Caspase 8 association with the focal adhesion complex promotes tumor cell migration and metastasis

Abstract

Caspase 8 (Casp-8) is a proapoptotic protease which suppresses neuroblastoma metastasis by inducing programmed cell death. Paradoxically, Casp-8 can also promote cell migration among nonapoptotic cells, and here we show that Casp-8 can promote metastasis when apoptosis is compromised. Migration is enhanced by Casp-8 recruitment to the cellular migration machinery following integrin ligation. Casp-8 catalytic activity is not required for Casp-8-enhanced cell migration; rather, Casp-8 interacts with a multiprotein complex that can include focal adhesion kinase (FAK) and calpain 2 (CPN2), enhancing cleavage of focal adhesion substrates and cell migration. Casp-8 association with CPN2/calpastatin disrupts calpastatin-mediated inhibition of CPN2. In vivo, knockdown of either Casp-8 or CPN2 disrupts metastasis among apoptosis-resistant tumors. This unexpected molecular collaboration provides an explanation for the continued or elevated expression of Casp-8 observed in many tumors.

Introduction

Caspase 8 (Casp-8) is an apical protease and initiator of the extrinsic programmed death pathway. The Casp-8 zymogen is recruited to the death inducing signaling complex following ligation of death receptors, such as Fas, where it undergoes activation. The loss of Casp-8 has been associated with increased malignancy of neuroendocrine tumors, including neuroblastoma (1–5). We previously reported that Casp-8-expressing neuroblastoma exhibit increased dependence upon integrins and the extracellular matrix (ECM) relative to Casp-8-lacking counterparts (6). Failure to maintain adequate integrin-mediated ECM contacts promoted Casp-8-dependent, and death receptor-independent, apoptosis among invasive cells (7). This process, which we termed “Integrin-mediated Death”, acted to limit metastasis in vivo. In turn, the results from those studies have supported the implementation of new therapeutic regimens to Casp-8 expression within neuroblastoma tumor cells in vivo as a potential therapeutic approach.

However, it remains unclear whether upregulation of caspase 8 would be universally beneficial for preventing metastasis. It is notable that a significant fraction of aggressive stage IV neuroblastoma (10–30%) maintain Casp-8 expression, and that Casp-8 is not frequently inactivated among adult cancers, such as carcinoma (8). Inactivating mutations are surprisingly rare (8, 9), although it is important to note that such tumors, which develop over many decades, frequently have other lesions that interfere with the apoptotic cascade (10, 11). Since Casp-8 is an initiator caspase, downstream mutations common in some cancers could well promote cell survival irrespective of Casp-8 (12). Under such circumstances, Casp-8 may play alternative physiological roles within the cell. Casp-8 has been linked to proliferation (13–16), and to the migration in a number of primary and tumor cells (17–20). Such observations imply that clinical strategies to up-regulate Casp-8 might not be universally beneficial, and may even contribute to tumor aggressiveness. A particular concern is the possibility that it could promote tumor cell dissemination among apoptosis-resistant tumor cells.

Although unligated or antagonized integrins promote Casp-8 activation (7), ligated integrins suppress caspase 8 activation (21). Ligated integrins promote assembly of the focal adhesion complex, a signaling complex anchored by the actin cytoskeleton (22). The focal adhesion complex contains an interacting matrix of numerous proteins which includes nonreceptor tyrosine kinases, such as Src and focal adhesion kinase (FAK), adaptor and actin-binding proteins, including talin and paxillin, as well as cytosolic phosphatases and proteases (23, 24). In particular, the calpain proteases have been implicated in the cleavage of focal adhesion proteins that promotes focal adhesion turnover (25, 26). The high degree of complexity of focal adhesion reflects its physiological versatility in promoting signaling, survival, anchorage and migration. Here, we have explored the interaction between the focal adhesion complex and Casp-8 in migration and metastasis. Surprisingly, the “normally proapoptotic” enzyme Casp-8 is found to be incorporated into focal adhesions, and promotes not only cell migration, but also metastasis of apoptosis-resistant cells.

Materials and methods

Chemicals reagents, cDNA and vectors

Calpastatin Peptide (Cat 208902), Calpain Inhibitor II ALLM (Cat 208721), were purchased from Calbiochem; Leupeptin (Cat L8511), PMSF (Cat P-7626), Fibronectin from bovine plasma (Cat F1141) and Laminin (L2020) from Sigma ; Collagen Type I (Cat 08-115) from Upstate; Vitronectin generated from human placenta was the kind gift of Dr David Cheresh; Complete mini Protease inhibitor (Cat 11836153001) and Fugene Transfection Reagent (Cat 11814443001) from Roche Diagnostics. cDNA of human calpastatin cloned into pCMV.SPORT6 vector was from ATCC (Gene Bank ID: BC013579; Cat 10700497); pcDNA3.1 myc-His mammalian expression vector from Invitrogen (Cat V88-20); Caspase 8, Caspase 3 or Calpain 2 lentiviral ShRNAs from Open Biosystem; Rat Calpain-2 recombinant protein (208718) from Calbiochem; 7-amino-4-chloromethylcoumarin, t-BOC-L-leucyl-L-methionine amide (t-BOC-Leu-Met; Molecular Pro Cat A6520)

