TRAIL-death receptor and apoptosis are selectively regulated by -1 activation

Carlos R. Reisa, Ping-Hung Chena, Nawal Bendrisa, and Sandra L. Schmida,1

aDepartment of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390

Edited by Ira Mellman, Genentech, Inc., South San Francisco, CA, and approved December 5, 2016 (received for review September 8, 2016) Clathrin-mediated endocytosis (CME) constitutes the major path- high curvature-generating properties when compared with Dyn2, way for uptake of signaling receptors into eukaryotic cells. As making it more suited for rapid compensatory endocytosis (13). such, CME regulates signaling from cell-surface receptors, but Hence, at the synapse, Dyn1 is well-suited to mediate rapid whether and how specific signaling receptors reciprocally regulate compensatory endocytosis and recycling following the CME machinery remains an open question. Although best neurotransmission (14). Unexpectedly, we have recently shown studied for its role in membrane fission, the GTPase dynamin also that Dyn1 can also be activated in nonneuronal cells, downstream regulates early stages of CME. We recently reported that dynamin-1 of an Akt/GSK3β signaling cascade to alter the rate and regulation (Dyn1), previously assumed to be neuron-specific, can be selectively of CME (15), linking endocytosis to signaling. activated in cancer cells to alter endocytic trafficking. Here we Activation of the apoptosis-signaling machinery by TRAIL report that dynamin isoforms differentially regulate the endocyto- (TNF-related apoptosis-inducing ligand) through the engage- sis and apoptotic signaling downstream of TNF-related apoptosis- inducing ligand–death receptor (TRAIL–DR) complexes in several ment of death receptors (DRs) has gained considerable interest – cancer cells. Whereas the CME of constitutively internalized trans- as a potential anticancer strategy (16 19); however, its efficacy is ferrin receptors is mainly dependent on the ubiquitously expressed limited by a variety of cancer cell-resistance mechanisms. TRAIL Dyn2, TRAIL-induced DR endocytosis is selectively regulated by ac- binding to its cognate death receptors (DR4 and DR5) triggers tivation of Dyn1. We show that TRAIL stimulation activates ryano- the formation of the death-inducing signaling complex by the dine receptor-mediated calcium release from endoplasmic reticulum recruitment of the adaptor FADD (Fas-associated death domain) CELL BIOLOGY stores, leading to calcineurin-mediated dephosphorylation and ac- and the initiator caspase-8 (20). Activated caspase-8 can then ac- tivation of Dyn1, TRAIL–DR endocytosis, and increased resistance to tivate effector caspase-3 and -7, leading to cell-extrinsic apoptosis. TRAIL-induced apoptosis. TRAIL–DR-mediated ryanodine receptor Binding of human recombinant TRAIL to its cognate apoptosis- activation and endocytosis is dependent on early caspase-8 activa- inducing DR (DR4 or DR5) stimulates their internalization via tion. These findings delineate specific mechanisms for the reciprocal CME (21, 22). However, there are conflicting reports as to the crosstalk between signaling and the regulation of CME, leading to effect of TRAIL-induced endocytosis of DRs on apoptotic sig- autoregulation of endocytosis and signaling downstream of surface naling (21–23). Initial experimental data suggested that CME of receptors. TRAIL–DR complexes has an inhibitory effect on TRAIL-induced apoptosis (21, 22). However, using overexpressed dominant-nega- clathrin-mediated endocytosis | calcineurin | ryanodine receptor | programmed cell death | caspases tive to block CME, others have shown that endocytosis of TRAIL–DR is required for apoptosis, and proposed possible cell-type–specific differences in TRAIL signaling (23). eceptor-mediated endocytosis plays a critical role in regulat- Ring signaling, by either promoting rapid endocytosis of ligand– receptor complexes and attenuating cell-surface signaling, or by Significance promoting the formation of endosomes that can serve as signaling platforms for these complexes (1, 2). Clathrin-mediated endocy- Clathrin-mediated endocytosis (CME) regulates