Download Prostate Cancer New Marker- Sarcosine Found in Urine and Tumor

The Prostate-cancer Metabolome
Prostate Cancer New MarkerSarcosine Found in Urine and
Tumor Tissue
Lei Wang
2/27/09
Introduction
cancer — the most frequently
diagnosed cancer in men — is a main
cause of morbidity and mortality
 Current clinical effective diagnoses:
 Prostate
Digital rectal examination
Measure the levels of the enzyme PSA in blood
serum
 Difficulty:
High variable examination in PSA among patients;
Hard to identify those at high risk of their disease
progressing to advanced stages
Nature: 12 February 2009

Sreekumar et al. report applying metabolomics
to discover biomarker Sarcosine in urine and
tissue (P&PCA) that concentration of a small
molecule could reveal how advanced a patient's
prostate cancer is.
 And could potentially be used for non-invasive
diagnosis and prognostic evaluation of prostate
cancer.

So, what is metabolomics?
Metabolomics

In the postgenomic era, cancer researchers
survey the genome, transcriptome, proteome, to
figure out molecular signatures that distinguish
tumors from normal tissues
 The metabolome is the latest ‘ome’. Analyses of
the various ‘omes’ are complementary, but
determining the metabolite content of cancer
cells — cancer metabolomics — is particularly
attractive because it can provide an accurate
read-out of tumors’ cellular physiology and
biochemical activity.
Sreekumar’s experiments

Method: both liquid and gas chromatography
coupled with mass spectrometry to interrogate the
relative levels of metabolites across 262 prostaterelated biospecimens
 Results: evaluation of the unbiased metabolomic
profiles of plasma or urine did not identify robust
differences between biopsy-positive and -negative
individuals


For plasma, 20 out of 478 (4%) metabolites were differential ( Wilcoxon
P<0.05), with a false discovery rate (FDR) of 99%
For urine, 36 out of 583 (6%) metabolites were differential ( Wilcoxon
P,0.05), with a FDR of 67%.
Tissue metabolomic profiles: method
This includes tissue procurement,
histopathological examination, metabolite
extraction and separation, mass spectrometrybased detection, spectral analysis, data
normalization, delineation of class-specific
metabolites and altered pathways, validation of
class-specific metabolites and their functional
characterization.
result
Metabolomic alterations of
prostate cancer progression.
a, Heat map showing 87 differential
metabolites in PCA relative to
benign samples ( Wilcoxon P<0.05,
23% FDR).
b, the relative levels of the 37
named metabolites that were
differential between benign
prostate and PCA samples.
Benign-based z-score plot of
named metabolites from a.
c, Displays the levels of the 91
named metabolites altered in
metastatic samples. Met samples
compared to the localized tumors is
4% FDR. As in b except for the
comparison between Mets (red)
and PCA (yellow), with data
represented relative to the mean of
the PCA samples.

They identify 87 metabolites that distinguish PCA from
benign prostate tissue. Of these, six metabolites
including sarcosine, uracil, kynurenine, glycerol-3phosphate, leucine and proline were significantly
increased on disease progression from benign to PCA to
metastatic PCA.

Notably, metastatic samples showed markedly increased
levels of sarcosine in 79% of the specimens analysed,
whereas 42% of the PCA samples showed an increase
in the levels of this metabolite and none of the benign
samples had detectable levels of sarcosine.

The authors further pursued one of these metabolites as
a possible biomarker for cancer progression:
sarcosine — a derivative of the amino acid glycine .
 To
confirm this pattern of sarcosine
increase in cancer progression, they
developed a highly sensitive and specific
isotope dilution gas chromatography–
mass spectrometry (GC–MS) method for
accurately quantifying the metabolite from
biospecimens
Sarcosine levels in prostate-cancer-related
tissue specimens (n=89)
 Next,
they monitored sarcosine levels in
urine specimens from biopsy-positive and
–negative individuals, most of whom have
increased levels of prostate-specific
antigen (PSA) (>4.0 ng ml-1) and in which
prostate needle biopsy was used for
diagnosis.
Sarcosine levels in urine sediments from men with
biopsy-proven prostate cancer (n=49) and prostatebiopsy negative controls (n=44).
Notably, an area under
the curve (AUC) of 1.0
indicates perfect
prediction and an AUC
of 0.5 indicates
prediction equivalent
to random selection.
The AUC for sarcosine and PSA for 53 restricted patients samples
used in this study having PSA levels in clinical grey zone of 2-10
ng/ml are 0.69 (95 % CI: 0.55, 0.84) and 0.53 (95 % CI: 0.37, 0.69)
respectively.
 To
determine whether the sarcosine
increase in PCA has biological relevance,
they measured its levels in:
PCA cell lines: VCaP, DU145, 22RV1 and LNCaP
Primary benign prostate epithelial cells: PrEC
Immortalized benign RWPE prostate cells
Sarcosine levels in invasive prostate cancer cells compared to
non-invasive benign prostate epithelial cell lines show
increasing

