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
The development of cancer is a multistep process involving initiation, progression, local-regional recurrence, tumour metastasis and the host anti-tumour response. We are also now aware that changes in the broad genetic and epigenetic landscape as well as molecular mechanisms beyond histology and clinical characteristics contribute to this process. One such mechanism is the relationship between the repertoire of proteases expressed by a tissue and their substrates, which was found to be important in all steps of tumour progression by interactions with tumour cells and the tumour milieu.
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.
References
1. Peccerella T, Lukan N, Hofheinz R, Schadendorf D, Kostrezewa M, Neumaier M, Findeisen P. Endoprotease profiling with double-tagged peptide substrates: a new diagnostic approach in oncology. Clin chem. 2010; 56(2): 272–280.
2. Fan J, Niu S, Dong A, Shi J, Wu HJ, Fine DH, et al. Nanopore film based enrichment and quantification of low abundance hepcidin from human bodily fluids. Nanomedicine. 2014; 10(5): 879–888.
3. Li Y, Li Y, Chen T, Kuklina AS, Bernard P, Esteva FJ, et al. Circulating proteolytic products of carboxypeptidase N for early detection of breast cancer. Clin Chem. 2014; 60(1): 233–242.
4. Deng Z, Li Y, Fan J, Wang G, Li Y, Zhang Y, et al. Circulating peptidome to indicate the tumor-resident proteolysis. Sci Rep. 2015; 5: 9327.
5. D’Eliseo D, Pisu P, Romano C, Tubaro A, De Nunzio C, Morrone S, et al. Granzyme B is expressed in urothelial carcinoma and promotes cancer cell invasion. Int J Cancer 2010; 127(6): 1283–1294.
6. Hu SX, Wang S, Wang JP, Mills GB, Zhou Y, Xu HJ. Expression of endogenous granzyme B in a subset of human primary breast carcinomas. Br J Cancer 2003; 89(1): 135–139.
7. Jezierska A, Motyl T. Matrix metalloproteinase-2 involvement in breast cancer progression: a mini-review. Med Sci Monit. 2009; 15(2): RA32–40.
8. Keefe D, Shi L, Feske S, Massol R, Navarro F, Kirchhausen T, et al. Perforin triggers a plasma membrane-repair response that facilitates CTL induction of apoptosis. Immunity 2005; 23(3): 249–262.
9. Zhang W, Zhang C, Li W, Deng J, Herrmann A, Priceman SJ, et al. CD8+ T-cell immunosurveillance constrains lymphoid premetastatic myeloid cell accumulation. Eur J Immunol. 2015; 45(1): 71–81.
10. Gondek DC, Lu LF, Quezada SA, Sakaguchi S, Noelle RJ. Cutting edge: contact-mediated suppression by CD4+CD25+ regulatory cells involves a granzyme B-dependent, perforin-independent mechanism. J Immunol. 2005; 174(4): 1783–1786.
11. Cao X, Cai SF, Fehniger TA, Song J, Collins LI, et al. Granzyme B and perforin are important for regulatory T cell-mediated suppression of tumor clearance. Immunity 2007; 27(4): 635–646.
12. Strauss L, Bergmann C, Whiteside TL. Human circulating CD4+CD25highFoxp3+ regulatory T cells kill autologous CD8+ but not CD4+ responder cells by Fas-mediated apoptosis. J Immunol. 2009; 182(3): 1469–1480.
13. Parks WC, Wilson CL, Lopez-Boado YS. Matrix metalloproteinases as modulators of inflammation and innate immunity. Nat Rev Immunol. 2004; 4(8): 617–629.
14. Ota I, Li XY, Hu Y, Weiss SJ. Induction of a MT1-MMP and MT2-MMP-dependent basement membrane transmigration program in cancer cells by Snail1. Proc Natl Acad Sci U S A. 2009; 106(48): 20318–2023.
15. Prakash MD, Munoz MA, Jain R, Tong PL, Koskinen A, Regner M, et al. Granzyme B promotes cytotoxic lymphocyte transmigration via basement membrane remodeling. Immunity 2014; 41(6): 960–972.
16. Wang H, Sun Q, Wu Y, Wang L, Zhou C, Ma W, et al. Granzyme M expressed by tumor cells promotes chemoresistance and EMT in vitro and metastasis in vivo associated with STAT3 activation. Oncotarget. 2015; 6(8): 5818–5831.
17. Zuo JH, Zhu W, Li MY, Li XH, Yi H, Zeng GQ, et al. Activation of EGFR promotes squamous carcinoma SCC10A cell migration and invasion via inducing EMT-like phenotype change and MMP-9-mediated degradation of E-cadherin. J Cell Biochem. 2011; 112(9): 2508–2517.
