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.
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).
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.
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 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.
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.
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.
TEM-8 stable transfectants (HECV
ΔTEM8a) 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
ΔTEM8a) significantly decreased the micro-vessels formation compared with controls (HECV
pControl) (mean
±
SE, 20.3
±
4.03
μm;
p
=
0.0086 vs. control 39.5
±
10.1
μm), and migration (38.52
±
2.17;
p
<
0.05 vs. control 80.23
±
3.19), and micro-vessel formation of HECV
ΔTEM8a cell was also reduced compared with wild-type (HECV
WT) (mean
±
SE, 20.3
±
4.03
μm;
p
=
0.0078 vs. wild-type 42.5
±
9.1
μm) and migration (38.52
±
2.17
μm;
p
<
0.05 vs. wild-type 82.4
±
4.45
μm). 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.
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.
Interleukin-7 (IL-7), a haematopoietic growth factor, is known to induce the differentiation and proliferation of some haematological malignancies including certain types of leukaemias and lymphomas. ...However, little is known about its role in solid tumours, including breast cancer. In this study, the expression level of IL-7, IL-7 receptor (IL-7R) and their downstream signalling molecules, including the Janus kinases (Jak-1 and Jak-3), phosphoinositide 3-kinase (PI3-K) and signal transducers and activators of transcription (Stat-5) were analysed using the reverse transcriptase-polymerase chain reaction (RT-PCR), real-time quantitative PCR and immunohistochemistry in a cohort of patients with breast cancer. The results were analysed in relation to tumour grade, TNM stage, patients' prognosis (using the Nottingham Prognostic Index (NPI)) and survival. The levels of expression of IL-7, IL-7R, Jak-1, Jak-3, PI3-K and Stat-5 were significantly higher in the most aggressive tumours. With the exception of Stat-5 expression, the transcript copies of IL-7 and all other signalling molecules were higher in patients with the worst prognoses (NPI3) and in patients who died from breast cancer after 72 months of follow-up. This aberrant expression of IL-7 and its signalling intermediates in invasive breast cancers could have significant diagnostic and prognostic implications. Measuring these molecules in breast cancer tissues may provide, for the first time, important molecular indicators of tumour differentiation, aggressiveness, nodal status, prognosis and patient survival.
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.
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).
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).
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.
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.
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).
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).
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.
The von Hippel-Lindau (VHL) gene is located on the short arm of chromosome 3, the mutations of which lead to the development of von Hippel-Lindau disease. The VHL gene is a putative tumour suppressor ...gene in VHL and a few other conditions, possibly by negative regulation of hypoxia- inducible factor-1 (HIF-1) and the stromal-derived factor-1 (SDF-1) receptor, CXCR4, via which the VHL protein negates angiogenesis and tumour cell migration. The current study investigated the expression of VHL at the mRNA and protein levels in clinical breast tumours and evaluated the impact of VHL on the invasion of human breast cancer cells in vitro. Primary breast cancer samples (n=124), adjacent non-cancerous breast tissues obtained from patients in cohort (n=33) and a panel of human breast cancer cells (n=12) were used. Tissue distribution of VHL protein in human breast cancer tissues was assessed using immunohistochemical analysis, and VHL transcript was determined using quantitative reverse transcription PCR. Breast cancer cell line MDA-MB-231 was transfected with a human VHL expression construct (pCR3-GFP/VHL) to allow forced overexpression of VHL in the cells. Invasiveness and migration of cancer cells were assessed using the Matrigel invasion and Cytodex-2 migration assays. Statistical analysis was performed using the Student's t-test. Our results showed that breast cancer cell lines MCF-7 and ZR-75-1 expressed very high levels of VHL transcripts, but the highly aggressive MDA-MB-231, MDA-MB-435 and MDA-MB-453 expressed either no VHL or a low level. The levels of VHL transcripts were significantly lower in grade 2 and grade 3 tumours (mean +/- SD, 1.36+/-0.55 and 0.9+/-0.37), compared with grade 1 tumours (12.3+/-7.6, p<0.002). Node-positive tumours had lower levels of VHL than node-negative tumours. Although tumours from patients with metastasis and from those who died of breast cancer had low levels of VHL, the most significant reduction in VHL was seen in tumours which developed local recurrence (p=0.03). The staining of VHL protein was most abundant in mammary epithelial cells and moderate in endothelial cells. Tumour cells in breast tissues had low to moderate VHL staining. pCR3-GFP/VHL-transfected MDA-MB-231 (MDA-MB-231VHL+) exhibited a reduced spontaneous in vitro invasiveness (14.8+/-2.7) compared with the control cells (18.4+/-1.4). MDA-MB-231VHL+ cells also lost their invasion response to HGF/SF, an invasion-inducing cytokine. The MDA-MB-231VHL+ cells had substantially reduced motility compared with that of the controls (14.8+/-0.7 for MDA-MB-231VHL+ and 20.7+/-1.2 for the control; p<0.001). Thus, VHL exerts inhibitory effects on the invasive and migratory capacity of breast cancer cells in vitro. Low levels of VHL occur in most aggressive breast tumours. Taken together, VHL is a powerful putative tumour suppressor gene in human breast cancer.
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