To determine whether sarcosine has a more direct role in this
process, they added the metabolite to non-invasive benign prostate
epithelial cells. Alanine, an isomer of sarcosine, was used as a
control for these experiments.
Assessment of cell
invasiveness of prostate
epithelial cells upon
exogenous administration of
alanine, glycine or sarcosine

Sarcosine treatment, however, did not affect the ability of these cells
to progress through the different stages of cell cycle or impair cell
proliferation
 Why
Glycine can induce cell invasiveness
of prostate epithelial cells?
This invasion could result from the conversion of
glycine to sarcosine by the enzyme glycine-N-methyl
transferase (GNMT)
In addition to GNMT, sarcosine levels are regulated by sarcosine
dehydrogenase (SARDH), this enzyme converts sarcosine back to
glycine, and dimethylglycine dehydrogenase (DMGDH), which
generates sarcosine from dimethylglycine
Knockdown of
SARDH in benign
prostate epithelial
cells (RWPE)
resulted in increase in
endogenous
sarcosine levels with
a concomitant 3.5fold increase in
invasion
sarcosine levels and
cell invasiveness after
knockdown of GNMT in
DU145.
Knock-down of dimethylglycine
dehydrogenase (DMGDH) attentuates
invasion in DU145 prostate cancer cells
Other reverse supporting rationale
 Mice
lacking GNMT develop liver cancer
with age (Hepatology 2008).
 Moreover, in a significant proportion of
human prostate cancers, GNMT
undergoes a phenomenon called loss of
heterozygosity — in which one copy of the
gene is lost — and the expression of this
gene was documented to decrease with
prostate-cancer progression (cancer res
2007).
Androgen signalling and ETS family of genes (ERG, ETV1)
fusions are key factors for PCA progression (Science 2005),
they investigated their role in regulating GNMT and SARDH.

Treatment with androgen for 48 h in VCaP (ERG-positive) and
LNCaP (ETV1-positive) prostate cancer cells resulted in a stepwise
increase in GNMT expression and a concomitant decrease in
SARDH levels, (qPCR)
qRT–PCR analysis of GNMT and
SARDH mRNA expression in
androgen-stimulated VCaP cells
Left, overexpression of ERG or ETV1 in RWPE cells increase
sarcosine levels and cell invasiveness.
Right, knockdown of TMPRSS2–ERG in VCaP cells decrease
sarcosine levels and cell invasiveness.
summary




Taken together, they explored the metabolome of
prostate cancer progression.
Identified sarcosine as a key metabolite increased in
metastatic PCA and detectable in the urine.
Sarcosine and its proximal regulatory enzymes seem to
modulate cell invasion and migration. The master
transcriptional regulators, AR and ETS gene fusions,
seem to regulate directly sarcosine levels by control of
its regulatory enzymes, GNMT etc..
Sarcosine pathway may have potential as biomarkers of
PCA progression and serve as new avenues for
therapeutic intervention.
weakness




This study just focus on sarcocine level in plasm, urine
and prostate (tumor) tissue sample in patients, and ignore
patients’ syndrome investigation.
At least, should check if cancers associated with GNMT
enriched gluconeogenic tissues such as from lung,
pancreas and liver. Also in diabetic animal such as sheep
and rat are showing increase in GNMT activity and
sarcosine change.
Animal model experiment or clinical investigation (sample
AR level) will enhance the cell culture results such as
sarcocine pathway, AR-ERG pathway.
AR-ERG pathway in cell culture experiments is not a so
strong evidence to modulate sarcocine because most of
metastasis PCA that are high sarcocine level, are
androgen independent, or just has low level AR.
Hypothesis
 If
there is sarcosine level change in tumor
tissue or serum in PCA animal model:
TRAMP, xenograft model (LNCaP,DU-145)
 If chemoprevention compounds can
modulate metabolism of sarcocine in
tumor cell or tissue or body system.
 Use animal graft model to ascertain which
tissue or organ or tumor can produce
sarcosine metabolite.