Tumour-associated angiogenesis in human colorectal cancer.

Tumour angiogenesis is a critical step in the growth, metastatic spread and regrowth of colorectal cancer. Angiogenesis specific to tumour is a complicated process, the mechanisms of which remain unclear. Metastasis of colorectal cancer may result from passive entry into the circulation secondary to the effect of angiogenic factors. The survival and growth of colorectal tumour and thus their metastases are dependent on the balance of endogenous angiogenic and anti-angiogenic factors such that the outcome favours increased angiogenesis. Angiogenesis has become an attractive target for anticancer drug development, based on its important roles in tumour growth, invasion and metastasis. Several growth factors have been identified that regulate angiogenesis in colorectal cancer; the most important of these are vascular endothelial growth factors (VEGF), and of the several angiogenic factors, VEGF expression at the deepest invasive site of tumour is the most statistically significant prognostic indicator in advanced colorectal carcinoma. In this review article, we provide an overview on angiogenic factors and their receptors, and discuss the role of newly identified tumour endothelial markers (TEMs) that are involved in tumour-associated angiogenesis in colorectal cancer.

TEM-8 and tubule formation in endothelial cells, its potential role of its vW/TM domains.

BACKGROUND AND AIMS: Tumour endothelial marker-8 (TEM-8) has been found to be selectively up regulated in tumour-associated endothelial cells, it is implicated in tumour specific angiogenesis, but its mechanism in angiogenesis is not defined. METHODS: A ribozyme transgene (TEM-8) was cloned into a suitable mammalian expression vector (pc DNA 3.1-GFP-NT) and transfected into HECV cells. Various domains of TEM-8 were designed and cloned into pEF6/V5-His TOPO TA vector and transfected into Chinese Hamster ovarian cells (CHO), which do not form tubules and do not express TEM-8 in general (CHO(vW), CHO(TM), CHO(vW/TM), CHO(AE), CHO(AC), CHO(IC), and CHO(FL) domains, respectively). The effect of TEM-8 knocked out HECV cells was tested (by angiogenesis and migration assays), and the effect of each cleavage domain of TEM-8 was tested by microtubule formation assay. RESULTS: TEM-8 stable transfectants (HECV(DeltaTEM8a)) manifested a complete loss of TEM-8 gene expression at mRNA and protein levels. In contrast, control GFP plasmid (HECV(pControl)) and wild-type HECV cells (HECV(WT)) had similar levels of TEM-8 expression. TEM-8 transfected cell (HECV(DeltaTEM8a)) significantly decreased the micro-vessels formation compared with controls (HECV(pControl)) (mean+/-SE, 20.3+/-4.03 microm; p=0.0086 vs. control 39.5+/-10.1 microm), and migration (38.52+/-2.17; p<0.05 vs. control 80.23+/-3.19), and micro-vessel formation of HECV(DeltaTEM8a) cell was also reduced compared with wild-type (HECV(WT)) (mean+/-SE, 20.3+/-4.03 microm; p=0.0078 vs. wild-type 42.5+/-9.1 microm) and migration (38.52+/-2.17microm; p<0.05 vs. wild-type 82.4+/-4.45 microm). vW together with transmembrane domains of TEM-8 (CHO(vW/TM)) and full-length CHO(FL) showed formation of tubule-like structure in CHO cells, whereas the other domains showed no effect. CONCLUSION: Targeting the TEM-8 gene by way of a hammerhead ribozyme knocks out TEM-8 cells, and is an effective way to reduce the micro-vessel formation or migration potential in tumour-associated endothelial cell through its vW domain. These results suggest that the vW domain together with the transmembrane domain of TEM-8 may play an important biological role in TEM-8 related tubule formation.

Prognostic values of tumor endothelial markers in patients with colorectal cancer.

AIM: Tumor endothelial markers (TEMs) are a newly discovered family of endothelial markers associated with tumor specific angiogenesis. This study sought to examine the levels of expression (qualitatively and quantitatively) for TEMs in human colon cancer. METHODS: Human colorectal cancer tissues (n = 48) and normal background tissues (n = 31) were obtained after surgery. RNA was extracted from frozen sections for gene amplification. The expression of TEMs (TEM-1 to TEM-8) was assessed using RT-PCR and their transcript levels were determined using real-time-quantitative PCR (Q-RT-PCR). RESULTS: TEM-1 (P = 0.01), TEM-7 (P = 0.04), TEM-7R (P = 0.03), TEM-8 (P = 0.001) significantly raised in colon cancer tissues compared with the levels detected in normal background tissues. The expressions of TEM-2 and TEM-6 were found to be not significantly different between tumor tissues and normal tissues (P > 0.05). Patients who had cancer penetrating into and through the muscularis propria of the bowel wall and developed nodal involvement (Dukes C) exhibited significantly higher levels of TEM -8 compared to patients who were node negative (P < 0.05). TEM-7 and TEM-7R showed high level of transcripts in Dukes C, but they were not statistically significant. CONCLUSION: The level of the expression of TEM-1, TEM-7, TEM-7R and TEM-8 (but not TEM-2 and TEM-6) were associated with both nodal involvement and disease progression, and may therefore, have a prognostic value in colorectal cancer.

Tumour endothelial marker 8 (TEM-8) in human colon cancer and its association with tumour progression.

BACKGROUND AND AIMS: Tumour endothelial marker-8 (TEM-8) is endothelial cell surface marker that may be specific to tumour endothelial cells. This study examined the role of TEM-8 in human colon cancer and its correlation with tumour prognosis. METHODOLOGY: Specimens of colorectal tissue (normal and cancer) were stained immunohistochemically with an anti-TEM-8 antibody, newly developed in our laboratory, and with anti-vonWillebrand Factor antibody. RNA was extracted from frozen sections for gene amplification. The anti-TEM-8 antibody specificity tested by using slot blotting with irrelevant antibody, and western blotting with different cell lines. The expression of TEM-8 was assessed using RT-PCR, and the level of TEM-8 was quantified using real-time-quantitative PCR (Q-RT-PCR). RESULTS: TEM-8 staining was primarily seen in endothelial cells. TEM-8 identified more micro-vessels in colon tumour tissue, than in normal colon tissues, (p=0.002). Whereas, fewer vessels were stained positive for TEM-8 in normal tissues stained positive for vonWillebrand Factor (factor-8), (p=0.008). Malignant cells in tumour tissues were found to be stained strongly positive for TEM-8 compared with the epithelial cells in normal colon tissues. The level of TEM-8 expression was significantly higher in the tumour tissues compared to the normal colon mucosa (p=0.001). TEM-8 mRNA expression was also found to be more elevated in patients with advanced tumour, Dukes C (Dukes A vs. Dukes C, p=0.01). CONCLUSION: TEM-8 is a marker that identifies tumour associated micro-vessels in colon cancer. The levels of expression of TEM-8 in invasive colon cancer are linked to disease progression. This suggests that TEM-8 has significant prognostic and therapeutic values in colon cancer.