Expression profiling of angiogenic genes for the characterisation of colorectal carcinoma
Introduction
Colorectal cancer is one of the leading cancer-related causes of death, responsible for approximately 400,000 deaths each year worldwide.1 Surgery remains the only curative treatment to date and, despite adequate intervention, disease recurs in nearly half of the patients within 5 years, mainly because of undetected metastatic spread.1, 2 Thus, a better understanding of the molecular mechanisms underlying tumour development and metastasis is essential for both diagnostic and prognostic purposes.
Angiogenesis is a crucial event in tumour progression and dissemination.3 When solid tumours grow, their core becomes hypoxic and activates a transcriptional cascade leading to new blood vessel formation,4 which is associated with poor prognosis and relapse of the disease.3 Amongst the genes responsible of tumour-associated angiogenesis, the members of the vascular endothelial growth factor (VEGF) family (VEGF-A, B, C, D, E and PlGF – placental growth factor) play a key role.5 Whereas it is generally agreed that vascular endothelial growth factor receptor 2 (VEGFR-2)/KDR/Flk-1 is the major receptor mediating the angiogenic effects of VEGF-A,5 the role of VEGFR-1/Flt-1 (a receptor shared by VEGF-A, VEGF-B and PlGF) is less understood and variably proposed as an inhibitor or as an inducer of angiogenesis; recently, a specific role of Flt-1 in mediating vascular permeability, as well as mononuclear and tumour cell recruitment has been suggested.5, 6 VEGF-C and VEGF-D are key regulators of lymphatic sprouting, mainly through their interaction with VEGFR-3.7 It has become increasingly apparent that VEGF receptors act in concert with other molecules, belonging to the integrin, cadherin, neuropilin and Ephrin receptor families.8, 9 Consistently, different ephrins and semaphorins (neuropilin ligands), originally identified as axonal guidance cues, have recently been shown to affect vascular development.10
In addition to VEGF, other growth factors participate to the angiogenic process, including members of the fibroblast growth factor (FGF) family11 and angiopoietins, which control vessel stability and maturation by interacting with their Tie receptors.12, 13, 14, 15
Chemokines also regulate both tumour angiogenesis and lymphangiogenesis, either by directly stimulating endothelial cell proliferation, as is the case of interleukin-8 (IL-8),16 or by mediating mononuclear cell recruitment (e.g. monocyte chemotactic protein-1 (MCP-1)17) and retention (e.g. stromal cell-derived factor 1 (SDF-1)8). Finally, growing vessels are invariably accompanied by extracellular matrix remodelling by matricellular proteins, such as thrombospondin-1 and osteopontin.19, 20, 21
As all these molecules regulate angiogenesis in experimental animal tumours, their use as clinical biomarkers of cancer stage and disease progression is particularly attractive. However, a reliable assay to clearly define their contribution to tumour progression in humans has never been established.22 Several of the above-mentioned factors have been studied by immunohistochemistry, which is notoriously poorly quantitative and only detects a few markers at a time.23, 24 On the other hand, microarrays provide genome-wide signatures expression profiles in cancer,24 but the ensuing information is often redundant, poorly reproducible and difficult to translate into useful clinical practise.25, 26
Here we wanted to assess whether colorectal carcinoma might be characterised and classified according to the expression levels of a series of selected, candidate genes, previously associated to tumour angiogenesis and lymphangiogenesis.
Section snippets
Patients selection and sample collection
Consecutive patients scheduled to undergo surgical resection of tumour mass at the Department of Surgery of the Azienda Ospedaliero-Universitaria ‘Ospedali Riuniti di Trieste’ of Trieste, Italy were recruited for this study, with no specific criteria for exclusion. All patients were enroled according to protocols approved by the Ethical Committee of the Azienda Ospedaliero-Universitaria ‘Ospedali Riuniti di Trieste’, after written informed consent was obtained. No patients enroled in the study
Experimental strategy
Seventy-eight patients with colorectal carcinoma, scheduled to undergo surgical resection, were enroled in this study. The major clinical features of the patients are shown in Table 1, along with the histological characterisation of their tumours. During surgery, different samples were harvested from the tumour, together with a biopsy of matched, apparently healthy mucosa at a distance of at least 15 cm from the tumour border. From these samples, we assessed, by real-time quantitative polymerase
Discussion
Our analysis entailed the assessment of expression levels of 27 genes in paired mucosa and colorectal carcinoma samples from 78 patients. From the methodological point of view, the introduction of an exogenous RNA normaliser allowed us to generate more robust results compared to the use of both GAPDH and risomboal 18S RNA (r18S) housekeeping genes. Another critical methodological issue addressed in our study was the analysis of homogeneity of gene expression within the same tumour mass. By
Conflict of interest statement
None declared.
Acknowledgments
This work was supported by a grant from ‘Fondazione CR Trieste’, Trieste, Italy. The authors are grateful to Suzanne Kerbavcic for precious editorial assistance.
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