Caspase-8: Fly Or Die Steven M

Review

Caspase-8: Fly or Die Steven M. Frisch

West Virginia University, Mary Babb Randolph Cancer Center, Morgantown, West Virginia

Abstract Caspase-8 Functions Upstream of Calpain-2 Recent studies have revealed that procaspase-8 has an Initial findings documented the cell motility/migration and important function in cell adhesion and motility. Src cell-matrix adhesion effects of caspase-8 in knockout mouse phosphorylation controls this function by preventing the embryo fibroblasts, the caspase-8–null neuroblastoma cell line conversion of procaspase-8, which is an adhesion/migration NB7, and other cell lines in which caspase-8 was inactivated by factor, to mature caspase-8, which is an apoptosis-inducing crmA protein (6). Interestingly, although cell motility and adhesion factor. This provides a mechanism to switch these opposing were found to be stimulated by caspase-8 in these cell lines, a functions. In its migratory role, procaspase-8 interacts with paradoxical effect—inhibition of cell adhesion in normal (i.e., the phosphatidylinositol-3-OH kinase regulatory subunit p85A nontransformed) epithelial cell lines—was noted, indicating that and c-src to modulate signaling by Rac and extracellular the effect of caspase-8 may differ in epithelial versus fibroblastic or signal-regulated kinase, and promote calpain activation. tumor cells. Here, I survey the findings of these studies and discuss A potential role for caspase-8 in regulating calpain activation potential mechanisms and ramifications for cancer prognosis was indicated (6). Calpain activation by the activated epidermal and therapy. [Cancer Res 2008;68(12):4491–3] growth factor (EGF) receptor is important in most or all aspects of cell migration: lamellipodial extension, rac activation, trailing edge A Cell Migration/Adhesion Function for Caspase-8 detachment, and focal adhesion turnover, as well as other aspects of cell behavior such as cell-matrix adhesion and high fidelity of Caspase-8 is well-characterized as an initiator of death receptor– cytokinesis, that is, suppression of multinucleate cell formation mediated apoptosis and has been implicated in other apoptotic (10, 11). The caspase-8-dependence of calpain activation was responses as well. As with other caspases, it has long been intuited indeed reflected in an effect of caspase-8 on those four of the six that caspase-8 may not have evolved solely to kill cells (1). In calpain-dependent processes listed above that were tested; the particular, the decision of a cell to undergo apoptosis versus cell aberrant protrusions found in spreading caspase-8–null cells migration might, in principle, be controlled by a molecular toggle, remarkably resembled those of calpain-2–null cells (12). due to the mutual exclusivity of these phenomena, i.e., migration How could caspase-8 regulate calpains? Calpain-2, an important involves transient cytoskeletal changes that might otherwise isoform for cell migration, is regulated in part by extracellular engage an anoikis response, and, conversely, suppressing the signal-regulated kinase (ERK)-mediated phosphorylation on migration of apoptotic cells may prove physiologically useful. serine-50 (13). In this connection, a contribution of caspase-8 Several recent observations have provided clues that caspase- to EGF-stimulated ERK activation was observed (5, 8). This 8 may possess a ‘‘moonlighting function’’ related to this molecular phenomenon—which is consistent with the stimulation of ERK toggle, albeit hypothetical. Caspase-8 knockout mice die during by TNFRI and FAS (14, 15)—suggests one potential mechanism early embryogenesis, and tissue-specific knockouts have develop- for caspase-8 to regulate calpain-2 as well as other migration/ mental defects that cannot be explained by deficits in apoptosis (2). adhesion pathways that are ERK dependent. It is not yet clear how Transcription through the nuclear factor-nB pathway and prolif- caspase-8 regulates ERK activation. One report attributed this eration of T cells is also downstream of caspase-8 (3). Moreover, regulation, surprisingly, to the Death Effector Domains (DED; i.e., although caspase-8 expression is compromised in a restricted prodomain) of caspase-8 (8). Consistent with this idea, a direct subset of human tumors, it is maintained or up-regulated in many association between ERK and a DED-containing fragment of common tumor types, consistent with a nonapoptotic, possibly caspase-8, and cotransport of an ERK-caspase-8-DED complex to prometastatic function (4, 5). the nucleus during apoptosis has been reported (16). Although Stimulated by these cues, caspase-8 was found to promote cell a corresponding interaction between the DEDs of (full length) migration and cell-matrix adhesion (6). These findings were caspase-8 and ERK in nonapoptotic cells is hypothetically pos- confirmed and extended by additional studies (5, 7, 8), which sible, we have neither observed this interaction nor evidence of defined a new function for caspase-8 that could explain its caspase-8 fragmentation in nonapoptotic cells.1 An alternative developmental effects as well as the observation that activated FAS possibility is that the DEDs localize caspase-8 such that it acti- promotes invasion (9). Thus, incorporating the new findings, the vates kinases upstream of ERK, using either a binding/confor- function of caspase-8 can be conceptualized as a unique ‘‘fly or die’’ mational switch mechanism (favored in one cell system where switch. Here, I summarize and critically evaluate the mechanisms the catalytic activity was dispensable; refs. 5, 8) or through the that have been proposed by the recent studies (5–8). proteolytic activation of kinases by caspase-8 (in other cell contexts) or calpains.