Antibodies

Anti-Talin N-Terminus (MAB 1676 clone TA205), anti-Calpain2 (AB1013), anti-Calpastatin (MAB3084) were purchased from Chemicon; anti-caspase 8 antibody from BD Bioscience (cat n 551242); Anti-Phospho-p44/42 MAP Kinase (Cat 9101) from Cell Signal; anti-P-FAK (P-Tyr 397; Cat .44–625G ) from BioSource International Antibody; anti-FAK (C-20; Cat sc-558) and anti-ERK2 (C-14 Cat sc-154) from Santa Crux Biotechnology; Anti-Actin (clone AC-15 Cat A5441) from Sigma; Anti-myc antibody (Cat. R950) from Invitrogen.

Cell lines

A549 lung carcinoma cells were acquired from ATCC. To create FAK-deficient and control cell lines, A549 cells were infected with a lentivirus encoding shRNA to FAK (Open Biosystems) or a control shRNA (Addgene, plasmid 1864). The neuroblastoma tumor lines NB7, NB5 and NB16 were established at St Jude Children's Hospital. Mouse embryo fibroblasts (MEF) from Src deficient mice (Src−/−) or FAK deficient mice (FAK−/−) mice were the kind gift of Dusco Ilic. FAK −/− fibroblasts reconstituted with GFP-FAK (FAK −/− + FAK*) cells were described previously (27). Cells or Cell lines were maintained in either DMEM (A549 and MEF cells) or RPMI medium 1640 (NB7, NB5 and NB16) supplemented with 10% FCS, 1% glutamine and minimal amino acids. Caspase-8-deficient NB7 neuroblastoma cells were reconstituted with Casp-8 by retroviral transduction with a caspase-8 expression construct as previously described (6). Knockdown of caspase 8 was accomplished by using adenovirus delivered caspase-8 ShRNA (6) or via stable lentiviral delivery of shRNA to caspase 8 as previously described (28) (Open biosystems). As a control ShRNA, cells were infected with a lentivirus encoding a nonspecific ShRNA sequence (plasmid 1864)(Addgene). Knockdown of caspase 3 was similalry performed using the lentivirus (Open biosystems). Knockdown of calpain 2 was performed by using lentivirus (Open Biosystems) in which the puro resistance cassette of the pLKO.1 vector was replaced with a neomycin-resistance cassette to permit double antibiotic selection. Calpastatin cDNA cloned in into pCMV.SPORT6 vector was amplified by polymerase chain reaction utilizing SP6 and 5’agtcatcttttggcttgg primers to remove the Stop codon. The PCR product was subcloned into the mammalian expression vector pcDNA3.1 myc-His using EcoRV restriction site, to allow expression of a C-terminal myc and polyhistidine-tagged protein. Plasmid was transfected into NB7 cells using the Fugene system (Roche) for 24h.

Immunoprecipitation and immunoblotting

Cells were lysed in either NP40 lysis buffer (150 mM NaCl, 50 mM Tris Base pH 7.4, 1% NP40) or RIPA buffer (50mM Tris pH 7.4, 100mM NaCl, 0.1 % SDS) supplemented with complete protease inhibitor mixture (Roche), 50 mM NaF and 1 mM Na₃VO₄ and centrifuged at 13,000g for 10 min at 4°C. Protein concentration was determined by Bicinchoninic Acid Assay (Pierce). For immunoprecipitation, 500µg of proteins were incubated with 2 µg of anti-Caspase-8 antibody (cat 551242, BD Bioscience) or anti-Calpain 2 (Cat AB1013, Chemicon) antibody overnight at 4°C. Complexes were precipitated with 30 µl of protein A/G (Pierce). Beads were washed five times with TBS 0.1% NP40, and bound proteins eluted in boiling Laemmli buffer, resolved on 10% SDS-PAGE and immunoblotted using specific antibodies for total FAK (anti-FAK C-20 from Santa Cruz Biotechnology) or Calpain (cat AB1013 from Chemicon) or caspase8 (cat 551242 from BD Bioscience). For the calpain-calpastatin interaction, NB7 cells were transfected with the calpastatin myc-His construct using Fugene System (Roche). Myc-his-calpastatin was immunoprecipitated with an anti-myc antibody, and immunoprecipitates were then incubated for 90 min at 37 °C with or without addition 100 ng or 1 µg of recombinant human caspase-8 catalytic domain (inactive C360A mutant). Lysates were washed once and assessed by SDS-PAGE and Western blotting with anti-calpain2 or anti-calpastatin antibodies

Adhesion-dependent signaling

Neuroblastoma cells were serum starved for 6 hours, placed in suspension for 15 min in ice (representing the zero time point) or plated into 100 mm non-tissue culture-treated dishes (1×10⁶ cells/100 mm dish) pre-coated with ECM proteins (2 µg/ml fibronectin or 2 µg/ml Vitronectin or 10 µg/ml Collagen). Cells were allowed to attach for the times as indicated. At each time point adherent cells were directly lysed in RIPA buffer as described above. Where indicated, cells were incubated in the presence of ALLM (50 µM), or calpastatin peptide (2 µM), before (20’) and during cell adhesion. PBS vehicle served as a control. The Adenovirus mediating delivering of Caspase-8 SiRNA, or an empty type 5 adenoviral vector (pAd Easy), was added to the cells 72 h before and during cell adhesion (6). Cells lysates were analyzed by western blot as above described for the specific antibodies.

