Hanrahan V, Currie M, Gunningham S, Morrin H, Scott P, Robinson B, Fox SThe angiogenic switch for vascular endothelial growth factor (VEGF)-A, VEGF-B, VEGF-C, and VEGF-D in the adenoma-carcinoma sequence during colorectal cancer progression. J Pathol 200: 183-194
Angiogenesis is essential for tumour growth and metastasis. It is controlled by angiogenic factors, one of the most important being vascular endothelial growth factor (VEGF)-A. Although its role has been demonstrated in many tumour types including colorectal carcinoma (CRC), the importance of the newer family members in adenoma, invasive tumour growth, and progression to a metastatic phenotype has been poorly characterized in CRC. The aim of this study was to determine the role and timing of the VEGF angiogenic switch during CRC progression. We measured the gene expression of VEGF ligands (VEGF-A, VEGF-B, VEGF-C, and VEGF-D) and their receptors (VEGFR-1, VEGFR-2, and VEGFR-3), in normal colorectal tissues (n = 20), adenomas (n = 10), and in CRC (n = 71) representing different Duke's stages using ribonuclease protection assay, semi-quantitative relative reverse transcriptase polymerase chain reaction, together with the pattern of their expression by immunohistochemistry. VEGF-A mRNA was the most abundant in colorectal tissue, followed by VEGF-B, VEGF-C, and VEGF-D. VEGF-A and VEGF-B mRNAs were significantly more abundant in adenomas (p = 0.0003 and p = 0.04 respectively) compared with normal tissues, while VEGF-A and VEGF-C were significantly increased in carcinomas compared with normal tissues (p = 0.0006 and p = 0.0009 respectively). A significantly greater amount of VEGF-C mRNA was present in carcinomas compared with adenomas (p = 0.03), whereas there was a significant reduction of VEGF-B in carcinomas compared with adenomas (p = 0.0002). VEGF-D mRNA was significantly more abundant in normal tissues than in adenomas (p = 0.0001) and carcinomas (p < 0.0001). In normal tissues distant from the primary tumour, there was a significantly greater amount of VEGF-A and VEGF-D mRNA in patients with Duke's B and Duke's C respectively, compared with Duke's A stage tumours (p = 0.04 and p = 0.01 respectively). Immunohistochemistry showed low basal levels of all ligands in histologically normal tissues and their expression in the epithelium of tumours reflected the levels of mRNA expression identified. VEGF-A and VEGF-C mRNA levels correlated significantly with tumour grade (p = 0.01 and p = 0.01 respectively) and tumour size (p = 0.001 and p = 0.01 respectively), but not with patient age, sex, presence of infiltrative margin, lymphocytic response, vascular invasion, Duke's stage, or lymph node involvement (p > 0.05). VEGF-B mRNA correlated with an infiltrative margin (p = 0.04) but no other clinicopathological variable, and expression of VEGF-D demonstrated no association with any parameter examined. VEGFR-1 was significantly correlated with tumour grade (p = 0.02), Duke's stage (p < 0.001), and lymph node involvement (p = 0.004), VEGFR-2 with lymph node involvement (p = 0.02), and VEGFR-3 did not correlate with any of the clinicopathological variables tested. These results suggest that VEGF-A and VEGF-B play a role early in tumour development at the stage of adenoma formation and that VEGF-C plays a role in advanced disease when there is more likelihood of metastatic spread. The finding of increased levels of VEGF-A and VEGF-D expression in normal tissues collected from a site distant from the primary tumour indicates changes in the surrounding tumour environment that may enhance the subsequent spread of tumour cells.
"They are also located in close proximity region in the VEGF gene, in which a high degree of linkage disequilibrium exists and haplotype effects might therefore be a possibility (Renner et al., 2000; Krippl et al., 2003; Hofmann et al., 2008; Dassoulas et al., 2009; Hansen et al., 2010a; Antonacopoulou et al., 2012). Although it has been indicated that VEGF could be a good marker for determining the risk of developing CRC and could be used as a therapeutic target for new molecular anticancer drugs (Hanrahan et al., 2003; Fernando and Hurwitz, 2004; Dassoulas et al., 2009; Vidaurreta et al., 2010), the data remain conflicting if VEGF gene polymorphisms are associated with the prognosis of CRC (Yamamori et al., 2004; Park et al., 2007; Bae et al., 2008; Maltese et al., 2009; Wu et al., 2009; Hansen et al., 2010b; Antonacopoulou et al., 2012; Jang et al., 2013b). Therefore, to test this hypothesis, we investigated possible associations between genetic variations at the -2578A > C, + 936C > T, and -460C > T polymorphic site in the VEGF gene, in patients who have had CRC compared to healthy individuals. "
[Show abstract][Hide abstract] ABSTRACT: Aims:
Colorectal cancer (CRC) is the third most common cancer in the world and its etiology involves the interaction of genetic and environmental factors. New blood vessels form through a process called angiogenesis and have an essential role in tumor growth, progression, and metastasis of malignant tumors. The vascular endothelial growth factor (VEGF), one of the most important angiogenic factors, is a specific mitogen for vascular endothelial cells. In the present case-control study, we carried out the study to evaluate whether the VEGF single-nucleotide polymorphisms play a role in modulating susceptibility to CRC.