Requests for reprints: Steven M. Frisch, West Virginia University, Mary Babb Randolph Cancer Center, 1 Medical Center Drive, Campus Box 9300 Morgantown, WV 26506. Phone: 304-293-2980; E-mail: [email protected]. I2008 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-08-0952 1 S.M. Frisch, unpublished data. www.aacrjournals.org 4491 Cancer Res 2008; 68: (12).June 15, 2008

Interactions of Caspase-8 with p85A and c-src: and suppressing FASL-induced apoptosis (19). The activity of c-src Mechanisms and Ramifications is frequently elevated in cancer cells, and is activated transiently by motility factors such as EGF (20); consistent with this, increased A second, nonmutually exclusive mechanism for cell migration/ caspase-8 tyrosine-380 phosphorylation was detected in cancer adhesion effects of caspase-8 involving novel caspase-8 protein cells with hyperactive src (19). These observations provide a novel interactions was posited. Caspase-8 exists as a f55 kDa precursor, mechanism by which c-src protects cells against apoptosis, by procaspase-8, which is activated by FADD or c-FLIP–mediated maintaining caspase-8 in the proform. dimerization (17). During an apoptotic response, dimeric caspase- Interestingly, the phosphorylation acceptor site, tyrosine-380, was 8 cleaves itself at three sites, first at D374 and D384, producing an required for cell migration and adhesion, elevating procaspase-8 enzymatically active, DISC-bound species, then at D216, releasing from a mere protease precursor to a phosphorylation-regulated the mature caspase-8 tetramer into the cytoplasm (Fig. 1). An signaling switch for fly or die (5, 7). uncleavable mutant form of caspase-8 (D374A/D384A) that These groups then pursued the function of tyrosine-380, constitutively remains in the procaspase-8 form—the form that is generating different but nonmutually exclusive results. The first mainly or solely detected in nonapoptotic cells—cannot support group showed that phosphorylated caspase-8 interacts with the FASL-induced apoptosis (18). Might procaspase-8 perform the p85a regulatory subunit of phosphatidylinositol-3-kinase, requiring nonapoptotic functions of caspase-8? tyrosine-380 and phosphorylation by c-src or c-fyn (5). They An uncleavable mutant of caspase-8 restored adhesion/motility hypothesized that this interaction might affect Rac activation as efficiently as wild-type caspase-8 (5) and these activities did not through a direct or indirect mechanism, perhaps involving the require the active-site cysteine (5, 8). These observations reveal the p85-Rac complex reported earlier (21). The second group showed first biological functions of procaspase-8, other than serving as a that c-src is in a complex with phosphorylated (but not precursor for the mature enzyme. How procaspase-8 might achieve unphosphorylated mutant) caspase-8, and postulated that this this activity was postulated as follows. complex is important for cell motility (7). This is plausible in that it Recently, c-src was reported to phosphorylate caspase-8 at is not unusual for src substrates to complex stably with c-src (20). tyrosine-380 (Fig. 1), blocking caspase-8 autocleavage at D374/D384 Phosphorylated caspase-8—but not a Y380F mutant—localized to

Figure 1. Model: Src phosphorylation switches caspase-8 function. Under apoptotic stimulation, procaspase-8 undergoes autocatalytic cleavage to generatethe proapoptotic mature tetramer. However, upon stimulation with motility factors such as EGF, tyrosine kinases including c-src phosphorylate caspase-8, preventing its autocatalysis and enabling an interaction with p85a. This interaction, as well as potential (direct or indirect) interactions with c-src (dotted lines), stimulates cell migration and adhesion through molecules including Rac, calpain-2, and ERK.

Cancer Res 2008; 68: (12).June 15, 2008 4492 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research. Caspase-8/Migration/Adhesion membrane ruffles associated with early cell attachment. Interest- Summary and Future Perspectives a ingly, src interacts directly with p85 (through an SH3-polyproline In summary, the four recent papers on cell migration/adhesion binding event), raising the possibility of either caspase-8-p85-src of procaspase-8 indicate that the processing of caspase-8, which is and/or caspase-8-src-p85 ternary complexes (22). In this connec- a controlled in part by tyrosine phosphorylation, is an important tion, a c-yes-mediated recruitment of p85 to CD95 that stimulates migration/adhesion versus apoptosis switch mechanism. Elucida- glioblastoma invasion has been reported recently (23). tion of the regulatory mechanisms involved will undoubtedly yield The third group reported no role for tyrosine-380 in cell new connections and new surprises. From a clinical point of view, adhesion (its role in cell migration was not addressed) and, as it may prove useful to characterize the expression and phosphor- mentioned above, attributed cell adhesion, ERK activation, and src ylation state of caspase-8 in tumors, to assess the feasibility of interaction to the DEDs (8). Although seemingly contradictory, using this protein as a prognostic marker or to pharmacologically these negative results (with regard to the cell adhesion effect of stimulate caspase-8 processing. tyrosine-380) arose from a conventional adhesion assay, which presumably reflects integrin expression and affinity, whereas the Disclosure of Potential Conflicts of Interest positive data arose from Electrical Cell-Substratum Impedance Sensing, which measures adhesion and cell spreading. Thus, these No potential conflicts of interest were disclosed. observations are not incompatible, in that different end points Acknowledgments were measured, although the same term was used to describe them. The attribution of caspase-8-src interaction and ERK acti- Received 3/12/2008; accepted 3/18/2008. Grant support: NIH grant CA123359 (S.M. Frisch) and a core administrative vation to the DEDs in one study (8), but tyrosine-380 in the other component of a COBRE grant (to D. Flynn). I thank Brooke Helfer, YoungJin Cho, (7) might well reflect the contributions of both elements. and George Prendergast for critical reading of the manuscript.

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Steven M. Frisch

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