18. Boivin WA, Cooper DM, Hiebert PR, Granville DJ. Intracellular versus extracellular granzyme B in immunity and disease: challenging the dogma. Lab Invest. 2009; 89(11): 1195–1220.
19. Martinez A, Oh HR, Unsworth EJ, Bregonzio C, Saavedra JM, Stetler-Stevenson WG, et al. Matrix metalloproteinase-2 cleavage of adrenomedullin produces a vasoconstrictor out of a vasodilator. Biochem J. 2004; 383(Pt. 3): 413–418.
20. Wang S, Dangerfield JP, Young RE, Nourshargh S. PECAM-1, alpha6 integrins and neutrophil elastase cooperate in mediating neutrophil transmigration. J Cell Sci. 2005; 118(Pt 9): 2067–2076.
21. Kwong GA, von Maltzahn G, Murugappan G, Abudayyeh O, Mo S, Papayannopoulos IA, et al. Mass-encoded synthetic biomarkers for multiplexed urinary monitoring of disease. Nat Biotech. 2013; 31(1): 63–70.
22. Ueda K, Saichi N, Takami S, Kang D, Toyama A, Daigo Y, et al. A comprehensive peptidome profiling technology for the identification of early detection biomarkers for lung adenocarcinoma. PLoS One. 2011; 6(4): e18567.
23. Hu Y, Peng Y, Lin K, Shen H, Brousseau LC, 3rd, Sakamoto J, et al. Surface engineering on mesoporous silica chips for enriching low molecular weight phosphorylated proteins. Nanoscale 2011; 3(2): 421–428.
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
E-mail: yhu@houstonmethodist.org
UriSed mini
, /in Featured Articles /by 3wmediaTime matters, results count
, /in Featured Articles /by 3wmediaTumour markers: why not VOCs?
, /in Featured Articles /by 3wmediaSince early diagnosis of cancer is crucial in reducing mortality, much research is directed towards finding minimally invasive methods of detecting early tumour growth. In 1971 the Nobel Prize winner Linus Pauling froze his exhaled breath to analyse the volatile organic compounds (VOCs) it contained by gas chromatography. Forty-six years later, although many studies have suggested that some of the over two hundred VOCs found in human breath and other body specimens such as urine and stool could be diagnostic for different cancers, routine VOC analysis has yet to reach clinical laboratories.
A fairly recent study involving over 300 subjects demonstrated that appropriately trained dogs could identify cases of colon cancer from breath or stool samples with 95% accuracy, and there is a plethora of anecdotal evidence suggesting that canines can identify breast, ovarian, bladder, skin and lung cancers by smelling appropriate samples. Last month an article in the journal of Urology reported that two female dogs had correctly identified prostate cancer in well over 90% of cases by sniffing urine samples from 900 men, 360 of whom had the disease. While packs of Labradors roaming clinic corridors would be neither practical nor affordable, this is surely evidence that signature VOCs for different tumours do exist, and that their detection would be an excellent non-invasive approach for early cancer diagnosis if suitable methods could be developed.
So what are the problems? Firstly much of the research on cancer in recent years has understandably focused on changes in DNA and proteins in tumour cells. In addition relevant VOCs are found in such low concentrations (parts per billion/trillion) that research laboratories with expensive instruments such as GC-MS, proton transfer reaction-MS and selected ion flow tube-MS have so far been necessary to extract and analyse them. A study published in last month’s Gut reported the analysis of VOC patterns in the exhaled breath of 484 subjects, 99 of whom had gastric cancer and 325 of whom had pre-cancerous conditions, by two methods: GC-MS and via a nanoarray sensor. The results were encouraging, indicating that an eight VOC signature had a specificity of 98%; sensitivity was 73%. And a small sorbent trap for breath collection has now been developed that captures more than two hundred VOCs for subsequent GC-MS analysis to find the elucidated signatures specific for various diseases including breast and lung cancer. Clinical trials are underway; the goal is a point-of-care system for early diagnosis. After nearly half a century there may be light at the end of the tunnel!
The promise and the challenges
, /in Featured Articles /by 3wmediaThere is growing interest in tumour markers as aids for the diagnosis, staging and management of cancer. Some are expected to succeed, after several years of evaluation to trials and eventual clinical use. A large number, however, are not likely to make it beyond development. On their part, physicians need to be aware of both opportunities and limitations in the clinical use of tumour markers.
Tumour markers are substances (antigens, proteins, enzymes or hormones) which indicate the presence of cancer or provide information about its likely course of development. They are present in cancerous tissue as well as in the bodily fluids of cancer patients.
