Current therapeutic approaches to cancer are designed to target molecules that contribute to malignant behavior but leave normal tissues intact. B 1 integrin is a candidate target well known for mediating cell-extracellular matrix (ECM) interactions that influence diverse cellular functions; its aberrant expression has been implicated in breast cancer progression and resistance to cytotoxic therapy. The addition of B 1 integrin inhibitory agents to breast cancer cells at a single-cell stage in a laminin-rich ECM (three-dimensional lrECM) culture was shown to down-modulate B 1 integrin signaling, resulting in malignant reversion. To investigate B 1 integrin as a therapeutic target, we modified the threedimensional lrECM protocol to approximate the clinical situation: before treatment, we allowed nonmalignant cells to form organized acinar structures and malignant cells to form tumor-like colonies. We then tested the ability of B 1 integrin inhibitory antibody, AIIB2, to inhibit tumor cell growth in several breast cancer cell lines (T4-2, MDA-MB-231, BT474, SKBR3, and MCF-7) and one nonmalignant cell line (S-1). We show that B 1 integrin inhibition resulted in a significant loss of cancer cells, associated with a decrease in proliferation and increase in apoptosis, and a global change in the composition of residual colonies. In contrast, nonmalignant cells that formed tissue-like structures remained resistant. Moreover, these cancer cell-specific antiproliferative and proapoptotic effects were confirmed in vivo with no discernible toxicity to animals. Our findings indicate that B 1 integrin is a promising therapeutic target, and that the threedimensional lrECM culture assay can be used to effectively distinguish malignant and normal tissue response to therapy.

B 1 Integrin signaling has been shown to mediate cellular resistance to apoptosis after exposure to ionizing radiation (IR). Other signaling molecules that increase resistance include Akt, which promotes cell survival downstream of B 1 integrin signaling. We previously showed that B 1 integrin inhibitory antibodies (e.g., AIIB2) enhance apoptosis and decrease growth in human breast cancer cells in threedimensional laminin-rich extracellular matrix (lrECM) cultures and in vivo. Here, we asked whether AIIB2 could synergize with IR to modify Akt-mediated IR resistance. We used three-dimensional lrECM cultures to test the optimal combination of AIIB2 with IR treatment of two breast cancer cell lines, MCF-7 and HMT3522-T4-2, as well as T4-2 myr-Akt breast cancer colonies or HMT3522-S-1, which form normal organotypic structures in three-dimensional lrECM. Colonies were assayed for apoptosis and B 1 integrin/Akt signaling pathways were evaluated using Western blot. In addition, mice bearing MCF-7 xenografts were used to validate the findings in three-dimensional lrECM. We report that AIIB2 increased apoptosis optimally post-IR by down-regulating Akt in breast cancer colonies in three-dimensional lrECM. In vivo, addition of AIIB2 after IR significantly enhanced tumor growth inhibition and apoptosis compared with either treatment alone. Remarkably, the degree of tumor growth inhibition using AIIB2 plus 2 Gy radiation was similar to that of 8 Gy alone. We previously showed that AIIB2 had no discernible toxicity in mice; here, its addition allowed for a significant reduction in the IR dose that was necessary to achieve comparable growth inhibition and apoptosis in breast cancer xenografts in vivo. [Cancer Res 2008;68(11):4398-405]

In eukaryotes, cyclin B-bound cyclin-dependent protein kinase 1 promotes mitotic entry but is held in check, in part, by Wee1 protein kinase. Timely mitotic entry in budding yeast requires inactivation of Swe1 (Wee1 ortholog). Perturbations of the septin collar at the bud neck lead to Swe1 stabilization, delaying the G2͞M transition. Swe1 is recruited to the neck and hyperphosphorylated before ubiquitin-mediated degradation. Hsl1 kinase (Nim1 ortholog), a negative regulator of Wee1, is required for efficient Swe1 localization at the neck but seems not to phosphorylate Swe1. Here, we show that two other kinases targeted sequentially to the neck, Cla4͞PAK and Cdc5͞Polo, are responsible for stepwise phosphorylation and down-regulation of Swe1. This mechanism links assembly of a cellular structure to passage into mitosis.Cdk1 regulation ͉ mitotic progression ͉ G2͞M transition ͉ Saccharomyces cerevisiae