Focal adhesion enrichment and isolation

The assay was performed after that described by Kaplan (29). Briefly, cells were plated onto fibronectin- coated plates as described for the cell signaling assay. At the times indicated, adherent cells were pre-extracted with 0.5% Triton X-100 lysis buffer for 0.5 hours; this fraction is clarified by centrifugation to remove nuclei, and is referred to as the cytosolic fraction (500g × 5 min). The focal adhesion enriched fraction was prepared from the remaining cell fraction bound (adhesion complex) on the plate, which was lysed in RIPA buffer for 5 min on ice, and scraped off the dish. This fraction is clarified by centrifugation (10 min ×14000g) to remove contaminating nuclear material. Lysates from the two different fractions were analyzed by western blot

Tumor growth and metastasis

5 × 10⁶ neuroblastoma cells suspended in 40 µl of complete medium were seeded on 11-day-old chick chorioallantoic membrane. Tumours were left to develop for 8 days and were then resected and weighed as previously determined (6). The metastasis assays were performed by seeding 7×10⁶ cells onto the surface of the chick chorioallantoic membrane, and assessing the presence of metastases in lungs and bone marrow by amplification of a human-specific Alu sequence, as previously reported (6)

Migration Assays

Cell migration was performed using a variant of the wounding as previously described (Barbero et al. 2008) or a transwell assay using modified Boyden chambers, 6.5 mm diameter, 8 µm pore size (Transwell from Costar Corp.) according to the protocol of the manufacturer. Briefly, the lower sides of the inserts were coated with fibronectin (0.2 µg/ml). Cells (5×10⁵) were plated in the upper chamber of transwell inserts and serum free medium was added to the bottom chamber. After 4 hours stationary cells were removed from the upper side of the membrane while migrated cells in the lower side of the inserts were stained with 0.1% crystal violet in 2% ethanol. Dye was eluted with methanol, and absorbance was measured at 600 nm.

Calpain activity assay in living cells

NB7 or NB7C8 cells were plated in 96 well-multi well plate precoated with fibronectin (2 µg/ml) at concentration of 25.000 cells/well for 10 min. Calpain activity was evaluated after the times as indicated by incubating the cells with the cell permeable calpain fluorogenic substrate t-BOC-Leu-Met (10 µM) and measuring the fluorescence with the TECAN Geniios Pro fluorometer at excitation and emission wavelength of 350 and 460 nm, respectively. As a control cells are incubated with the solubilizing vehicle (DMSO)

Immunofluorescence Studies

Cells were permitted to attach to cover slips coated with fibronectin (2 µg/ml) for 1 h, such that they were confluent. Cells monolayer was then wounded with a pipette tip and cells allowed to begin to migrate into the wound for 1 hour. Alternatively, cells were plated at subconfluence and allowed to migrate randomly. In either case, cells were fixed with 4% PFA for 10 minutes, permeabilized in PBS containing 0.1% Triton-X100 for 3 minutes, and blocked for 60 minutes, at room temperature with 2% BSA in PBS. Cells were then stained with monoclonal antibody to caspase 8 (BD Biosciences Cat 551242) (1:100) for 1 h. After washing several times in PBS/BSA, the cells were exposed to secondary antibody specific for mouse (1:300) (Alexa 488, green, or Alexa 565, red (Invitrogen). Samples were mounted in Vectashield hard set mounting media (Vector Laboratories) and imaged on a Nikon Eclipse C1 confocal microscope.

1D-gel-based ABPP of focal adhesion and cytosolic fractions

Standard conditions for ABPP reactions were as follows: cytosolic and focal adhesion proteomes were adjusted to a final protein concentration of 0.6 mg/ml in 25 µL of PBS pH 7.4) and treated with 1 µM AB19-bodipy-TMR (Caspase 8 probe) or 1µM DCG-04-bodipy-TMR (calpain probe) for 30 min at 37°C as described previously (30, 31). Reactions were quenched with one volume of standard 4x SDS-PAGE loading buffer (reducing), and separated by 1D SDS-PAGE gels (10% acrylamide). Fluorescently labeled proteins were visualized in-gel with a Hitachi FMBio IIe flatbed fluorescence scanner (MiraiBio)

Statistical analysis

Where pertinent, results were compared using unpaired t tests of at least 3 independent experiments or ANOVA as indicated. A p value <0.05 was considered significant. For the in vivo studies, statistical power was realized by evaluating cohorts including all animals from all experiments by Chi Square and Mann-Whitney statistical tests, as previously described (6).

