Tumor Endothelial Marker 8 Expression Research Articles

Tumor Endothelial Marker 8 Promotes Proliferation and Metastasis via the Wnt/β-Catenin Signaling Pathway in Lung Adenocarcinoma.

Tumor endothelial marker 8 (TEM8), also known as ANTXR1, was highly expressed in cancers, and was identified as a biomarker for early diagnosis and prognosis in some cancers. However, the clinical role and molecular mechanisms of TEM8 in lung adenocarcinoma (LUAD) are still unclear. The present study aimed to explore its clinical value and the molecular mechanisms of TEM8 underlying the progression of LUAD. Our study found the elevation of TEM8 in LUAD cell lines and tissues. What’s more, we observed that the TEM8 expression level was associated with tumor size, primary tumor, and AJCC stage, and LUAD patients with high TEM8 expression usually have a poor prognosis. Then, we conducted a series of experiments by the strategy of loss-of-function and gain-of-function, and our results suggested that the knockdown of TEM8 suppressed proliferation, migration, and invasion and induced apoptosis in LUAD whereas overexpression of TEM8 had the opposite effect. Molecular mechanistic investigation showed that TEM8 exerted its promoting effects mainly through activating the Wnt/β-catenin signaling pathway. In short, our findings suggested that TEM8 played a crucial role in the progression of LUAD by activating the Wnt/β-catenin signaling pathway and could serve as a potential therapeutic target for LUAD.

Targeted silencing of TEM8 suppresses non‑small cell lung cancer tumor growth via the ERK/Bcl‑2 signaling pathway.

Non-small cell lung cancer (NSCLC) is one of the most common malignancies with high rates of mortality. Although great progress has been made with the development of novel immunotherapies and targeted therapeutic strategies, the 5-year total survival rate of lung cancer has remained unchanged over the past few decades. Therefore, more effective therapeutics are urgently needed. Tumor endothelial marker 8 (TEM8) is an integrin-like cell surface transmembrane protein that has been demonstrated to be upregulated in numerous cancer types and previously showed promise for targeted cancer therapy. However, the role of TEM8 in NSCLC remains poorly understood. The present study aimed to investigate the effects of silencing TEM8 on expression and regulation of extracellular signal-regulated kinase (ERK)1/2 signaling pathways in NSCLC. In the present study, a lentiviral vector that encoded a short hairpin RNA targeting TEM8 was designed and transfected into Xuanwei Lung Cancer (XWLC)-05 lung cancer cells to silence TEM8 expression. Male BALB/c-nu/nu mice were then given subcutaneous injections in the right dorsal flank with XWLC-05 cells. Microvessel density was measured using an anti-CD34 antibody. The mRNA and protein levels of ERK1/2 and Bcl-2 in XWLC-05 cells or xenograft tumor tissues were detected by reverse transcription-quantitative polymerase chain reaction and western blotting. TEM8 knockdown was found to significantly inhibit tumor growth and conferred an anti-angiogenic ability in vivo. Furthermore, TEM8 knockdown suppressed the expression of Bcl-2 mediated by ERK1/2 activity in XWLC-05 cells or tissues from mice with NSCLC. To conclude, these results suggest that the targeted silencing of TEM8 may serve as an effective method of treating NSCLC.

Low doses of deoxynivalenol inhibit the cell migration mediated by H3K27me3-targeted downregulation of TEM8 expression

Deoxynivalenol (DON) is a mycotoxin produced by multipleFusariumspecies that often contaminates cereals and threatens human and animal health. A wide range of cytotoxic effects, such as the induction of DNA damage, an increase in mitochondrial permeability and the inhibition of macromolecule synthesis, have been reported. However, the effects of DON on cell migration—a fundamental process in living cells critical for normal development, immune responses, and disease processes—and the mechanism underlying these effects are still unclear. Here, we showed that DONsignificantly inhibited the migration of MRC-5, CCD-18Co, HCT116 and WM793 cells at 50 ng/ml, 50 ng/ml, 400 ng/ml and 250 ng/ml, respectively, which maintained cell viability at 90%. Further analysis showed that DON inhibited the expression of tumour endothelial marker 8 (TEM8), a key gene in cell migration. Furthermore, we showed that DON inhibited the expression of TEM8 through increasing the level of H3K27me3 in the TEM8 promoter. Finally, overexpression of TEM8 or treating by H3K27me3-specific inhibitor GSK126 attenuated the inhibitory effect of DON on cell migration. In summary, low doses of DON at approximately dietary exposure significantly inhibited cell migration by downregulating the expression of TEM8 in a manner mediated by H3K27me3, which may generate increasing concerns for the risk of DON exposure.