Range of applications
Tumour markers have shown their potential for several applications. These range from the differential diagnosis of benign and malignant conditions to prognostic assessments, postoperative surveillance, the prediction of drug response or resistance, and the monitoring of therapy in
advanced disease.
The key advantage of tumour markers in the above applications is convenience. Inexpensive automated assays allow for fast processing of samples.
The case for tumour markers
The best known tumour markers include Her2/neu for breast cancer, which has an established economic case. Her-2/neu is a target for trastuzumab, whose use as an adjuvant has been shown to decrease cancer recurrence rates by 50%. However, up to one in 20 trastuzumab recipients develop cardiac dysfunction. Given that the cost of one year of therapy is close to 100,000 Euros, the need for accurately and precisely assaying every tissue sample is evidently strong.
Work in progress
Tumour markers are, however, still a work in progress and expected to remain so. In the US, the National Cancer Institute (NCI) states that “more than 20 tumour markers are currently in use.” It however lists over 30. The European Group on TumoUr Markers (EGTM) has a list of 16.
Despite the number of tumour markers in development, only ‘traditional’ markers are used in diagnosis, prognosis and monitoring. For example, at least six urine tumour marker kits are approved by the US Food and Drug Administration for bladder cancer. However, none are backed by data from clinical trials that increased survival time, improved quality of life or decreased cost of treatment.
Appropriate use, caution urged
Many experts urge caution with respect to tumour markers. Inappropriate use, according to an article in the ‘British Medical Journal’, can cause patients unnecessary anxiety and distress, and may also delay correct diagnosis and treatment. The authors cite one hospital audit which found “that only about 10% of requests for tumour markers were appropriate.”
The European Group on Tumor Markers attributes part of this problem to the growing availability of automated immunoassays. This makes tumour marker tests available in routine rather than specialist laboratories. “Results are consequently more readily available to non-specialist clinicians, who may be less familiar with their interpretation.”
Challenges of sensitivity and specificity
Only some markers, known as tumour-specific markers, are produced exclusively by a particular tumour As a result, most tumours cannot be detected by a single test, and tests for multiple markers are often required.
Tests are therefore often accompanied by the risk of both false positives and false negatives. The Cancer Information & Support Network (CISN) sums up the picture: False positives may occur because most tumour markers “can be made by normal cells, as well as cancer cells,” and markers “can be associated with noncancerous conditions.” On the other hand, the reason for false negatives is that “tumour markers are not always present in early stage cancers” and because “people with cancer may never have elevated tumour markers.”
For example, the level of CA-125, a marker for ovarian cancer, is also elevated in a variety of non-malignant disorders such as cirrhosis, pancreatitis, endometriosis, and pelvic inflammatory disease. In addition, medications appear to alter the results of a varied range of tests. So too do pregnancy, menstruation, cigarette smoking and various benign disorders.
Biopsy remains only definitive way for diagnosis
The above lack of sensitivity and specificity has been a major limitation facing the use of tumour markers in clinical practice. As with imaging, the use of tumour markers has been limited to supporting the diagnostic process, and the gold standard for diagnosis still remains a biopsy.
Although difficult to access areas such as the brain are likely to result in more use of tumour markers, a biopsy remains “the only definitive way” for diagnosis of a tumour” even in the brain.
NACB Guidelines
In 2008, the National Academy of Clinical Biochemistry (NACB) in the US released updated Laboratory Medicine Practice Guidelines for the use of tumour markers.
The guidelines made cross-referrals to efforts by numerous professional and regulatory best-practices bodies, including the American Society of Clinical Oncology (ASCO) and the National Comprehensive Cancer Network (NCCN), Britain’s National Institute for Health and Clinical Excellence (NICE), the European Group on Tumor Markers (EGTM), the International Federation of Gynecology and Obstetrics (FIGO) and the Gynecologic Cancer Intergroup (GCIG).
The NACB guidelines cover five cancer sites: testicular, prostate, colorectal, breast, and ovarian.
Testicular cancer
For testicular cancer, α-fetoprotein (AFP), human chorionic gonadotropin and lactate dehydrogenase are recommended for diagnosis, staging, prognosis determination, recurrence detection and the monitoring of therapy. AFP is also recommended for the differential diagnosis of tumours
Prostate cancer
Prostate-specific antigen (PSA) is considered to be potentially useful for detecting prostate cancer recurrence and monitoring therapy. Free PSA is considered useful for distinguishing malignant from benign prostatic disease.