Results

Casp-8 promotes metastasis among Casp-3-deficient cells

Casp-8 has been implicated in the suppression of neuroblastoma metastasis via the induction of apoptosis among invasive cells (6), consistent with loss of Casp-8 in the majority of aggressive NB (4). However, Casp-8 can promote cell migration via localization to the cell periphery and activation of small GTPases and calpain (17). These results suggest that Casp-8 may promote metastasis, particularly when apoptosis is compromised. To test this, we used an shRNA approach to suppress expression of caspase 3 (Casp-3), a critical downstream effector of Casp-8-mediated killing (32) and other forms of apoptosis (Supplementary Figure 1). Casp-3 expression was suppressed in NB7 neuroblastoma cells reconstituted with Casp-8 (NB7C8) and then tumor growth and metastasis assessed in the chorioallantoic membrane (CAM) model that previously established a metastasis-suppressor role for Casp-8 (6).

Casp-3 knockdown (Fig. 1A) did not significantly impact neuroblastoma proliferation in vitro (unpublished data) or tumor growth in vivo (Fig.1B). Loss of Casp-3 did not appreciably affect metastasis of NB7 neuroblastoma deficient for Casp-8 (Fig. 1C, compare the right open bar with left filled), but rescued metastasis among tumors expressing Casp-8 (+)(p<0.001, comparing center filled and open bars). Surprisingly, a disproportionate increase in metastasis was observed; the Casp8+Casp3- cells disseminated more efficiently than neuroblastoma lacking both caspases (p<0.05)(Fig. 1C). As expected, the Casp8+Casp3+ tumors exhibited the lowest overall incidence of metastasis (Fig. 1C, left open bar), confirming that Casp-8 blocks metastasis when an intact caspase cascade is present (6). Together, the results indicated that Casp-8, a putative metastasis suppressor, could act to promote tumor dissemination among populations of “apoptosis-compromised” cells.

Figure 1.

Impact of Caspase Expression on Neuroblastoma Progression. NB7 Neuroblastoma cells reconstituted for caspase 8 (Casp-8) and/or caspase 3 (Casp-3) were seeded in the chorioallantoic membrane (CAM) of chick embryos and tumors were allowed to develop for 8 days. (A) An immunoblot analysis of the expression of Casp-3 and Casp-8 and actin, as control of proteins loading is shown. (B) Tumors were resected from the CAM and the wet weight determined, with the mean and standard error shown. (C) The total cumulative incidence of detection of human Alu sequence from genomic DNA isolated from lung and bone marrow of the tumor-seeded chick cohorts. DNA isolated from lung and bone marrow was used as a template for PCRbased detection of human cells using primers specific for the human Alu sequence. The limit of sensitivity of the assay is ~200 tumor cells. The incidence with which human genomic DNA was detected in the cohort is shown, with statistical comparisons between cohorts representing three similar but separate experiments (N = 38, 40, 70 and 72) performed via Mann-Whitney test, (** < 0.001, * < 0.05).

Supporting this notion, Casp-8 promotes neuroblastoma migration under nonapoptotic condition in vitro (Fig. 2A) (18–20). Similarly, shRNA-based knockdown of Casp-8 transgene expression in the NB7C8 cells, or knockdown of endogenous Casp-8 expression in A549 carcinoma cells decreases cell migration relative to cells treated with control shRNA (Fig. 2B). The Casp-8 shRNA had no impact on Casp-8-deficient cells (Fig. 2B). Collectively, the results support a general role for Casp-8 in supporting cell migration, and accordingly we find that Casp-8 is enriched in the leading edge among randomly migrating cells (Fig. 2C)(20). These results were extended using confocal microscopy; Casp-8 was found to be enriched among cells entering a wound in both pseudopods (Fig. 2D, upper image) and lamella (Fig. 2D lower image).

Figure 2.

Caspase 8 influences cell migration. (A) NB7 Neuroblastoma cells deficient in caspase 8 (− Caspase 8) or reconstituted for caspase 8 expression (+ Caspase 8) were assessed for their ability to migrate on increasing concentrations of collagen substrate in a transwell Boyden chamber system, as described in the Material and Methods section. Migrated cells were fixed, stained with crystal violet and extracted dye quantitated (absorbance 600nm). Data shown are the means ± S.E. from six determinations in a representative assay. (B) NB7 cells expressing or lacking caspase 8, or A549 carcinoma cells expressing endogenous Casp-8, were infected with either control nonspecific shRNA lentivirus or lentivirus encoding shRNA to Casp-8 to knock down caspase 8 (immunoblots shown). Cells were evaluated for their capacity to migrate using a wound assay, and alterations in cell migration plotted. NB7 did not significantly vary in migration when treated with the C8-shRNA, while NB7-C8 and A549 exhibited decreased wound closure (p<0.05 and p<0.01, by t-test, respectively). (C) We evaluated the localization of caspase 8 among A549 cells migrating randomly on a cover slip; cells were fixed, permeabilized and caspase 8 localization determined via immunofluorescence using a monoclonal antibody to caspase 8 (Green channel). Note the localization at the peripheral ruffles of the moving cells (arrows). (D) Confocal microscopy assessment of caspase 8 among migrating cells. NB7C8 cells seeded on fibronectin coated coverslips (2µg/ml) were wounded and allowed to migrate into the wound for 4h. Confocal thin sections were captured at the cell:slide interface to assess caspase 8 localization at the front of migrating cells. (Bar = 10µM.)

