Connective tissue growth factor (CTGF) is a member of an emerging family of immediate-early gene products that coordinate complex biological processes during differentiation and tissue repair. Here we describe the role of CTGF in integrin-mediated adhesive signaling and the production of extracellular matrix components in human mesangial cells. The addition of CTGF to primary mesangial cells induced fibronectin production, cell migration, and cytoskeletal rearrangement. These functional responses were associated with recruitment of Src and phosphorylation of p42/44 MAPK and protein kinase B. The inhibition of CTGF-induced p42/44 MAPK or phosphatidylinositol 3-kinase (PI3K)/protein kinase B pathway activities abrogated the induction of fibronectin expression. In addition, anti-␤ 3 integrin antibodies attenuated the activation of both the p42/44 MAPK and protein kinase B and the increase in fibronectin levels. CTGF also induced mesangial cell migration via a ␤ 3 integrin-dependent mechanism that was similarly sensitive to the inhibition of the p42/44 MAPK and PI3K pathways, and it promoted the adhesion of the mesangial cells to type I collagen via up-regulation of ␣ 1 integrin. Transient actin cytoskeletal disassembly was observed following treatment with the ligand over the course of a 24-h period. CTGF induced the loss of focal adhesions from the mesangial cell as evidenced by the loss of punctate vinculin. However, these processes are p42/44 MAPK and PI3K pathway-independent. Our data support the hypothesis that CTGF mediates a number of its biological effects by the induction of signaling processes via ␤ 3 integrin. However, others such as actin cytoskeleton disassembly are modulated in a ␤ 3 integrin/MAPK/PI3K-independent manner, indicating that CTGF is a complex pleiotropic factor with the potential to amplify primary pathophysiological responses.

Significant caspase-8 activity has been found in normal and certain tumor cells, suggesting that caspase-8 possesses an alternative, nonapoptotic function that may contribute to tumor progression. In this article, we report that caspase-8 promotes cell motility. In particular, caspase-8 is required for the optimal activation of calpains, Rac, and lamellipodial assembly. This represents a novel nonapoptotic function of caspase-8 acting at the intersection of the caspase-8 and calpain proteolytic pathways to coordinate cell death versus cell motility signaling. (Cancer Res 2006; 66(8): 4273-8)

3-dimensional (3D) culture models have the potential to bridge the gap between monolayer cell culture and in vivo studies. To benefit anti-cancer drug discovery from 3D models, new techniques are needed that enable their use in high-throughput (HT) screening amenable formats. We have established miniaturized 3D culture methods robust enough for automated HT screens. We have applied these methods to evaluate the sensitivity of normal and tumorigenic breast epithelial cell lines against a panel of oncology drugs when cultured as monolayers (2D) and spheroids (3D). We have identified two classes of compounds that exhibit preferential cytotoxicity against cancer cells over normal cells when cultured as 3D spheroids: microtubule-targeting agents and epidermal growth factor receptor (EGFR) inhibitors. Further improving upon our 3D model, superior differentiation of EC50 values in the proof-of-concept screens was obtained by co-culturing the breast cancer cells with normal human fibroblasts and endothelial cells. Further, the selective sensitivity of the cancer cells towards chemotherapeutics was observed in 3D co-culture conditions, rather than as 2D co-culture monolayers, highlighting the importance of 3D cultures. Finally, we examined the putative mechanisms that drive the differing potency displayed by EGFR inhibitors. In summary, our studies establish robust 3D culture models of human cells for HT assessment of tumor cell-selective agents. This methodology is anticipated to provide a useful tool for the study of biological differences within 2D and 3D culture conditions in HT format, and an important platform for novel anti-cancer drug discovery.