Colorectal cancer
In colorectal cancer, carcinoembryonic antigen is recommended (with some caveats) for prognosis determination, post-operative surveillance, and therapy monitoring in advanced disease. Fecal occult blood testing is considered useful for screening asymptomatic adults who are older than 50 years.
Breast cancer
For breast cancer, estrogen and progesterone receptors predict response to hormone therapy, human epidermal growth factor receptor-2 predicts response to trastuzumab, while urokinase plasminogen activator/ plasminogen activator inhibitor 1 is used for determining prognosis in lymph node-negative patients. CA15-3/BR27–29 or carcinoembryonic antigen can be used for therapy monitoring in advanced disease.
Ovarian cancer
CA125 is recommended (with transvaginal ultrasound) for early detection of ovarian cancer in women at high risk for this disease. CA125 is also recommended for differential diagnosis of suspicious pelvic masses in post-menopausal women, as well as for detection of recurrence, monitoring of therapy, and determination of prognosis in women with ovarian cancer.
Future research to target higher specificity and sensitivity
In the future, research is expected to focus on finding markers which are specific of one pathology, have higher sensitivity with a low cut-off and deliver results which correlate to tumour mass and growth potential. Ideal candidates would also have a short life duration to permit efficient follow-up; in other words, their presence should decrease during treatment and increase before a relapse.
The promise and challenges of screening
Given that tumour markers can aid in assessing the response to cancer treatment and making prognoses, many public health professionals have hoped they might also be used for screening tests which would detect cancer before the presence of symptoms.
Indeed, many tests have both screening and diagnostic uses, with only the context of use determining whether the test is one or the other. “A screening test is done on asymptomatic individuals who receive the test principally because they are of the age or sex at risk for the cancer. A diagnostic test is done on an individual because of clinical suspicion of disease.”
However, no tumour marker identified to date is sufficiently sensitive or specific to be used on its own for screening, demonstrating a survival benefit in randomized controlled trials in the general population.
One of the best known examples is the prostate-specific antigen (PSA) test. Although it is now accepted that most men with elevated PSA levels do not have prostate cancer, the implications of this remain mired in controversy and also illustrate the kind of limitations which other tumour biomarkers may face in the future for use in screening.
PSA screening has been the subject of two large randomized controlled trials in the US and Europe in the 2000s. However, as the Mayo Clinic notes, in spite of the size of the trials, there were “no clear conclusions.” This is because their diversity of methodology allows for significant flexibility in interpretation. As a result, “the decision of whether to screen or not screen – using PSA testing or other means or both – is a decision best made between physicians and their individual patients.”
The two trials were PLCO (Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial) conducted by the National Cancer Institute in the US, and the European Randomized Study of Screening for Prostate Cancer (ERSPC), billed as “the largest randomized trial of screening for prostate cancer” with 162,388 subjects.
In 2011, a US government task-force concluded that healthy men should not be screened for prostate cancer. The finding, which “drastically changed the standard of care for middle-age American men who had grown accustomed to annual screenings,” was largely based on 10 years data from the two studies, which found risk of over-diagnosis and over-treatment.
Problems began when follow-on data from ERSPC two years later showed screening was associated “with a 21% reduction in risk of prostate cancer mortality.” However, this was accompanied by a still-sizeable risk of over-diagnosis and over-treatment. As a result, the authors said that “population-based screening could not yet be recommended.
In April 2015, an article in ‘The Lancet’ re-confirmed “a substantial 21% reduction” from PSA screening. However, due to access restrictions to the ERSPC trial data, the authors called this figure into serious question.
The controversy is unlikely to go away for some time. An Op Ed in The New York Times called the PSA test “hardly more effective than a coin toss.” Although the date of publication was 2010, the author of the commentary was Dr. Richard Ablin, who discovered PSA in 1970.
Such challenges are also likely to accompany screening for other conditions. For instance, data from the PLCO trial show that screening for CA-125 (recommended by the National Academy of Clinical Biochemistry for women with ovarian cancer, along with transvaginal ultrasound), does not reduce ovarian cancer mortality. Instead, false-positive screening test results have been associated with complications.
Circulating peptidomics: a promising approach for the diagnosis and treatment of human cancers
, /in Featured Articles /by 3wmediaRecently, 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
The development of cancer is a multistep process involving initiation, progression, local-regional recurrence, tumour metastasis and the host anti-tumour response. We are also now aware that changes in the broad genetic and epigenetic landscape as well as molecular mechanisms beyond histology and clinical characteristics contribute to this process. One such mechanism is the relationship between the repertoire of proteases expressed by a tissue and their substrates, which was found to be important in all steps of tumour progression by interactions with tumour cells and the tumour milieu.
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
E-mail: yhu@houstonmethodist.org
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