Casp8-dependent alterations are observed in talin following substrate adhesion

Casp-8 localizes to different cellular compartments, and may be targeted based on post-translational modification (9, 33, 34). Casp-8 can colocalize with integrins (7), and can be activated (promoting apoptosis) when integrins are antagonized or unligated (6, 7). Since integrin-mediated adhesion protects cells form Casp-8-mediated apoptosis (21), and Casp-8 is phosphorylated following attachment to fibronectin (20), we speculated that Casp-8 might also associate with nascent focal adhesions thereby influencing cell migration. Therefore, NB7 cells deficient or reconstituted for Casp-8 were replated onto fibronectin substrate to activate integrin signaling, and the activation of integrin downstream signaling molecules was evaluated as a function of time by immunoblot analysis. However, we detected no differences in the activation of the integrin-proximal nonreceptor tyrosine kinase FAK or in the activation of the downstream target ERK (Fig. 3A).

Figure 3.

Impact of caspase 8 on integrin signaling. (A) NB7 cells lacking (− Caspase 8) or reconstituted for caspase 8 (+ Caspase 8) were plated on fibronectin-substrate (2µg/ml) for times as indicated, lysed in RIPA, and the expression of FAK, ERK and phosphorylated forms of these proteins evaluated by immunoblotting (upper panel). Immunoblotting was similarly performed to assess the presence of talin as well as the amino-terminal FERM cleavage product (lower panel). (B) Immunoblot analysis of talin was performed among NB7 caspase 8-reconstituted and then shRNA knock down cells, as described above. (C) Focal adhesion fractions were purified from NB7 neuroblastoma cells lacking or expressing Casp-8. Cells were allowed to attach for 30 or 60 minutes, and then 25µg of protein subjected to immunoblot analysis for the presence of FAK and pFAK, ERK and pERK, and Talin.

In contrast, differences were evident in the appearance of the integrin-associated cytoskeletal protein talin during substrate adhesion to fibronectin (Fig. 3A) or collagen or vitronectin substrates (Supplementary Fig. 2a) in the Casp-8 expressing or deficient NB7 cells. In particular, Casp-8-expressing cells showed enhanced production of the N-terminal talin fragment (35) during substrate attachment. This fragment contains the integrin-binding region of talin known as the FERM domain. Binding of the FERM domain to integrins enhances their binding to ligand, thus influencing cell migration (25, 36–38). Accordingly, knockdown of Casp-8 expression via shRNA blocked the production of the FERM domain fragment following substrate adhesion (Fig. 3B, Supplementary Fig. 2B), and inhibited cell migration (see Fig. 2B). Together, the results implicate Casp-8 as a talin-dependent regulator of cell motility. Nonetheless, Casp-8 did not appear to cleave talin directly, as we were unable to demonstrate cleavage of talin immunoprecipitates by recombinant Casp-8 in vitro (data not shown). This was not completely surprising since Casp-8 activity is influenced by steric or allosteric factors as well as post-translational modifications (18, 20, 39–41). For example, Src-mediated phosphorylation inhibits Casp-8 activation (41). Moreover, these results were consistent with our prior observations that a proteolysis-deficient mutant of Caspase 8, in which the tyrosine in position 360 has been substituted to alanine (Caspase 8 C360A), promotes Casp-8 targeting to the cell periphery and migration (20).

Talin is an integral member of the focal adhesion complex assembled following integrin ligation, and competition for available talin within a cell limits integrin activity (38). When focal adhesion(FA)-containing fractions were purified (29) from cells expressing or lacking Casp-8, we found no obvious differences in FA-associated talin holoprotein, but selectively observed accumulation of the FERM domain fragment specifically within FA of Casp-8-expressing cells (Fig. 3C). The Casp-8 zymogen was also observed (~56kDa), while FAK and ERK accumulated independent of Casp-8 expression (Fig. 3C).