receptor traf- tosis (CME) is one of the most important and well-characterized ficking, thereby affecting several cellular signaling pathways. endocytic pathways in eukaryotes (3, 4). The CME core compo- We discovered that dynamin-1 is selectively activated down- nents—clathrin, dynamin, and adaptor protein 2 (AP2)—interact stream of TNF-related apoptosis-inducing ligand–death recep- with several endocytic accessory proteins to initiate, stabilize, and tors (TRAIL–DRs) to self-regulate their endocytosis, attenuate promote the maturation of clathrin-coated pits (CCPs). Following apoptotic signaling, and increase cell survival. Activation of 2+ maturation, CCP scission is catalyzed by the large GTPase dyna- initiator caspase-8 by TRAIL–DRs triggers spikes of Ca min, leading to the formation of cargo-containing vesicles (5, 6). through ryanodine receptor calcium channels, activating calci- Once thought to be a constitutive process, it is now recognized neurin, and in turn dephosphorylating dynamin-1 to promote – that CME can be highly regulated (7), but many questions remain cargo-selective endocytosis of TRAIL DR. This study delineates as to the molecular mechanisms underlying the regulation of specific mechanisms linking signaling downstream of cell-sur- CME. Moreover, recent data have suggested that signaling G face receptors to the regulation of cargo-selective CME, and protein-coupled receptors (GPCRs) can directly regulate CCP thus their signaling properties. Cancer cell-specific adaptation dynamics through selective recruitment of dynamin and endocytic of this bidirectional crosstalk between signaling and CME has accessory proteins (8, 9). The extent of possible crosstalk between implications for tumor progression and metastasis. signaling receptors and CME has not been explored. Author contributions: C.R.R. and S.L.S. designed research; C.R.R., P.-H.C., and N.B. per- Dynamins are master regulators of CME. In addition to their formed research; C.R.R. contributed new reagents/analytic tools; C.R.R., P.-H.C., and N.B. role in promoting the fission of invaginated CCPs, dynamins analyzed data; and C.R.R. and S.L.S. wrote the paper. control earlier rate-limiting steps of clathrin-coated vesicle for- The authors declare no conflict of interest. – mation (10 12). There are three dynamin isoforms in vertebrates: This article is a PNAS Direct Submission. dynamin-1 (Dyn1) is predominantly expressed in neurons, dyna- 1To whom correspondence should be addressed. Email: sandra.schmid@utsouthwestern. min-2 (Dyn2) is ubiquitously expressed, and dynamin-3 (Dyn3) is edu. expressed in neurons, lung, and testis. Dyn1 and Dyn2 are distinct This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. in their curvature sensing/generating properties: Dyn1 exhibits 1073/pnas.1615072114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1615072114 PNAS Early Edition | 1of6 Downloaded by guest on September 23, 2021 In this study, we sought to determine the contribution of CME AP2 or CHC (Fig. 1A and Fig. S1 A and B). This finding was true to the regulation of signaling via TRAIL–DR. Experimental for both DR4- and DR5-triggered apoptosis, as assessed using down-regulation of all of the core components of the CME DR-selective variants of TRAIL (24, 25) (Fig. S1 C and D). machinery revealed that dynamin isoforms differentially regulate Western blotting confirmed that the differential effects of Dyn1 CME of selected cargoes: whereas CME of constitutively in- and Dyn2 were not a result of differential efficiencies of ternalized transferrin receptors (TfnR) requires Dyn2, CME of knockdown or because of compensatory up-regulation of the TRAIL–DR complexes is Dyn1-dependent. We discovered that nontargeted isoform (Fig. S2 A and B). Indeed, depletion of TRAIL-induced early activation of caspase-8 results in ryano- Dyn1 did not impact the protein levels of Dyn2 in A549 cells and + dine receptor (RyR)-mediated calcium release, causing the Ca2 MDA-MB-231 cells and Dyn2 knockdown only moderately (by and calcineurin-dependent activation of Dyn1, Dyn1-dependent ∼20%) increased the protein levels of Dyn1 in