Christopher Beecher

Metabolomic Profiles Delineate Potential Role for Sarcosine in Prostate Cancer Progression

Multiple, complex molecular events characterize cancer development and progression. Deciphering the molecular networks that distinguish organ-confined disease from metastatic disease may lead to the identification of critical biomarkers for cancer invasion and disease aggressiveness. Although gene and protein expression have been extensively profiled in human tumours, little is known about the global metabolomic alterations that characterize neoplastic progression. Using a combination of high-throughput liquid-and-gas-chromatography-based mass spectrometry, we profiled more than 1,126 metabolites across 262 clinical samples related to prostate cancer (42 tissues and 110 each of urine and plasma). These unbiased metabolomic profiles were able to distinguish benign prostate, clinically localized prostate cancer and metastatic disease. Sarcosine, an N-methyl derivative of the amino acid glycine, was identified as a differential metabolite that was highly increased during prostate cancer progression to metastasis and can be detected non-invasively in urine. Sarcosine levels were also increased in invasive prostate cancer cell lines relative to benign prostate epithelial cells. Knockdown of glycine-N-methyl transferase, the enzyme that generates sarcosine from glycine, attenuated prostate cancer invasion. Addition of exogenous sarcosine or knockdown of the enzyme that leads to sarcosine degradation, sarcosine dehydrogenase, induced an invasive phenotype in benign prostate epithelial cells. Androgen receptor and the ERG gene fusion product coordinately regulate components of the sarcosine pathway. Here, by profiling the metabolomic alterations of prostate cancer progression, we reveal sarcosine as a potentially important metabolic intermediary of cancer cell invasion and aggressivity.

PTH/PTHrP receptor is temporally regulated during osteoblast differentiation and is associated with collagen synthesis

The temporal sequence of PTH/PTHrP receptor mRNA, binding, biologic activity, and its dependence on matrix synthesis was determined using MC3T3‐E1 preosteoblast‐like cells and primary rat calvarial cells in vitro. Osteoblastic cells were induced to differentiate and form mineralized nodules with the addition of ascorbic acid and β‐glycerophosphate, and samples were collected from 0–26 days of culture. DNA levels as determined by fluorometric analysis increased 12‐ and 17‐fold during the collection period for both MC3T3‐E1 and primary calvarial cells respectively. Steady state mRNA levels for the PTH/PTHrP receptor as determined by northern blot analysis, were initially low for both cell types, peaked at day 4 and 5 for MC3T3‐E1 and primary calvarial cells respectively, and declined thereafter. Competition binding curves were performed during differentiation using 125I‐PTHrP. The numbers of receptors per μg DNA were greatest at days 3 and 5 for MC3T3‐E1 and primary calvarial cells respectively. The biologic activity of the receptor was evaluated by stimulating the cells with 10 nM PTHrP and determining cAMP levels via a binding protein assay. The PTHrP‐stimulated cAMP levels increased 5‐fold to peak values at day 5 for MC3T3‐E1 cells and 6‐fold to peak values at day 4 for the primary calvarial cells. Ascorbic acid was required for maximal development of a PTH‐dependent cAMP response since ascorbic acid‐treated MC3T3‐E1 cells had twice the PTH‐stimulated cAMP levels as non‐treated cells. When the collagen synthesis inhibitor 3,4‐dehydroproline was administered to MC3T3‐E1 cultures prior to differentiation, there was a subsequent diminution of the PTH/PTHrP receptor mRNA gene expression and numbers of receptors per cell; however, if administered after the initiation of matrix synthesis there was no reduction in PTH/PTHrP receptor mRNA. These findings indicate that the PTH/PTHrP receptor is associated temporally at the level of mRNA, protein, and biologic activity, with a differentiating, matrix‐producing osteoblastic cell in vitro.

Transforming growth factor-β1 regulates steady-state PTH/PTHrP receptor mRNA levels and PTHrP binding in ROS 17/2.8 osteosarcoma cells

The effect of transforming growth factor beta1 (TGF-beta1) on the expression of mRNA for the parathyroid hormone receptor and binding of iodinated parathyroid hormone-related protein in ROS 17/2.8 osteosarcoma cells was evaluated. TGF-beta1 stimulated a 2-7-fold increase in steady state mRNA levels for the parathyroid hormone receptor at a maximal dose of 5 ng/ml, with increased levels of expression at 6 h of TGF-beta1-incubation, and peak levels at 8-24 h. Receptor binding studies revealed a significant increase in PTHrP-specific binding with TGF-beta1 doses as low as 0.5 ng/ml and a 55% increase in numbers of receptors with no alteration in binding affinity with 5.0 ng/ml TGF-beta1. Time course studies indicated that receptor binding was increased at 24 h with peak levels reached at 48 h of treatment. PTH-stimulated cAMP levels were significantly increased in ROS 17/2.8 cells treated with TGF-beta1 (0.5 ng/ml) for 48 h. These data indicate that TGF-beta1 upregulates steady-state mRNA, ligand binding and PTH/PTHrP receptor signaling in rat osteosarcoma cells. The effects of TGF-beta1 on bone may be attributed in part to regulation of the PTH/PTHrP receptor at the molecular level.

Re: Florian Jentzmik, Carsten Stephan, Kurt Miller, et al. Sarcosine in Urine after Digital Rectal Examination Fails as a Marker in Prostate Cancer Detection and Identification of Aggressive Tumours. Eur Urol 2010;58:12–8

In the paper published by Jentzmik et al. [1], the authors address the importance of urine-derived sarcosine based on public interest in our report [2], which describes elevated levels of the metabolite in urine of biopsy-proven prostate cancer (PCa) patients. We found especially elevated sarcosine levels in tumor specimens from patients with metastatic PCa, compared with organ-confined tumors. In their paper [1], the authors have examined urine supernatants collected after digital rectal examination (DRE) from 139 patients with prostate-specific antigen (PSA) levels between 2 and 20 ng/ml. These patients included 106 patients with PCa and 33 individuals with no evidence of malignancy (NEM), as assessed by biopsy. A total of 99 patients in this cohort had PSA levels between 0 and 10 ng/ml.

Corrigendum: Metabolomic profiles delineate potential role for sarcosine in prostate cancer progression

This corrects the article DOI: 10.1038/nature07762

Erratum: Metabolomic profiles delineate potential role for sarcosine in prostate cancer progression (Nature (2009) 457 (910-914) DOI: 10.1038/nature07762)

This corrects the article DOI: 10.1038/nature07762

Erratum: Corrigendum: Metabolomic profiles delineate potential role for sarcosine in prostate cancer progression

This corrects the article DOI: 10.1038/nature07762

Abstract ED01-03: Metabolomics of disease progression

While the total potential of a biological systems response is defined by their genes, and the organism9s desired response (to its current status) may be embodied in their transcripts, the metabolome embodies what an organism is actually of doing at a biochemical level. For this reason the metabolomic data may be quite diagnostic of a given disease state, that may or may not be dictated by either the genetic or transcriptomic levels. In this presentation we will discuss an application of metabolomics to define a series of biomarkers. The subsequent explorations of the details of this biomarker have revealed mechanisms that are likely to be significant to the progression of cancer. Citation Information: Cancer Prev Res 2010;3(12 Suppl):ED01-03.