Casp-8 promotes calpain activity within focal adhesions

To identify the protease responsible for mediating talin cleavage, we used an activity-based profiling approach, probing for the activity of caspases and calpains (42). The caspase-selective probes used (such as Casp-8-selective probe AB19-BTMX) detected no signal in either the cytosolic or focal adhesion cell fractions (Supplementary Fig. 3). However, the activity-based probe DCG-04, a calpain selective probe, was incorporated strongly within FA fractions of NB7 cells reconstituted for Casp-8 expression, identifying a putative protease of ~72 kDa (Fig. 4A, arrow). Similar results were obtained in NB5 neuroblastoma cells expressing endogenous Casp-8, and the 72kDa signal was eliminated by knockdown of Casp-8 expression (Supplementary Fig. 3). These results are in agreement with reports that (a) calpain 2 (CPN2)-mediated talin cleavage regulates focal complex turnover and cell migration (35), and (b) that calpains regulate Casp-8-mediated motility (19). Supporting the concept that CPN2 activity was elevated in FAs, we observed cleavage of calpain substrates such as α-II spectrin, and an auto-processed form of CPN2 selectively in FAs (open arrowhead), but not in the cytosolic fraction (Fig. 4B). Fluorometric substrate-cleavage assay using live cells showed increased calpain activity selectively among NB7C8 cells during substrate attachment (Fig. 4C). The peptidyl protease inhibitor, ALLM, can block cell migration via inhibition of CPN2 (43) (and data not shown). In agreement with the notion that talin cleavage facilitates migration, we find that ALLM treatment also suppressed substrate attachment-induced cleavage of talin (Fig. 4D). Similar results were seen with calpeptin, another peptidyl inhibitor of calpain (derived from the endogenous calpain inhibitor, calpastatin) (Supplementary Fig. 4A). These results support the proposed role of calpain as an effector of Casp-8-mediated motility (19), and extend these results by localizing the activity to nascent focal adhesion/cytoskeletal complexes initiated by integrin-substrate ligation.

Figure 4.

Calpain is activated selectively in caspase 8 expressing cells. (A) Isolated focal adhesion fractions (FA) or cytosolic fractions (Cyt) were incubated with the cysteine protease probe DCG-04 which reacts with active calpain, and 20µg of each lysate was then resolved by electrophoresis and DCG-04 reactive proteins resolve. (B) The focal adhesion (FA) and cytoslic (Cyt) fractions were assessed to detect calpain substrate cleavage. Calpain substrates tested included calpain 2 (to evaluate production of the autocatalytic fragment) and the calpain 2 substrate α-II spectrin. (C) NB7 cells lacking (− Caspase 8; open) or reconstituted for caspase 8 (+ Caspase 8; filled) were plated on fibronectin (2µg/ml) in the presence of a calpain-activated fluorescent substrate (t-BOCLeu- Me), and fluorescence recorded as a function of time. Calpain activity induced by attachment from a representative experiment is shown. (D) Immunoblot analysis of the effect of ALLM, an inhibitor of calpain 2, on talin cleavage induced by substrate adhesion was performed as described in Fig. 3 above.

Casp-8 associates with CPN2 and FAK

The focal adhesion targeting of CPN2 has been proposed to occur via a scaffolding function of focal adhesion kinase (FAK), with CPN2 binding near the FAK-Y397 phosphorylation site (24). Since Casp-8 associates with FAK-associated SH2-containing proteins such as Src and p85α (18, 20), we tested whether Casp-8 and CPN2 might be present within the same molecular complex. NB7 cells reconstituted for caspase 8 expression were kept in suspension or attached to fibronectin (2 µg/ml) for 30 min and cell lysates were immunoprecipitated with anti-casp 8 antibody (cat 551242 from BD Bioscience) and subjected to immunoblot analysis for total FAK (anti-FAK C-20 from Santa Cruz Biotechnology) or Calpain (cat AB1013 from Chemicon) or caspase8 (cat 551242 from BD Bioscience).

Co-precipitation analysis revealed that CPN2 and Casp-8 associated with each other and with FAK selectively following substrate adhesion, but not among suspended cells (Fig. 5A). The induced association following substrate adhesion suggested a role for the focal adhesion complex in assembling the Casp-8/CPN2 containing complex. Surprisingly, however, we found that Casp-8 and CPN2 could associate in FAK−/− cells, suggesting FAK was not essential (Fig. 5B). Similarly, the kinase c-Src, which associates with both Casp-8 and FAK, was not necessary for Casp-8/CPN2 association or the formation of the FAK/Casp-8/CPN2 complex (Fig. 5B). However, FAK was important for Casp-8 distribution, since MEF cells lacking FAK had disrupted localization of Casp-8 in the periphery (Supplementary Fig. 5A) while reconstituted cells exhibited normal peripheral localization of Casp-8. Similarly, we assessed the distribution of caspase 8 among A549 cells in which FAK has been knocked down (~80–90%)(Insert, Fig. 5C). Among similarly spread cells (Supplementary Figure 5B), we found that the distribution of caspase-8 in the membrane ruffles was compromised in FAK knockdown cells relative to control A549 (Fig 5C), and this was not simply time-dependent, since FAK−/− cells do not show enhanced peripheral casp-8 localization at later time points (data not shown, and Supplementary Fig. 5B). Thus, FAK appears to play a role in localizing Casp-8 to the periphery among spreading cells.

Figure 5.

Caspase 8 is targeted to the periphery in a FAK-dependent manner.

(A) NB7 cells reconstituted for Casp-8 in suspension or those allowed to attach to FN (2µg/ml) for thirty minutes were lysed and subjected to immunoprecipitation of caspase 8. Whole cell lysate and nonspecific beads + antibody control was performed in parallel, and all samples were subjected to immunoblot analysis for total FAK, caspase 8 (Casp-8) and calpain 2 (CPN2).

