Identification of proteomic biomarkers predicting prostate cancer aggressiveness and lethality despite biopsy-sampling error.

BACKGROUND: Key challenges of biopsy-based determination of prostate cancer aggressiveness include tumour heterogeneity, biopsy-sampling error, and variations in biopsy interpretation. The resulting uncertainty in risk assessment leads to significant overtreatment, with associated costs and morbidity. We developed a performance-based strategy to identify protein biomarkers predictive of prostate cancer aggressiveness and lethality regardless of biopsy-sampling variation. METHODS: Prostatectomy samples from a large patient cohort with long follow-up were blindly assessed by expert pathologists who identified the tissue regions with the highest and lowest Gleason grade from each patient. To simulate biopsy-sampling error, a core from a high- and a low-Gleason area from each patient sample was used to generate a 'high' and a 'low' tumour microarray, respectively. RESULTS: Using a quantitative proteomics approach, we identified from 160 candidates 12 biomarkers that predicted prostate cancer aggressiveness (surgical Gleason and TNM stage) and lethal outcome robustly in both high- and low-Gleason areas. Conversely, a previously reported lethal outcome-predictive marker signature for prostatectomy tissue was unable to perform under circumstances of maximal sampling error. CONCLUSIONS: Our results have important implications for cancer biomarker discovery in general and development of a sampling error-resistant clinical biopsy test for prediction of prostate cancer aggressiveness.

Delineation of a region in the B2 bradykinin receptor that is essential for high-affinity agonist binding.

We have made mutations in the predicted sixth transmembrane segment of a rat B2 bradykinin receptor and analyzed the variant proteins by expressing them in COS-1 cells. Two amino acid substitutions reduced the affinity of the receptor for bradykinin (Phe261-->Val by 1600-fold; Thr265-->Ala by 700-fold) with comparatively little effect on the affinity for the bradykinin antagonists NPC17731 and D-Arg-[Hyp3,D-Phe7]bradykinin (where Hyp is hydroxyproline). Three other substitutions (Gln262-->Ala, Asp268-->Ala, and Thr269-->Ala) modestly reduced the affinity for bradykinin and for the antagonist D-Arg-[Hyp3,D-Phe7]bradykinin. Even the most dramatically affected mutated receptors were still able to couple, after bradykinin binding, to phosphatidylinositol turnover. The data suggest that bradykinin directly contacts the face of the sixth transmembrane helix formed by the residues Phe261, Gln262, Thr265, Asp268, and Thr269 or that this face of the helix is the site of intraprotein contacts that serve to stabilize the agonist-binding conformation of the receptor.

Identification of a B2 bradykinin receptor expressed by PC12 pheochromocytoma cells.

We have used rat PC12 pheochromocytoma cells, a clonal cell line closely related to sympathetic neurons, to investigate reports that the bradykinin receptor expressed in the peripheral nervous system is distinct from the well-characterized B2 bradykinin receptor of smooth muscle. Although there have been reports that [Thi5,8,D-Phe7]bradykinin [where Thi is beta-(2-thienyl)alanine] is a full agonist at some sites in the peripheral nervous system, we find that in PC12 cells [Thi5,8,D-Phe7]bradykinin behaves as a competitive antagonist of bradykinin-stimulated phosphatidylinositol turnover. In particular, sufficient concentrations of [Thi5,8,D-Phe7]bradykinin completely block the increase in inositol bisphosphate and trisphosphate in response to 100 nM bradykinin; [Thi5,8,D-Phe7]bradykinin alone, at up to 10 microM, does not appreciably increase inositol bisphosphate and trisphosphate. In contrast to the absence of evidence for a distinctive neuronal receptor, we have found convincing evidence that the bradykinin receptor previously identified in smooth muscle is present in PC12 cells. Using the polymerase chain reaction, we have isolated a full-length cDNA encoding a bradykinin receptor that is expressed in PC12 cells and verified that its nucleotide sequence is identical except at a single position to that of the rat uterine B2 bradykinin receptor. When expressed in COS cells this uterine bradykinin receptor exhibits the same high affinity for [3H]bradykinin (Kd 4.4 nM), the same relative affinities for a series of kinin antagonists, and the same efficient coupling to phosphatidylinositol turnover (EC50 2.5 nM) as the receptor in PC12 cells. We interpret our data, and the findings of a number of pharmacological studies, as strengthening the view that the B2 receptor expressed in PC12 cells and in certain cells of the peripheral nervous system is identical to the receptor in rat uterine smooth muscle.