Expression of Tumour Endothelial Marker 8 in Canine Mammary Gland Tumour Cells

The aim of this study was to investigate the expression of tumour endothelial marker 8 (TEM8) in canine mammary gland tumours (MGTs) by immunohistochemistry and to evaluate the association between tumour cell TEM8 expression and tumour histological features, histological grades and expression of luminal and basal/myoepithelial cell markers. TEM8 expression was detected in >60 % of neoplastic epithelial cells in all simple adenomas (n=25), simple carcinomas (n=43) and invasive micropapillary carcinomas (n=5) studied. Six of the 18 solid carcinomas studied showed TEM8 expression in >60% of carcinoma cells present in solid structures and in 12 of the 18 solid carcinomas, <30% of the luminal structure-forming carcinoma cells showed TEM8 expression. TEM8 expression in the neoplastic cells was not associated with histological malignancy in canine MGTs. TEM8+ tumour cells frequently showed the luminal-like phenotype cytokeratin (CK)19+/p63-/α-smooth muscle actin (SMA)-, while most TEM8- tumour cells exhibited the basal-like phenotype CK19-/p63+/αSMA-. These findings indicate that TEM8 may be involved in maintaining the characteristics of luminal cells in canine MGTs and that TEM8 would be useful in identifying the type of neoplastic epithelial cell in MGTs.

In situ targeting TEM8 via immune response and polypeptide recognition by wavelength-modulated surface plasmon resonance biosensor.

There is an increasing interest in real-time and in situ monitoring of living cell activities in life science and medicine. This paper reports a whole cell sensing protocol over the interface of Au film coupled in a wavelength-modulated surface plasmon resonance (WMSPR) biosensor. With dual parabolic mirrors integrated in the sensor, the compact and miniaturized instrument shows satisfactory refractive index sensitivity (2220 nm/RIU) and a high resolution of resonance wavelength shift of 0.3 nm to liquid samples. The affinity interactions between the biomarker of human tumor endothelial marker 8 (TEM8) and antibody (Ab) or specific polypeptide (PEP) were firstly introduced to WMSPR biosensor analysis. Both the interaction events of Ab-cell and PEP-cell over the Au film interface can be recognized by the sensor and the balance time of interactions is about 20 min. The concentration range of Ab for quantitative monitoring of the TEM8 expression on human colon carcinoma SW620 cells was investigated. The present low-cost and time-saving method provides a time resolution of binding specificity between Ab/PEP and TEM8 for real-time analysis of antigen on living tumor cell surface.

Immuno-PET imaging of tumor endothelial marker 8 (TEM8).

