Recently,
low-molecular-weight-peptide enrichment from blood samples by on-chip
fractionation with nanopore platforms has been established successfully for the
quantification and phenotypic characterization of the substrate degradome – the
peptide products generated by the protease activity of a tumour environment.
This article will provide evidence for this peptidomics-based approach and the
clinical relevance in future therapeutic benefits will also be discussed.
by
Dr Xu Qian and Dr Tony Y. Hu
Introduction
Considering
the systematic role of proteases in malignant tumour development, it was
thought that it might be possible to detect signature products of substrate
proteolysis – the substrate degradome – in the patient’s blood samples that are
the result of protease dysregulation. This might then function as a diagnostic
marker for tumour progression and a surrogate marker for monitoring the effects
of protease-inhibitor therapy. This approach, called ‘exogenous peptidomics’
[1], based on mass spectrometry (MS) has proven its usefulness in the discovery
of peptides from biofluids.
Challenges
remain in this field as a consequence of the low molecular weight, low
concentrations and quick degradation of such peptides in the peripheral blood
of cancer patients. We recently developed an MS-based on-chip fractionation
method assisted by nanopore technology, which has the advantages of being
simple, high-throughput, high-resolution, and non-invasive [2]. We successfully
identified circulating carboxypeptidase N (CPN)-catalysed C3f-fragments in a
breast cancer mouse model as well as in patients with breast cancer [3] and
matrix metalloproteinases (MMP)-9-catalysed C3f-fragments in an ovarian cancer
mouse model [4]. This review discusses the applications of this new approach
for studying peptide profiling in relation to tumour-resident proteases as
biomarkers and potential therapy target.
Proteases
and the substrate degradome in cancer development
The
developing tumour microenvironment is composed of proliferating tumour cells,
blood vessels, infiltrating inflammatory cells, a variety of associated tissue
cells and tumour stroma, as well as secreted cytokines, chemokines, growth
factors and matrix-degrading proteases. Intracellular and extracellular
proteases that can function as signalling molecules play an indispensable role
in this neoplastic process by enhancing cell proliferation, survival, adhesion,
migration, angiogenesis, senescence, autophagy, apoptosis and evasion of the
immune system in the tumour microenvironment [5–7]. For example, intracellular
granzyme B is a well-known protease facilitating the ability of NK cells and
CD8+ T-cells to kill their targets in the tumour milieu [8]. Elevated levels of
granzyme B were also found in pre-metastatic niches presenting a novel role for
the activation of CD8+ T-cells in constraining myeloid cell activity through
direct killing [9]. Interestingly, recent publications suggested that granzyme
B has a double-edged function. Regulatory T (Treg)-cells derived from the
tumour environment may induce NK and CD8+ T-cell death in a granzyme B- and
perforin-dependent fashion [10, 11] or kill CD4+ effector T-cells via granzyme
B in the presence of IL-2 [12]. These findings indicate that granzyme B is
relevant for Treg-cell-mediated suppression of tumour clearance in vivo.
Extracellular
matrix (ECM) degradation by proteolysis is critical for tumour invasion and
metastasis. Many proteases such as matrix metalloproteinases (MMPs), cathepsin
and the urokinase-type plasminogen activator system play roles in the
degradation of ECM in tumour progression [13, 14]. Extracellular granzyme B
released from migrating cytotoxic lymphocytes was found to participate in the
remodelling of vascular basement membranes (BMs) by cleaving BM constituents
and enabling chemokine-driven movement through BMs in vitro [15]. Recently,
another granzyme family member, granzyme M, was reported to be an inducer of
epithelial-mesenchymal transition (EMT) in cancers associated with STAT3
activation [16]. Cancer cells with EMT features were capable of changing their
shape, polarity and motility in a malignant manner. In the same study,
overexpression of granzyme M in cancer cells was found to promote
chemoresistance. The EMT phenotype of cancer cells was also achieved by
increased MMP-9 production and MMP-9-mediated degradation of E-cadherin,
involving ERK1/2 pathways [17].
In
elucidating the role of proteases in cancer development, it is also important
to gain a better understanding of the substrate degradome, which consists of
the terminal peptide products of the activities of the multistage proteases. We
have, therefore, identified hundreds of substrates of granzyme B, affecting
cell lysis, receptors, cytokines and growth factors, as well as
extracellular-matrix-structural proteins and intracellular proteins involved in
cell signalling and cycle regulation [15, 18]. Besides granzyme B, MMPs cleave
an increasingly large set of substrates, such as elastin, fibronectin, laminin
and collagen IV [14, 19, 20]. However, given the broad range of substrate
function, the mechanism of protease temporal and spatial regulation remains
largely unknown. The exact role of proteases and substrates in cancer biology
both at tissue level and in circulation is still needs to be clearly defined.