A549 cells (Fig. CME of TRAIL–DR, and suppression of TRAIL-induced S2 A and B). apoptosis. To determine whether the isoform-specific functions of Dyn1 and Dyn2 were related to their roles in endocytosis, we assessed Results and Discussion the effect of their down-regulation on TRAIL uptake. siRNA- Dynamins Differentially Regulate Cargo-Selective Endocytosis and mediated depletion of Dyn1 potently reduced TRAIL–DR en- TRAIL-Induced Apoptosis. Given the existence of conflicting re- docytosis to the same extent as depletion of AP2 or CHC (Fig. ports (21–23), we directly tested the role of CME in regulating 1B), resulting in increased levels of surface-bound TRAIL (Fig. TRAIL-induced apoptosis by small-interfering RNA (siRNA)- S2 C and D). Depletion of Dyn2 was much less effective in re- mediated knockdown of core components of the CME machin- ducing TRAIL–DR endocytosis. The opposite was true for uptake ery, namely the coat protein clathrin heavy chain (CHC), the of the classic CME cargo, TfnR, which was mainly dependent on AP2, and the ubiquitously expressed isoform of dynamin, Dyn2. Dyn2 but not on Dyn1 (Fig. 1C). To confirm these effects, we TRAIL-sensitive MDA-MB-231 human breast adenocarcinoma generated CRISPR-Cas9n Dyn1 knockout (Dyn1KO)A549cells cells and TRAIL-resistant A549 human lung adenocarcinoma (Fig. S3A). As seen in siRNA-mediated knockdown cells, A549 cells depleted for CHC or AP2 showed a significant sensitization Dyn1KO cells exhibited increased sensitivity to TRAIL-induced to TRAIL-induced cell death, compared with control siRNA- apoptosis, as measured by cell viability (Fig. 1D) and caspase-3/7 treated cells (Fig. 1A and Fig. S1 A and B). Unexpectedly, activity (Fig. 1E), and showed a strong impairment in TRAIL–DR depletion of Dyn2 showed a relatively modest effect on TRAIL- endocytosis (Fig. 1F). Importantly, both TRAIL resistance and induced cell death. Because Dyn1 has recently been shown to be TRAIL–DR endocytosis were restored by reconstitution of activated in nonneuronal cancer cells (15), we also tested the Dyn1KO cells with Dyn1-EGFP (Fig. 1 D–F). effect of Dyn1 knockdown. Strikingly, Dyn1-depleted cells were Knockdown of Dyn1, AP2, and CHC resulted in an increase in as sensitized to TRAIL-induced apoptosis as those depleted of cell surface binding of TRAIL, presumably because of inhibition

Fig. 1. Differential regulation of cargo-receptor endocytosis and TRAIL-induced apoptosis by Dyn1. (A) TRAIL activity (100 ng/mL) in MDA-MB-231 (Upper) and A549 cells (Lower) treated with the indicated siRNAs. (B) FLAG-tag TRAIL uptake or (C)TfnR uptake in MDA-MB-231 (Upper)andA549cells(Lower). (D) TRAIL activity in A549 WT, Dyn1KO,andDyn1KO cells reconstituted with Dyn1-EGFP. (E) TRAIL-induced cas- pase-3/7 activation in parental A549 cells, Dyn1KO and Dyn1KO cells reconstituted with Dyn1-EGFP. (F)FLAG- tag TRAIL uptake in the indicated cell lines. Reduction in cell viability (%) was calculated relative to control wells containing no ligand. All internalization rates were calculated relative to surface-bound TfnR or FLAG-tag TRAIL. All of the results are mean values ± SD (n = 3). Two-tailed Student’s t tests were used to assess statistical significance. *P < 0.05, **P < 0.005, ***P < 0.0005; n.s., nonsignificant.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1615072114 Reis et al. Downloaded by guest on September 23, 2021 of constitutive DR endocytosis. Thus, prolonged inhibition of GSK3β (28). Thus, we examined whether inhibition of GSK3β, CME could have increased TRAIL-mediated apoptosis simply which activates Dyn1 (15), might alter the cell sensitivity to by increasing the basal levels of surface DR before TRAIL ex- TRAIL-induced apoptosis in the context of Dyn1 expression, us- posure. To test this theory, we titrated cell surface binding of ing A549 WT and Dyn1KO cells. Pretreatment with Chir99021, TRAIL in A549 WT and Dyn1KO cells. As seen in Fig. 2A,at which potently inhibits GSK3β, did not alter the sensitivity to subsaturating concentrations of TRAIL, and within the range TRAIL-induced apoptosis in either cell line (Fig. S3 C and D). that showed enhanced