Tumor endothelial marker 8 (TEM8) is a cell surface receptor that is highly expressed in a variety of human tumors and promotes tumor angiogenesis and cell growth. Antibodies targeting TEM8 block tumor angiogenesis in a manner distinct from the VEGF receptor pathway. Development of a TEM8 imaging agent could aid in patient selection for specific antiangiogenic therapies and for response monitoring. In these studies, L2, a therapeutic anti-TEM8 monoclonal IgG antibody (L2mAb), was labeled with 89Zr and evaluated in vitro and in vivo in TEM8 expressing cells and mouse xenografts (NCI-H460, DLD-1) as a potential TEM8 immuno-PET imaging agent. 89Zr-df–L2mAb was synthesized using a desferioxamine–L2mAb conjugate (df–L2mAb); 125I-L2mAb was labeled directly. In vitro binding studies were performed using human derived cell lines with high, moderate, and low/undetectable TEM8 expression. 89Zr-df–L2mAb in vitro autoradiography studies and CD31 IHC staining were performed with cryosections from human tumor xenografts (NCI-H460, DLD-1, MKN-45, U87-MG, T-47D, and A-431). Confirmatory TEM8 Western blots were performed with the same tumor types and cells. 89Zr-df–L2mAb biodistribution and PET imaging studies were performed in NCI-H460 and DLD-1 xenografts in nude mice. 125I-L2mAb and 89Zr-df–L2mAb exhibited specific and high affinity binding to TEM8 that was consistent with TEM8 expression levels. In NCI-H460 and DLD-1 mouse xenografts nontarget tissue uptake of 89Zr-df–L2mAb was similar; the liver and spleen exhibited the highest uptake at all time points. 89Zr-L2mAb was highly retained in NCI-H460 tumors with <10% losses from day 1 to day 3 with the highest tumor to muscle ratios (T:M) occurring at day 3. DLD-1 tumors exhibited similar pharmacokinetics, but tumor uptake and T:M ratios were reduced ∼2-fold in comparison to NCI-H460 at all time points. NCI-H460 and DLD-1 tumors were easily visualized in PET imaging studies despite low in vitro TEM8 expression in DLD-1 cells indicating that in vivo expression might be higher in DLD-1 tumors. From in vitro autoradiography studies 89Zr-df–L2mAb specific binding was found in 6 tumor types (U87-MG, NCI-H460, T-47D MKN-45, A-431, and DLD-1) which highly correlated to vessel density (CD31 IHC). Westerns blots confirmed the presence of TEM8 in the 6 tumor types but found undetectable TEM8 levels in DLD-1 and MKN-45 cells. This data would indicate that TEM8 is associated with the tumor vasculature rather than the tumor tissue, thus explaining the increased TEM8 expression in DLD-1 tumors compared to DLD-1 cell cultures. 89Zr-df–L2mAb specifically targeted TEM8 in vitro and in vivo although the in vitro expression was not necessarily predictive of in vivo expression which seemed to be associated with the tumor vasculature. In mouse models, 89Zr-df–L2mAb tumor uptakes and T:M ratios were sufficient for visualization during PET imaging. These results would suggest that a TEM8 targeted PET imaging agent, such as 89Zr-df–L2mAb, may have potential clinical, diagnostic, and prognostic applications by providing a quantitative measure of tumor angiogenesis and patient selection for future TEM8 directed therapies.

Imaging tumor endothelial marker 8 using an 18F-labeled peptide

Tumor endothelial marker 8 (TEM8) has been reported to be upregulated in both tumor cells and tumor-associated endothelial cells in several cancer types. TEM8 antagonists and TEM8-targeted delivery of toxins have been developed as effective cancer therapeutics. The ability to image TEM8 expression would be of use in evaluating TEM8-targeted cancer therapy. A 13-meric peptide, KYNDRLPLYISNP (QQM), identified from the small loop in domain IV of protective antigen of anthrax toxin was evaluated for TEM8 binding and labeled with 18F for small-animal PET imaging in both UM-SCC1 head-and-neck cancer and MDA-MB-435 melanoma models. A modified ELISA showed that QQM peptide bound specifically to the extracellular vWA domain of TEM8 with an IC50 value of 304 nM. Coupling 4-nitrophenyl 2-(18)F-fluoropropionate with QQM gave almost quantitative yield and a high specific activity (79.2±7.4 TBq/mmol, n=5) of 18F-FP-QQM at the end of synthesis. 18F-FP-QQM showed predominantly renal clearance and had significantly higher accumulation in TEM8 high-expressing UM-SCC1 tumors (2.96±0.84 %ID/g at 1 h after injection) than TEM8 low-expressing MDA-MB-435 tumors (1.38±0.56 %ID/g at 1 h after injection). QQM peptide bound specifically to the extracellular domain of TEM8. 18F-FP-QQM peptide tracer would be a promising lead compound for measuring TEM8 expression. Further efforts to improve the affinity and specificity of the tracer and to increase its metabolic stability are warranted.

