Objective Endometrial tumors with non-functional p53, such as serous uterine endometrial carcinomas, are aggressive malignancies with a poor outcome, yet they have an Achilles’ heel: due to loss of p53 function, these tumors may be sensitive to treatments which abrogate the G2/M checkpoint. Our objective was to exploit this weakness to induce mitotic cell death using two strategies: (1) EGFR inhibitor gefitinib combined with paclitaxel to arrest cells at mitosis, or (2) BI2536, an inhibitor of polo-like kinase 1 (PLK1), to block PLK1 activity. Methods We examined the impact of combining gefitinib and paclitaxel or PLK1 inhibitor on expression of G2/M checkpoint controllers, cell viability, and cell cycle progression in endometrial cancer cells with mutant p53. Results In cells lacking normal p53 activity, each treatment activated CDC25C and inactivated Wee1, which in turn activated cdc2 and sent cells rapidly through the G2/M checkpoint and into mitosis. Live cell imaging demonstrated irreversible mitotic arrest and eventual cell death. Combinatorial therapy with paclitaxel and gefitinib was highly synergistic and resulted in a 10-fold reduction in the IC50 for paclitaxel, from 14 nM as a single agent to 1.3 nM in the presence of gefitinib. However, BI2536 alone at low concentrations (5 nM) was the most effective treatment and resulted in massive mitotic cell death. In a xenograft mouse model with p53-deficient cells, low dose BI2536 significantly inhibited tumor growth. Conclusions These findings reveal induction of mitotic cell death as a therapeutic strategy for endometrial tumors lacking functional p53.

A defective tight junction (TJ) barrier is a key pathogenic factor for inflammatory bowel disease. Previously, we have shown that autophagy, a cell survival mechanism, enhances intestinal epithelial TJ barrier function. Autophagy-related protein-6 (ATG6/beclin 1), a key protein in the autophagy pathway, also plays a role in the endocytic pathway. The constitutive role of beclin 1 in the intestinal TJ barrier is not known. In Caco-2 cells, beclin 1 was found to be coimmunoprecipitated with the TJ protein occludin and colocalized with occludin on the membrane. Treatment of Caco-2 cells with beclin 1 peptide [transactivating regulatory protein (Tat)-beclin 1] reduced TJ barrier function. Activation of beclin 1 increased occludin endocytosis and reduced total occludin protein level. In contrast, beclin 1 siRNA transfection enhanced Caco-2 TJ barrier function. In pharmacologic and genetic autophagy inhibition studies, the constitutive function of beclin 1 in the TJ barrier was found to be autophagy independent. However, de novo induction of autophagy with starvation or rapamycin prevented Tat-beclin 1-induced increase in TJ permeability and reduction in occludin level. Induction of autophagy also resulted in reduced beclin 1-occludin association. In mouse colon, beclin 1 colocalized with occludin on the epithelial membrane. Perfusion of mouse colon with beclin 1 peptide caused an increase in colonic TJ permeability that was prevented by in vivo induction of autophagy. These findings show that beclin 1 plays a constitutive, autophagy-independent role in the regulation of intestinal TJ barrier function via endocytosis of occludin. Autophagy terminates constitutive beclin 1 function in the TJ barrier and enhances the TJ barrier.

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To understand how type I and II endometrial tumors uniquely respond to tyrosine kinase inhibitor treatments, we evaluated the signaling pathways of epidermal growth factor (EGF) receptor (EGFR) under the effects of EGF and

We identified a major peptide signaling target of EGF/EGFR pathway and explored the consequences of blocking or activating this pathway in the first trimester extravillous trophoblast cells, HTR-8/SVneo. A global analysis of protein phosphorylation was undertaken using novel technology (Kinexus Kinetworks) that utilizes SDS-polyacrylamide minigel electrophoresis and multi-lane immunoblotting to permit specific and semiquantitative detection of multiple phosphoproteins. Forty-seven protein phosphorylation sites were queried, and the results reported based on relative phosphorylation at each site. EGF- and Iressa-(gefitinib, ZD1839, an inhibitor of EGFR) treated HTR-8/SVneo cells were subjected to immunoblotting and flow cytometry to confirm the phosphoprotein screen and to assess the effects of EGF versus Iressa on cell cycle and apoptosis. EGFR mediates the phosphorylation of important signaling proteins, including PKBα/AKT. This pathway is likely to be central to EGFR-mediated trophoblast survival. Furthermore, EGF treatment induces proliferation and inhibits apoptosis, while Iressa induces apoptosis.