serum of
prostate cancer patients. A preliminarily validation showed that Mac-2
binding protein has discriminatory ability in
prostate cancer diagnosis. However, an extended validation did not confirm this.
Based on our proof of principle study we optimized our workflow and extended our analysis by culturing three different
prostate cell lines [
PC3 (
bone metastasis),
LNCaP (
lymph node metastasis), and 22Rv1 (localized to
prostate)]. We conducted a bottom-up analysis of each
cell line by 2-dimensional
liquid chromatography and tandem
mass spectrometry. Of the 2124 proteins identified, 12% (329) were classified as
extracellular and 18% (504) as
membrane-bound among which were known
prostate cancer biomarkers such as PSA and
KLK2. To select the most promising candidates for further investigation, tissue specificity, biological function, disease association based on literature searches, and comparison of protein overlap with the
proteome of seminal plasma and serum were examined. Based on these results, several candidates were
selected for validation in serum of patients with and without
prostate cancer. Of these four novel candidates:
follistatin,
chemokine (C-X-C motif) ligand 16,
pentraxin 3 and spondin 2 showed discriminatory ability.
Of the four candidates,
follistatin was further studied in an extended validation in serum of patients with
biopsy confirmed
prostate cancer and tissues of
prostate cancer patients of low and high grade
tumours by
immunohistochemistry. In addition,
follistatin was also investigated in the tissue of colon and
lung cancer where intense
staining was observed in one specimen of
lung squamous carcinoma.