Tumor endothelial marker 8 enhances tumor immunity in conjunction with immunization against differentiation Ag

We have previously shown that xenogeneic DNA vaccines encoding rat neu and melanosomal differentiation Ag induce tumor immunity. Others have developed vaccines targeting tumor neovasculature. Tumor endothelial marker 8 (TEM8) is expressed in the neovasculature of human tumors, and in the mouse melanoma B16, but its expression is limited in normal adult tissues. We describe a DNA vaccine combining xenogeneic tumor Ag and TEM8. In-situ hybridization was used to detect TEM8 RNA in mouse tumors. Mice transgenic for the rat neu proto-oncogene were immunized with DNA vaccines encoding TEM8 and the extracellular domain of rat neu and challenged with the 233-VSGA1 breast cancer cell line. In parallel experiments, C57BL/6 mice were immunized with TEM8 and human tyrosinase-related protein 1 (hTYRP1/hgp75) and challenged with B16F10 melanoma. TEM8 was expressed in the stroma of transplantable mouse breast and melanoma tumors. In both model systems, TEM8 DNA had no activity as a single agent but significantly enhanced the anti-tumor immunity of neu and hTYRP1/hgp75 DNA vaccines when given in concert. The observed synergy was dependent upon CD8+ T cells, as depletion of this cell population just prior to tumor challenge obviated the effect of the TEM8 vaccine in both tumor models. A local immune response to TEM8 may increase inflammation or tumor necrosis within the tumor, resulting in improved Ag presentation of HER2/neu and hTYRP1/hgp75. Alternatively, TEM8 expression in host APC may alter T-cell interactions or homing. In this way, TEM8 may act more as an adjuvant than an immunologic target.

Elevated levels of tumour endothelial marker-8 in human breast cancer and its clinical significance

Tumour endothelial marker-8 (TEM-8) belongs to a family of endothelial markers that are raised during tumour angiogenesis. We have recently reported aberrant expression of TEMs at the mRNA level in human breast cancer. This study sought to examine the level of TEM-8 expression at the protein and mRNA level in human breast cancer tissue, and in a panel of human breast cancer cell lines. We also wished to determine if TEM-8 can be used as a suitable marker for identifying tumour associated micro-vessels. At the mRNA level more tumours showed positive TEM-8 expression compared to normal background tissue. TEM-8 was detected in a variety of breast cancer cell lines, endothelial cells (HECV) and in a human fibroblast cell line (MRC5) at both the mRNA and protein level. Using immunohistochemistry the distribution of TEM-8 staining was more widespread in invasive breast cancer tissue compared to normal background tissue. Furthermore, the TEM-8 marker was found to be more discriminatory in identifying micro-vessels in tumour endothelium (2.8+/-0.83 vs. normal 1.66+/-0.52; P<0.011), compared to the vWFA marker (1.61+/-0.54 vs. normal 2.71+/-0.76; P<0.009). Raised levels of TEM-8 were associated with shorter survival outcome, but were not correlated to disease-free survival as shown by Kaplan-Meier and Cox regression analysis. We conclude that TEM-8 is a useful marker for identifying tumour associated micro-vessels and that elevated levels are associated with disease progression, which may have some bearing on the prognostic outcome in breast 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 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. 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.

Upregulation of tumour endothelial marker-8 by interleukin-1β and its impact in IL-1β induced angiogenesis

Tumour endothelial marker-8 (TEM-8) has been found to be selectively upregulated in tumour-associated endothelial cells and is implicated in tumour specific angiogenesis. Specific factors, indigenous to tissues and tumours that regulate the TEM-8 mechanism in angiogenesis are not defined. We report for the first time that interleukin-1beta induces the expression of TEM-8 in endothelial cells. Human vascular endothelial cells (HECV), which strongly express IL-1beta receptor (as revealed by RT-PCR, Western blotting), increased the level of TEM-8 expression following stimulation with IL-1beta (as revealed by conventional and quantitative RT-PCR). Using a newly developed antibody to human TEM-8, we have further demonstrated that IL-1beta significantly raised the level of TEM-8 at the protein level, as revealed by Western blotting. In vitro tubule forming assay, revealed that IL-1beta significantly induced the formation of capillary-like tubules from the HECV cells, accompanied by an increase in TEM-8 expression. It is concluded that IL-1beta is a powerful regulator of the expression of TEM-8 in vascular endothelial cells. Our results suggest an important pathway through which IL-1beta regulates tumour-associated angiogenesis.

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.