Serum Biomarkers for Prostate Cancer Metastasis Figure 4. The expression of eEF1A1 and eEF1A2 isoforms in human prostate cancer cell lines. (A), Western blotting performed on prostate cancer cell lines using an antibody reactive to both the eEF1A1 and eEF1A2 isoforms. GAPDH was used as a loading control. Twenty-five micrograms of protein were loaded in each lane. Lanes 1–11: 1 = LNCaP; 2 = LNCaP-LN3; 3 = LNCaP-Pro5; 4 = LNCaP-C42; 5 = LNCaP-C4-2B; 6 = DuCaP; 7 = VCaP; 8 = Du145; 9 = PC-3; 10 = PC-3M; 11 = PC-3M-LN4. (B), Reverse-transcription PCR specific for the eEF1A1 and eEF1A2 isoforms, performed using mRNA extracted from prostate cancer cell lines: L = LNCaP; P = PC-3; V = VCaP; D = DuCaP. Control (Co), PCR was performed without mRNA. Relative equal expression of eEF1A1 and eEF1A2 mRNA can be seen in all cell lines tested (i.e. relative intensity ratio of 1.0, using Quantity One). (C), Sections of the DNA sequence chromatograms generated by sequencing the PCR products from the LNCaP cell line, confirming the specificity of the PCR primers used. Nucleotide bases unique to each isoform are marked by arrows. doi:10.1371/journal.pone.0030885.g004 reported in patients with bone metastatic prostate cancer, and have been associated with an adverse outcome for men with castration resistant prostate cancer [32]. Thus, our finding of elevated levels of CRP in metastatic cases is consistent with previous studies. In addition to CRP, many other proteins seen to be differentially expressed between patient groups were found to class to the APR protein family and include fibrinogen, alpha-2- macroglobulin, ceruloplasmin, haptoglobin, alpha-1- acid glyco- protein, alpha-1-antitrypsin and alpha-1-antichymotrypsin (Figure 2). Pathway analysis of the differentially expressed proteins showed that a number of these such as C4, C4a, C5, C5b, C9 and C6 mapped to the classical inflammatory pathway (Figure S1). Although this finding is perhaps not surprising as it is well documented that the presence of a tumour activates an inflammatory response, another possibility is that at least some of these APR proteins could have been secreted ‘ectopically’ by the tumour cells themselves. In support of this possibility, previous studies have reported that renal cell carcinoma, squamous cell carcinoma and breast cancer cell lines may produce and secrete common plasma proteins such as albumin, prealbumin, alpha-1- antitrypsin, ceruloplasmin, alpha-2-macroglobulin, haptoglobin, transferrin and alpha-1-antichymotrypsin [35,36]. Furthermore, the potential of assessing APR proteins as cancer biomarkers has already been reported in a previous study showing that levels of APR proteins in patients with prostate cancer could aid diagnosis and staging, and allowed the correct identification of metastatic disease in 88.6% of patients [37]. Thus, our data support the possibility of assaying a combination of APR proteins secreted by the tumour cells themselves, as well as APR proteins produced by the liver during an immune response. Although, the detection of APR proteins in serum may provide valuable diagnostic/ prognostic information these proteins could also potentially hamper the identification of bona fide cancer specific biomarkers due to their relatively higher abundance in the serum of cancer patients. For instance it has been shown that circulating concentrations of serum amyloid A are transiently increased as much as 1000-fold in response to inflammation [38]. Thus, any future biomarker discovery programmes may additionally benefit from the prior removal of major APR proteins in an effort to improve the detection of relatively lower abundance biomarkers. Another candidate identified as being relatively up-regulated in metastatic cases compared with the other groups was Beta-2- microglobulin (B2M), (Table S2). B2M is a component of the MHC I complex and has been shown to be released by LNCaP prostate cancer cells in culture in response to androgen stimulation [39]. There have been a number of reports implicating B2M as a candidate prostate cancer biomarker. For instance, elevated levels have been detected in prostatic secretions of patients with PLoS ONE | www.plosone.org 7 February 2012 | Volume 7 | Issue 2 | e30885