We evaluated the VEGF -2578A>C, +936C>T, and -460C>T genotypes obtained from 103 patients with CRC and 129 healthy controls by using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. Also, haplotype analysis was determined. Odds ratios (ORs) and 95% confidence intervals (CI) were estimated.
-2578A>C was significantly associated with CRC risk (OR 1.81; 95% CI 0.94-3.47; p=0.0495), while distribution of +936C>T and -460C>T genotypes in cases and controls did not significantly differ. The VEGF A2578-T936-T460 haplotype might be associated with the development of CRC (OR 8.77; 95% CI 1.05-73.36; p=0.0434). There was significant haplotype effect for all eight haplotypes (p=0.02).
These results suggest that the VEGF polymorphisms might play a role in the development of CRC. Therefore, the VEGF polymorphisms might be further investigated to use in the determination of risk factors for CRC and to have a predictive value for anti-VEGF-targeted cancer therapies.
"There are also conflicting reports between VEGF expression and tumor differentiation. High levels of VEGF expressions were significantly associated with poorly differentiated tumors in colorectal carcinomas [24,25] and soft tissue sarcomas . In this study, however, VEGF expression was elevated in well differentiated tumors in comparison with poorly differentiated ones by immunohistochemistry (P = 0.04) and protein array (P = 0.04). "
[Show abstract][Hide abstract] ABSTRACT: Background
Vascular endothelial growth factor (VEGF) is a critical pro-angiogenic factor, found in a number of cancers, and a target of therapy. It is typically assessed by immunohistochemistry (IHC) in clinical research. However, IHC is not a quantitative assay and is rarely reproducible. We compared VEGF levels in colon cancer by IHC and a quantitative immunoassay on proteins isolated from formalin fixed, paraffin embedded tissues.
VEGF expression was studied by means of a well-based reverse phase protein array (RPPA) and immunohistochemistry in 69 colon cancer cases, and compared with various clinicopathologic factors. Protein lysates derived from formalin fixed, paraffin embedded tissue contained measurable immunoreactive VEGF molecules. The VEGF expression level of well differentiated colon cancer was significantly higher than those with moderately and poorly differentiated carcinomas by immunohistochemistry (P = 0.04) and well-based RPPA (P = 0.04). VEGF quantification by well-based RPPA also demonstrated an association with nodal metastasis status (P = 0.05). In addition, the normalized VEGF value by well-based RPPA correlated (r = 0.283, P = 0.018). Furthermore, subgroup analysis by histologic type revealed that adenocarcinoma cases showed significant correlation (r = 0.315, P = 0.031) between well-based RPPA and IHC.
The well-based RPPA method is a high throughput and sensitive approach, is an excellent tool for quantification of marker proteins. Notably, this method may be helpful for more objective evaluation of protein expression in cancer patients.
"During tumorigenesis, the balance of proangiogenic factors, growth factors, and cytokines that regulate angiogenesis is disrupted and the “angiogenic switch” is increasingly recognized as a rate-limiting secondary event in multistage carcinogenesis [2–5]. Vascular endothelial growth factor-A (VEGF-A) is a key growth factor for endothelial cells in patients with CRC [6,7]. The addition of the monoclonal antibody, bevacizumab, has improved the overall survival of patients with mCRC [8,9]. "
[Show abstract][Hide abstract] ABSTRACT: Circulating angiogenic factors are altered in patients with mCRC receiving bevacizumab. Evaluation of alterations in levels of VEGF ligands may provide insights into possible resistance mechanisms.
PlGF, VEGF-A, VEGF-C, and VEGF-D were measured from two cohorts of patients. Sequential plasma samples were obtained from a discovery cohort of 42 patients treated with chemotherapy and bevacizumab. A validation cohort included plasma samples from a cross-sectional of 403 patients prior to chemotherapy, or after progression on a regimen with or without bevacizumab.
In the discovery cohort, VEGF-C was increased prior to progression and at progression (+49% and +95%, respectively, p<0.01), consistent with previously reported elevations in PlGF. Levels of VEGF-D were increased (+23%) at progression (p=0.05). In the validation cohort, samples obtained from patients after progression on a regimen with bevacizumab had higher levels of PlGF and VEGF-D (+43% and +6%, p=0.02, p=0.01, respectively) compared to untreated patients, but failed to validate the increase in VEGF-C seen in the first cohort. Patients who progressed on chemotherapy with bevacizumab had significantly elevated levels of PlGF (+88%) but not VEGF-C and VEGF-D compared to patients treated with chemotherapy alone. Elevations of PlGF and VEGF-D appeared transient and returned to baseline with a half-life of 6 weeks.
Increases in PlGF and VEGF-D were observed after progression on chemotherapy with bevacizumab. These changes appear to be reversible after discontinuing therapy. These ligands are associated with resistance to bevacizumab-containing chemotherapy in mCRC, but causation remains to be established.
PLoS ONE 10/2013; 8(10):e77117. DOI:10.1371/journal.pone.0077117 · 3.23 Impact Factor
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