Molecular Mechanisms of Resistance to Anti-VEGF Therapies: Il-8 and its Receptors
Authors: Heinz-Josef Lenz
Journal: Clinical Cancer Research (2007)

PL07-01  Tumor angiogenesis plays an important role in tumor development, tumor progression and metastases. The switch to the angiogenic phenotype involves a change in the local equilibrium between positive and negative regulators of the growth of microvessels. This “angiogenic switch” is considered a hallmark of the malignant process and is required for tumor propagation and progression. Vascular endothelial growth factor (VEGF) and its receptors VEGFR-1 and VEFGR-2 are critical activators of tumor associated angiogenesis. Recent research identified interleukin-8 (IL-8) and adrenomedullin (AM) as critical mediators of VEGF independent tumor angiogenesis. Induction of IL-8 preserved the angiogenic response in HIF1-α deficient colon cancer cells, suggesting that IL-8 mediates angiogenesis, independently of VEGF. IL-8 exerts its potent angiogenic properties on endothelial cells through interaction with its receptors CXCR1 and CXCR2. Induction of IL-8 preserved the angiogenic phenotype in HIF1-α deficient colon cancer cells, suggesting a critical role of IL-8 in tumor associated angiogenesis, independently of VEGF. Overexpression of IL-8 has been found to be associated with VEGF independent angiogenesis, advanced disease stage, lymphovascular invasion, poor prognosis and tumor recurrence in several different malignancies, including non-small cell lung cancer and rectal cancer. Additionally, colorectal cancer patients with lung- and liver metastases, have been found to have elevated plasma levels of IL-8 indicating a potential role in VEGF independent angiogenesis and tumor metastases. Hull et al. identified a common single nucleotide polymorphism 251 base pairs upstream of the IL-8 transcription start site. In their in vivo models, they demonstrated, that the IL-8 -251A allele was associated with increased plasma levels of IL-8.  Our pilot study that a germline polymorphism in the IL-8 gene, which consist of a T to A substitution at position -251 of the gene, was associated with response to low dose, metronomic cyclophospahmide and bevacizumab based chemotherapy in patients with recurrent ovarian cancer or primary peritoneal carcinoma. Previously, Hull et al 24 had found that the A variant of this polymorphism was associated with increased IL-8 production. Other studies reported that IL-8 plays a significant role in mediating human ovarian carcinoma-derived angiogenesis and tumorigenesis26. Il-8 exerts its potent angiogenic properties on endothelial cells through interaction with its cognate receptors CXCR1 and CXCR2. Induction of IL-8 preserved the angiogenic response in HIF1a ( transcription factor for genes that control angiogenesis, especially VEGF ) deficient colon cancer cells, suggesting that IL-8 mediates angiogenesis, independently of VEGF. These studies support our hypothesis that overexpression of IL-8 may be responsible for resistance to Bevacizumab based chemotherapy. Therefore, Il-8 may be a promising novel target for drug development. In vitro and in vivo data suggest that the combination of VEGF and IL-8 antibodies has a synergistic effect28, further supporting the significance of IL-8. In addition, we showed that the CXCR2 +785 polymorphism was associated with PFS. CXCR2 functions as receptor for Il-8 on endothelial cells, thus providing more evidence for IL-8 induced angiogenesis. In human intestinal microvascular endothelial cells,Il-8 mediated its angiogenic potential by signaling via CXCR2. CXCR2 knock out mice, in a murine model of lung cancer, demonstrated reduced growth, increased tumor associated necrosis and reduced metastatic potential in an angiogenesis-dependent manner, again supporting the role of IL-8 and CXCR2 in angiogenesis. Inhibition of this chemokine receptor decreased pancreatic cancer cell-induced angiogenesis. Strong expression of CXCR2 was found on ovarian cancer cells, indicating that IL-8 induced angiogenesis plays a role in ovarian cancer. So far, the functional significance of the CXR2 +785 polymorphism remains unclear. Previous studies have reported an association between this polymorphism and systemic sclerosis and crytogenic fibrosing alveolitis. However this polymorphism may be in linkage disequilibrium with other functional polymorphisms which are responsible for this significant association. Taken together, these data suggest the importance of the IL-8 pathways in angiogenesis of ovarian tumors.  We could also show an association between a polymorphism in the gene encoding for Adrenomedullin and PFS, indicating a potential role of Adrenomedullin in ovarian cancer angiogenesis. Adrenomedullin (AM), which was originally identified as a vasodilatatory peptide, was later described as an angiogenic factor. Previously, Nikitenko et al described the role of AM in tumor angiogenesis. AM mediates its signals via a G-protein coupled receptor, calcitonin receptor-like receptor(CRLR), and receptor activity modifying proteins (RAMPs) have been recognized as integral components of the AM signaling system. Recent studies have shown that AM activates, independent of VEGF, the PIK/Akt-dependent pathway in vascular endothelial cells. This pathway is considered to regulate endothelial cell survival, proliferation, migration and capillary-like structure formation. VEGF and AM have been found to act synergistically to induce angiogenic-related effects on endothelial cells in vitro. However, blockade of VEGF with antibodies could not inhibit AM-induced capillary tube formation by HUVECs, suggesting the VEGF independent role of AM in angiogenesis. AM-overexpressing tumors are characterized by increased vascularity. Additionally, increased expression of AM mRNA in ovarian tumors has been associated with poor prognosis35 and histological grade. In our study, we examined a CA dinucleotide repeat polymorphism in the 3’end of the gene. This polymorphism was found to be associated with a genetic predisposition to essential hypertension and type 2 diabetes in patients with renal failure. So far, there is no data available concerning the exact function of this repeat. Nevertheless, our data indicates that AM may play a role in VEGF-independent angiogenesis of ovarian tumors.  As previously demonstrated by our group IL-8 T-251A polymorphism and its receptor CXCR-1 were significantly associated with clinical outcome in colorectal cancer patients. IL-8 T-251A polymorphism was found to be significantly associated with risk of recurrence in rectal cancer patients in both univariate and regression tree analysis. IL-8 affects such downstream processes as apoptosis inhibition and showed in vitro data suggested that IL-8 is also associated with oxaliplatin sensitivity. One hundred fifty-two patients in a phase II prospective study of oxaliplatin in combination with continuous infusion 5-FU in colorectal cancer patients refractory to 5-FU/CPT-11 chemotherapy (3C-01-7) were genotyped. Median overall survival was 10.3 months, and median progression free survival was 4.2 months. Response rate was 19%. IL-8 A-251T polymorphism was significantly associated with time to tumor progression (p=0.04, logrank test). CXCR2 C+785T polymorphism was significantly associated with tumor response (p=0.034). These results suggest that the IL-8 signaling pathway plays an important role in colorectal cancer progression. Specifically, we show here that gene polymorphisms of IL-8 and its receptor CXCR2 are associated with oxaliplatin sensitivity in metastatic colorectal cancer patients treated with 5-FU/oxaliplatin.We have also data in 140 patients with stage III colon cancer showing that colon cancer patients harboring high expression variants of IL-8 -251 polymorphism (A/A genotype) were at higher risk of developing tumor recurrence, supporting our hypothesis that increased angiogenic potential is critical for tumor relapse.