(B) Mouse embryo fibroblasts from Src deficient mice (Src −/−), FAK deficient mice (FAK −/−), or FAK −/− fibroblasts reconstituted with GFP-FAK (FAK −/− + FAK*) were allowed to attach to fibronectin substrate, lysed, subjected to Casp-8 immunoprecipitation. Immunoprecipitates were analyzed for the presence of total FAK, caspase 8 (Casp-8) and calpain 2 (CPN2) (tagged FAK is resolved as a higher MW species) (C) A549 cells were transduced with lentivirus encoding a scrambled shRNA (control) or a FAK shRNA (FAK) to knock down FAK (left panel, immunoblot inset). Cells were allowed to attach and spread on fibronectin-coated (2µg/ml) surfaces, and stained for actin (Blue channel, arrows indicate actin ruffles) and caspase 8 (Red channel, to show caspase 8 distribution) and image by confocal microscopy (Bar = 10µm). Spreading cells were scored for the presence of enriched caspase 8 by blinded observers (150 cells/group, left panel, p <0.012). (D) NB7 expressing (+ Caspase 8) or lacking (− caspase 8) caspase 8 were allowed to attach, lysed and calpain 2 complexes immunoprecipitated. The complexes were resolved by PAGE and assessed for the presence of calpastatin and caspase 8. (E) His-Calpastatin was over-expressed in NB7, then pull-downs performed with Nickel NTA resin. Lysates were incubated in the presence of GST (con) or with 100ng or 1ug of the catalytic domain of caspase 8 (catalytically inactive C360A mutant), washed and resolved. The presence of calpastatin and calpain 2 was determined by immunoblotting.

We next examined how Casp-8 association with CPN2 or with focal adhesions might influence calpain activity. The principle regulator of calpain activity in living cells is calpastatin (37). Interestingly, calpastatin cleavage was enhanced in the FA fraction of NB7C8 cells (Supplementary Fig. 4B). Although Casp-8 did not cleave calpastatin in vitro (data not shown), active calpains can cleave calpastatin, and the observed products were consistent with those previously described for calpain cleavage. Calpastatin binds calpain via its amino terminal domain (44) and via three distinct conserved peptide sequences within its “calpastatin repeats”, each of which is required for effective inhibition of the enzyme (45). Physiologically, the activation of calpain requires displacement of calpastatin and association with targeting or anchoring proteins (46). Therefore, CPN2 or calpastatin binding to Casp-8 might act to disrupt the calpastatin-CPN2 interaction.

To test this possibility, we examined immunoprecipitates of calpain from cells expressing or lacking Casp-8. Calpastatin was readily detected co-precipitating with CPN2 in lysates from cells lacking Casp-8, but was nearly absent in lysates derived from cells expressing Casp-8 (Fig. 5D). This suggested that Casp-8 prevented formation of a CPN2-calpastatin complex. To determine if this was a direct effect, we then added back recombinant Casp-8 (C360A mutant, inactive) to the precipitated calpastatin complexes. The addition of recombinant Casp-8 disrupted the pre-existing calpain-calpastatin complex (Fig. 5D, right two lanes), indicating that Casp-8 antagonizes calpastatin:CPN2 interaction, and further suggesting that Casp-8-enhanced migration and metastasis was effected by CPN2.

CPN2 is crucial for promotion of migration and metastasis by Casp-8

To address this, we first knocked down the expression of CPN2 (via shRNA) in Casp-8-deficient or expressing NB7 cells already bearing a C3-knockdown (creating a double knockdown phenotype)(Fig. 6A). Assessing these cells, we found that the Casp8+Casp3-CPN2- cells exhibited decreased talin cleavage following substrate attachment, similar to Casp-8-deficient cells, while Casp8+Casp3- cells expressing a control shRNA exhibited talin cleavage following substrate attachment (Fig. 6B). To determine whether there was a selective impact on migration, we next assessed migration in vitro among the CPN2 knockdown cells (Fig 6B, left panel). Interestingly, the knockdown of CPN2 had a greater effect on the migration on fibronectin substrate of Casp-8 expressing cells relative to Casp-8 deficient cells. This suggested that knockdown of CPN2 might also decrease tumor metastasis of Casp-8-expressing cells in vivo. Evaluating this possibility, we found that suppression of CPN2 decreased the incidence of metastasis selectively among Casp-8-expressing cells (Fig. 6C, left panel). Together, these results extend prior suggestions that Casp-8-induced migration was dependent upon CPN2 (19), and demonstrate an important synergy with Casp-8 in metastasis in vivo among apoptosis-resistant tumors.

Figure 6.

Calpain 2 knockdown compromises talin cleavage, migration and metastasis.