apoptotic signaling in Dyn1 knockdown Dyn1 can also be activated through dephosphorylation by the + cells (i.e., 1–100 ng/mL), there were no differences in TRAIL calcium (Ca2 ) and calmodulin-dependent phosphatase, calci- binding when comparing WT to Dyn1KO cells. The same was neurin (29, 30). Inhibiting calcineurin by either cyclosporin A true even after 4 h of incubation at 4 °C with 100 ng/mL TRAIL (CsA) (Fig. 2B) or FK506 (Fig. S4), resulted in a significant in- (Fig. S3B). The increased surface levels of DR in Dyn1KO cells crease in sensitivity to TRAIL-induced apoptosis in A549 WT were only apparent at high, saturating concentrations of TRAIL cells, comparable to the levels seen in Dyn1KO cells. Previous (>500 ng/mL). Although DR–ligand interactions will be different studies have shown that high concentrations of CsA can cause at 4 °C vs. 37 °C, these results nonetheless suggest that apoptosis endoplasmic reticulum (ER) stress (31), which in turn can lead occurs primarily through the inhibition of TRAIL-induced DR to apoptosis via DR5 independently of TRAIL (32). However, in endocytosis, and not because of differences in the surface ex- the cell lines tested and at the incubation times and concentra- pression of DRs, in agreement with previous reports wherein DR tions of CsA used (below 40 μM), cytotoxicity remained abso- expression was not predictive of clinical responses (26, 27). lutely dependent on TRAIL (Fig. S5). Moreover, CsA did not Taken together, our findings confirm previous reports (21, 22) further sensitize Dyn1KO cells to TRAIL-induced cell death (Fig. of an important role for TRAIL–DR endocytosis in the negative 2 B and C); however, sensitivity to CsA was fully restored in regulation of TRAIL-induced apoptosis, and furthermore, es- A549 Dyn1KO cells by reintroducing Dyn1 WT. Importantly, tablish the existence of cargo-selective, dynamin isoform-specific Dyn1KO cells reconstituted with a nonphosphorylatable, and mechanisms for endocytosis. hence constitutively active mutant Dyn1S774A/S778A (KO+AA), were both resistant to TRAIL-induced apoptosis and insensitive TRAIL Induces Calcineurin-Mediated Dephosphorylation of Dyn1 and to CsA (Fig. 2C). Similar results were obtained in H1299 Dyn1KO DR Endocytosis. We next investigated the mechanism by which cells reconstituted with the above-mentioned proteins (Fig. S6A).

Dyn1 is activated downstream of DRs. At the synapse, Dyn1 ac- These results establish that the effects of calcineurin inhibition CELL BIOLOGY tivity is regulated by cycles of phosphorylation/dephosphorylation, on TRAIL-induced apoptosis are mediated primarily through including its phosphorylation at Ser778 and Ser774, the latter by Dyn1 activation. The effects of Dyn1 depletion and calcineurin

Fig. 2. TRAIL induces calcineurin-mediated de- phosphorylation of Dyn1 and TRAIL–DR endocytosis to reduce apoptosis. (A) TRAIL binding to WT and Dyn1KO A549 cells measured at 4 °C. (B) Effects of calcineurin inhibition by CsA (20 μM) on TRAIL ac- tivity in A549 WT and Dyn1KO cells. (C) TRAIL activity (100 ng/mL) in A549 WT, Dyn1KO, and Dyn1KO reconstituted with Dyn1 WT (KO + WT) or the non- phosphorylatable mutant, Dyn1 S774A/S778A (KO + AA), in the presence or absence of CsA. (D) FLAG-tag TRAIL uptake in control and CsA pretreated cells. (E) TfnR uptake in the presence or absence of CsA. (F) Caspase-3/7 activation in TRAIL-treated MDA-MB- 231 cells in the presence or absence of CsA. (G) Dyn1 phosphorylation (pSer774) profiles upon TRAIL stimulation (100 ng/mL) in MDA-MB-231 cells in the presence or absence of CsA, and quantification of pS774-Dyn1/total-Dyn1. (H) TRAIL-induced apoptosis in MDA-MB-231 WT and Dyn1 overexpressing cells in the presence or absence of CsA. All internalization rates were calculated relative to surface bound TfnR or FLAG-tag TRAIL. Reduction in cell viability by TRAIL (%) was calculated relative to control wells incubated with solvent alone or 20 μM of CsA. All of the results are mean values ± SD (n = 3). Two-tailed Student’s t tests were used to assess statistical sig- nificance. *P < 0.05, **P < 0.005, ***P < 0.0005; ns, nonsignificant.