A
new tool for the detection of circulating tumour-associated peptides as
biomarkers
The
proteolytic products of a protease are a useful indicator of the protease
concentration in serum. This is necessary, in part, because of the low
concentration of protease itself in serum compared with high detection limits,
the quick degradation of the protease, and the inaccuracy of detection
methodology such as enzyme linked immunosorbent assay (ELISA) as a result of
irreversible non-specificity. New protocols are currently being developed for
the detection and validation of substrate/peptides via peptidomics [21, 22].
Our
group developed a platform mainly including peptide on-chip fractionation
followed by a matrix-assisted laser desorption/ionization time-of-flight
(MALDI-TOF) MS analysis [23]. Briefly, nanoporous silica (NPS) thin films with
nanotextures are used to capture and preserve low-molecular-weight peptides
from 5 μl serum or plasma samples, whereas high-molecular-weight proteins are
excluded by a wash step (Fig. 1). By this on-chip fractionation,
low-molecular-weight peptides are enriched and separated. The peptides appear
as m/z peaks that could be detected by MALDI-TOF MS analysis. The main
advantage of this platform is that it allows specific profiling of the
peptidome with high-throughput, high-resolution and a simple loading step.
Using
this unique platform, we tested one hypothesis that, in breast cancer, CPN
activity and its proteolytic products could be detected in interstitial fluid
and blood [3]. CPN together with its substrate/peptide product levels may vary
during tumour initiation and progression, indicating different disease states.
We confirmed by ex vivo peptide cleavage assay and in vivo validation that the
previously identified substrate C3f_S1304-R1320 was cleaved by CPN specifically
at the C-terminal arginine. Moreover, six fragments generated from
C3f_S1304-R1320 cleavage by CPN increased significantly in mouse sera at 2
weeks after orthotopic implantation relative to normal controls. The most
important finding, however, documented that the plasma levels of
substrate/peptide products of CPN were apparently elevated in patients with
early stage breast cancer relative to controls, but levels of CPN protein
itself were unchanged. These observations indicate that there may be additional
regulation of CPN at different stages of tumour development. It is likely that
inhibition of CPN protease activity is included within this additional regulation.
However, the presence and frequency of substrate/peptides of CPN in the early
stage of breast cancer makes them potential biomarkers for early diagnosis.
Recently,
we investigated MMP-9 activity with the aim of monitoring a novel therapeutic
strategy. To do this we used a HeyA8-MDR-induced ovarian cancer mouse model,
where HeyA8-MDR cells are a human drug-resistant ovarian cancer cell line [4].
This study provided two major observations: (1) C3f was cleaved by MMP-9 in the
tumour microenvironment. Two fragments generated specifically by this
proteolysis were released and were detectable in mouse serum. (2) Treatment
with ephrin type-A receptor 2-siRNA-multistage vectors (MSV-EphA2) induced
apoptosis of tumour cells and a down-regulation of MMP-9 in tumour tissue.
Moreover, the decreased level of circulating C3f cleavage fragments correlated
with MSV-EphA2 treatment. Therefore, this change could be tracked and used to
monitor treatment efficiency in real-time by a simple on-chip blood test. Taken
together, these data suggest that the effect of EphA2 treatment extends to the
peripheral blood, well beyond the tumour microenvironment at the tissue level,
and thus can be easily assessed.
We
reasoned that, from these two experimental approaches, it might be possible to
gain information about the dynamic processes of proteases and their
substrate/peptide products in patients with cancer. Consequently, further
research in this field combined with other investigations aimed at improving
the management of patients with cancer by early diagnosis, accurate
characterization of disease, focused, treatment efficiency, and prognosis is
essential.
Conclusion
In
summary, MS-based on-chip fractionation assisted by nanopore platforms has been
shown to be a highly sensitive and practical tool for the quantification and
characterization of the circulating degradome. The combination of cellular
protease function as well as substrate/peptide analysis provides a biologically
meaningful picture of a specific tumour-entity at the level of the single
peptide. Analysis of the circulating pepidome will be complementary to the
standard diagnostic or prognostic procedure, such as routine blood test and
tissue biopsy, for patients with cancer. This approach also holds great promise
as a tool for monitoring novel therapeutic targets. For further development of
this technique, many predicted targets still await validation as direct
protease substrates and clarification of biological relevance in the network of
protease and its inhibitors. We should pay much attention to the potential
pitfalls.
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The
authors
Xu
Qian MD1,2, Tony Y. Hu PhD*1,3
1Dept
of Nanomedicine, Houston Methodist Research Institute, Houston, TX 77030, USA
2Key
Laboratory of Laboratory Medicine, Ministry of Education, Zhejiang Provincial
Key Laboratory of Medical Genetics, Wenzhou Medical University, Zhejiang, PR
China
3Dept
of Cell and Developmental Biology, Weill Cornell Medical College of Cornell
University, NY 10065, USA
*Corresponding
author
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