(A) Calpain 2 was knocked down in the paired C3-silenced NB7 cells expressing (+ Caspase 8) or deficient (− Caspase 8) for caspase 8. (B) The calpain 2 knockdown cells were allowed to attach to fibronectin substrate and talin cleavage assessed by immunoblotting, as described above. (C) The change in wound closure after 24 hours of migration following calpain knockdown among NB7 cells expressing (+ Caspase 8) or deficient (− Caspase 8) for caspase 8 was assessed via Boyden chamber assay for their ability to migrate on fibronectin substrate (2 µg/ml) (left panel). Data shown are the means ± S.E. from six determinations in a representative assay (*p<0.05). Metastasis assays were performed in the chick CAM, as described above, and the relative change in metastasis incidence plotted. Data shown is cumulative and represents the absolute incidence of metastsis for all animals used in the study;(+ Caspase 8, N = 54) (− Caspase 8, N=36) (p<0.05 for inhibition among Casp-8-expressing cells, Mann-Whitney test).

Discussion

Tumors can become apoptosis resistant via many mechanisms, including the expression of mitochondrial gatekeeper proteins of the bcl-2 family, the over-expression of IAPs (inhibitors of apoptosis) or lost expression of caspases, such as caspase 8, 9 or 3. Here, we examined the metastasis of neuroblastoma cells in which we compromised Casp-8–mediated killing by silencing the expression of the downstream effector Casp-3. The studies demonstrated that disruption of Casp-3 in the apoptotic cascade could not only relieve the metastastasis-suppressing activity of Casp-8, but they further revealed an unexpected metastasis-enhancing property cue to Casp-8-expression. Examining the mechanism by which this occurred, we found that Casp-8 promoted cell migration independent of its proteolytic activity, via recruitment to a complex that contained focal adhesion kinase, and calpain 2 (CPN2). Casp-8 disrupted the interaction of calpastatin with calpain, and permitted activation of CPN2. In turn, this promoted CPN2 cleavage of focal adhesion substrates, and subsequent cell migration (43). Accordingly, knockdown of CPN2 inhibited Casp-8-initiated metastasis. Our results demonstrate that the recruitment of Casp-8 to the focal complex regulates both cell migration and calpain activity (19). The capacity of Casp-8 to increase migration and metastasis may be clinically relevant; these nonapoptotic roles of Casp-8 suggest caution be used in strategies which seek to amplify Casp-8 expression.

The lack of apoptosis induced enriched peripheral Casp-8 may be due to allosteric limitations present within the tightly packed focal adhesion complex, or may result from posttranslational modifications such as phosphorylation of Casp-8 in Y380 by Src (41). Indeed, these events may not be easily dissociable, since recruitment of Casp-8 to the periphery of cells attaching to substrate is abnormal in the absence of FAK (Figure 5). Together with previous studies, our results suggest that ECM adhesion may trigger post-translational modification of Casp-8, permitting Casp-8 to play a nonapoptotic role as a promoter of cell migration. This is of particular interest, since SAGE analysis suggests increased caspase 8 expression may occur in Bladder, Liver, Ovarian, Pancreatic, Prostate and (non SCLC) Lung cancers (47).

The capacity for Casp-8 to interact with a cytoskeletal complex and influence cell behavior may be noteworthy with respect to prior studies. Many “nonapoptotic” cellular processes that have been found to be disrupted in Casp-8-deficient animals, such as T cell activation (48, 49) have well-documented requirements for talin and integrin (50). Other “nonapoptotic” Casp-8 activities, such as activation of NF-κb (51) or the small GTPase Rac (19), similarly link Casp-8 signaling to integrins and the cytoskeleton. Further, cell adhesion and cytoskeletal rearrangements are linked with resistance to apoptosis (34, 52, 53). While resistance can be related to transcriptional events and downstream modulation of apoptosis-regulating proteins (at least in some cases), our results provide a basis for exploring how early signaling events elicited by integrin-ligand interactions can directly contribute to the regulation of caspase cascade initiation. The apparent linkage between apoptosis and cellular cytoskeletal dynamics appears to be physiologically convenient; integrins act as biosensors that physically interrogate the local microenvironment, and thus are well-poised to help guide cell fate decisions.

It is also important to consider that caspases represent clinically relevant targets. While current strategies are focused on stimulating or inhibiting the caspase catalytic activity, the potential for noncatalytic function is likely to be important in future therapeutic considerations. Our results would strongly suggest that retention of Casp-8 may be “contextually” advantageous to a tumor cell, particularly those bearing downstream disruptions within the programmed cell death pathway. With respect to this, it is possible that current clinical trials which seek to up-regulate Casp-8 expression might, under some circumstances, exacerbate disease and promote metastasis. In addition to placing patients at risk, this could act to mask efficacy within statistical cohorts. However, an increased understanding of the molecular mechanisms involved in regulating this process would be predicted to provide new targets for use in personalized, and combinatorial, therapeutic approaches.

Abbreviations

ALLM

Acetyl-L-Leucyl-L-Leucyl-L-Methionyl

Casp-8

Caspase 8

CAM

Chorioallaontoic membrane

CPN2

Calpain 2

Cyt

Cytosol

FAK

Focal Adhesion Kinase

FA

Focal Adhesion; 7-amino-4-chloromethylcoumarin t-BOC-L-leucyl-L-methionine amide, t-BOC-Leu-Me