Anthrax toxin receptor (ANTXR1)/TEM 8 expression and sensitivity to protective antigen plus lethal factor.
Authors: Cecile Rouleau, Paula Boutin, Cheryl Guyre, Hitoshi Yoshida, Shiro Kataoka, Srinivas Shankara, Han-Mo Koo, Nicholas S. Duesbery, George Vande Woude, Hans-Peter Biemann, Beverly A. Teicher
Journal: Cancer Research (2004)

Proc Amer Assoc Cancer Res, Volume 45, 2004

3816 

The combination of two components of Anthrax Lethal Toxin (LeTx), composed of Protective Antigen (PA) plus Lethal Factor (LF), has reported cytotoxicity in vitro and anti-tumor activity in vivo. Current models of the mechanism by which the toxin affects cells involve binding of PA to the Anthrax Toxin Receptor (ANTXR) on the cell surface to mediate the entry of LF into cells. Inside the cell, LF inhibits MAPK signaling and triggers apoptosis of human melanoma cells but not that of normal human melanocytes. Multiple genes have been described to be ANTXR, the transcripts for one of these were found to be expressed at higher levels in freshly isolated human colon tumor endothelial cells compared to donor-matched normal colon endothelial cells and was designated tumor endothelial marker 8 (ANTXR1/TEM 8). We examined the expression of ANTXR1/TEM8 mRNA by real-time PCR and that of ANTXR1/TEM8 protein by flow cytometry on ten different human cell types including MEL 624, WM115, A375 and SKMEL28 human melanoma cell lines, SKNAS human neuroblastoma cell line, HT29 and SW480 human colorectal cancer cell lines, MCF7 and SKBR3 human breast carcinoma cell lines and normal primary human umbilical vein endothelial cells (HUVEC). The real-time PCR data indicated that eight out of ten cell types expressed ANTXR1/TEM8 mRNA, with HT29 and SKBR3 cells expressing no ANTXR1/TEM8 mRNA. In contrast, the flow cytometry data showed that only the SKNAS neuroblastoma cells expressed ANTXR1/TEM8 protein on the cell surface. We studied the effect of PA plus LF on the nine human cancer cell lines and HUVEC. Growth inhibition was assessed in a 96-well format with 2,000 cells per well and 2% FBS for 72 hrs using PA (100 ng/ml) and a concentration range of LF (0.1 –10 ng/ml). Six of ten cell types responded to PA plus LF growth inhibition. The six sensitive cell types were the HUVEC, the SKMEL28, A375 and WM115 human melanoma cell lines, the SKNAS human neuroblastoma cell line and the HT29 colon carcinoma cell line. The four resistant cell types were the MEL624 human melanoma cell line, the MCF7 and SKBR3 human breast carcinoma cell lines and the SW480 colon carcinoma cell line. Maximum growth inhibition was achieved at a LF concentration of 3 ng/ml and 70–100% growth inhibition was observed. Thus, PA plus LF may be useful in treating cancers in addition to melanoma. The sensitivity of HUVEC to PA plus LF suggests that this toxin might also be useful to treat highly angiogenic malignant tumors. While PA plus LF inhibited the growth of six cell types in culture, only one had detectable ANTXR1/TEM 8 protein on the cell surface, and the HT29 cell line which had neither ANTXR1/TEM 8 mRNA nor protein was inhibited by LF plus PA in a concentration-dependent manner. There was no association between ANTXR1/TEM 8 protein expression and sensitivity to PA plus LF therefore other ANTXRs may be required for toxin-induced growth inhibition in these cell types.