The Ubiquitin-Conjugating Enzyme E2-EPF Is Overexpressed in Primary Breast Cancer and Modulates Sensitivity to Topoisomerase II Inhibition1

We identified the ubiquitin-conjugating enzyme E2-EPF mRNA as differentially expressed in breast tumors relative to normal tissues and performed studies to elucidate its putative role in cancer. We demonstrated that overexpression of E2-EPF protein correlated with estrogen receptor (ER) negativity in breast cancer specimens and that its expression is cell cycle-regulated, suggesting a potential function for E2-EPF in cell cycle progression. However, reduction of E2-EPF protein levels by > 80% using RNAi had no significant effects on the proliferation of HeLa cervical cancer cells or ER- MDA-MB-231 or MDA-MB-453 breast cancer cells. Because E2-EPF protein levels were elevated during the G2/M phase of the cell cycle and because E2-EPF mRNA in tumor specimens was frequently coexpressed with genes involved in cell cycle control, spindle assembly, and mitotic surveillance, the possibility that E2-EPF might have a function in the cellular response to agents that induce a G2 checkpoint or an M checkpoint was investigated. E2-EPF knockdown sensitized HeLa cells to the topoisomerase (topo) II inhibitors etoposide and doxorubicin and also increased topo IIα protein levels. These data suggest that combined administration of topo II-directed drugs and E2-EPF inhibitors may enhance their clinical effectiveness

Anti-EphA2 Antibodies Decrease EphA2 Protein Levels in Murine CT26 Colorectal and Human MDA-231 Breast Tumors But Do Not Inhibit Tumor Growth

The EphA2 receptor tyrosine kinase has been shown to be over-expressed in cancer and a monoclonal antibody (mAb) that activates and down-modulates EphA2 was reported to inhibit the growth of human breast and lung tumor xenografts in nude mice. Reduction of Eph A2 levels by treatment with anti-EphA2 siRNA also inhibited tumor growth, suggesting that the anti-tumor effects of these agents are mediated by decreasing the levels of EphA2. As these studies employed human tumor xenograft models in nude mice with reagents whose cross-reactivity with murine EphA2 is unknown, we generated a mAb (Ab20) that preferentially binds, activates, and induces the degradation of murine EphA2. Treatment of established murine CT26 colorectal tumors with Ab20 reduced EphA2 protein levels to ∼ 12% of control tumor levels, yet had no effect on tumor growth. CT26 tumor cell colonization of the lung was also not affected by Ab20 administration despite having barely detectable levels of EphA2. We also generated and tested a potent agonistic mAb against human EphA2 (1G9-H7). No inhibition of human MDA-231 breast tumor xenograft growth was observed despite evidence for >85% reduction of EphA2 protein levels in the tumors. These results suggest that molecular characteristics of the tumors in addition to EphA2 over-expression may be important for predicting responsiveness to EphA2-directed therapies