Expression profiling of angiogenic genes for the characterisation of colorectal carcinoma

Abstract

The development of new blood and lymphatic vessels is a crucial event for cancer growth, metastatic spread and relapse after therapy. In this work, the expression levels of chemokines, angiogenic and angiostatic factors and their receptors were determined in paired mucosal and tumour samples of patients with colorectal carcinoma and correlated with clinical and histological parameters by advanced multivariate analyses. The most important predictors to discriminate between tumour and paired normal mucosa turned out to be the levels of expression of plexin-A1 and stromal cell-derived factor 1 (SDF-1), the former overexpressed and the latter downregulated in tumours. The levels of osteopontin and Tie-2 transcripts discriminated between the presence and absence of lymph node infiltration, the former overexpressed in the presence of infiltration whilst the latter providing a protective role. These results add support to the notion that the expression levels of selected genes involved in new blood and lymphatic vessel formation represent trustable biomarkers of tumour development and invasion and contribute to the identification of novel molecular classifiers for 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.