Caspase-8 as a regulator of tumor cell motility

Abstract

The caspases are a family of ubiquitously expressed cysteine proteases best known for their roles in programmed cell death. However, caspases play a number of other roles in vertebrates. In the case of caspase-8, loss of expression is an embryonic lethal phenotype, and caspase-8 plays roles in suppressing cellular necrosis, promoting differentiation and immune signaling, regulating autophagy, and promoting cellular migration. Apoptosis and migration require localization of caspase-8 in the periphery of the cells, where caspase-8 acts as part of distinct biosensory complexes that either promote migration in appropriate cellular microenvironments, or cell death in inappropriate settings. In the cellular periphery, caspase-8 interacts with components of the focal adhesion complex in a tyrosine-kinase dependent manner, promoting both cell migration in vitro and metastasis in vivo. Mechanistically, caspase-8 interacts with components of both focal adhesions and early endosomes, enhancing focal adhesion turnover and promoting rapid integrin recycling to the cell surface. Clinically, this suggests that the expression of caspase-8 may not always be a positive prognostic sign, and that the role of caspase-8 in cancer progression is likely context-dependent.

Figure 1

Tyrosine residues in caspase-8 and caspase-10. The structure of the catalytic domain of procaspase-8 (pdb code: 2K7Z [46]) is shown in grey with tyrosine residues displayed in black (left). Sequence alignment of caspase-8 and caspase-10 catalytic domains with the tyrosines highlighted in black (right). The secondary structure elements as present in caspase-8 are displayed above the alignment.

Figure 2

Tyrosine residues in the procaspase-8 | FLIP heterodimer (pdb code: 3H11 [48]) are shown. The catalytic domains of procaspase-8 and FLIP are shown in light and dark grey, respectively, with the tyrosines indicated in black. Note that several tyrosine residues fall within the interface between the two proteins. Phosphorylation at any of these could be sufficient to disrupt heterodimer formation.

Figure 3

A representation of interacting SH2 domains and corresponding phosphorylated regions of caspase-8 using the Panomics^™ SH2 screen. The size of the SH2 domain name indicates the relative strength of interaction. Non-hits are listed outside of the ovals. Shown is a compilation of data from Senft et al. 2007 and from our lab. Senft and colleagues probed the Panomics^™ SH2 screen with a short peptide corresponding to the flexible loop linker surrounding the Y380 site on caspase-8, with a phospho-tyrosine substituted for the tyrosine on residue Y380. The data is complemented by probing the same Panomics^™ SH2 screen with the whole catalytic domain of caspase-8 phosphorylated by Src kinase in vitro as described in [11].

Figure 4

Interactions of caspase-8 with calpastatin. Calpastatin or constructs containing only calpastatin domains or the L domain plus a single calpastatin domain depicted in the upper panel were expressed in NB7C8 cells, immunoprecipitated with anti-Myc antibodies, and probed for the presence of coprecipitating caspase-8, as shown in the middle panel. Protein A/G beads and empty vector controls were used for specificity. Shown is an example experiment. The bottom pair of images shows confocal images of COS-7 cells spreading on fibronectin. The left panel shows staining for caspase-8, originally resolved in the 520nm channel, and the right panel shows staining for calpastatin resolved at 568nm. Arrows indicate regions of colocaliation in the cellular lamella/processes.

Figure 5

A working model for the mechanism of caspase-8 promoted migration. Phosphorylated procaspase-8 is recruited to the leading edge of migrating cells, where it is incorporated into an insoluble complex of focal adhesion proteins. From there, procaspase-8 promotes calpain activity, and turnover of focal adhesion components. Later, procaspase-8 promotes the internalization of integrins by enhancing Rab5-GTP loading, which enhances endocytosis but prevents hand-off to later endosomal compartments. This permits integrins to recycle back to nascent focal contacts to complete the “conveyor belt” of focal adhesion turnover required for mesenchymal-type cellular migration.