Reis et al. PNAS Early Edition | 3of6 Downloaded by guest on September 23, 2021 + Fig. 3. TRAIL-mediated Ca2 release by RyR regulates TRAIL–DR endocytosis and apoptosis in MDA-MB-231 cells. (A) The effect of TRAIL on cell viability in control, BAPTA-AM (10 μM) and EGTA (1 mM) -treated cells. Cell viability after cells are pretreated or not with (B) XesC, an IP3R inhibitor (20 μM), or (C) with different concentrations of Ry, a RyR inhibitor, followed by TRAIL incubation. (D) TRAIL induced caspase-3/7 ac- tivation in the presence or absence of 40 μMRy. (E) FLAG-tag TRAIL uptake in control and Ry pre- treated cells. (F) TfnR uptake in control and Ry pre- treated cells. Internalization rates were calculated relative to surface bound TfnR or FLAG-tag TRAIL. All of the results are mean values ± SD (n = 3). Two- tailed Student’s t tests were used to assess statistical significance. *P < 0.05, ***P < 0.0005.

inhibition on sensitivity to TRAIL-induced apoptosis were also of Dyn1-EGFP in MDA-MB-231 cells increased their resistance observed in several other cancer cell lines, including HCT116 to TRAIL-induced cell death. The effect of Dyn1 overexpression colorectal carcinoma, SCC61 squamous carcinoma, HT1080 fi- was abrogated by treatment with CsA, which dramatically en- brosarcoma, and MV3 melanoma cells, but not, as expected, in hanced sensitivity to TRAIL-induced cell death in both Dyn1 the caspase-3–deficient TRAIL-resistant MCF-7 mammary car- overesspression and control cells (Fig. 2H). cinoma cells (Fig. S6 B and C). These results establish that cal- cineurin-mediated dephosphorylation of Dyn1 on Ser774/778 Calcium Release by RyRs Regulates TRAIL–DR Endocytosis. Given that + plays a critical role in TRAIL sensitization in diverse cancer calcineurin is a Ca2 -dependent serine-threonine phosphatase, + cell lines. we explored the source of Ca2 needed for its activation. Pre- + We next tested whether inhibition of calcineurin activity effected treatment of MDA-MB-231 cells with the intracellular Ca2 TRAIL–DR endocytosis. Treatment of A549 WT cells with the chelator BAPTA-AM increased their sensitivity to TRAIL- + calcineurin inhibitor CsA significantly inhibited TRAIL–DR up- induced apoptosis, whereas chelating extracellular Ca2 by EGTA + take, comparable to the reduced kinetics of TRAIL–DR uptake had no effect, suggesting that intracellular Ca2 is involved in seen in Dyn1KO cells. CsA had no effect on the residual TRAIL enhanced sensitization to TRAIL-mediated apoptosis (Fig. 3A). + uptake in Dyn1KO cells (Fig. 2D). In marked contrast, TfnR uptake Intracellular Ca2 ions are generally stored in the ER, and re- remained unaltered under all these conditions (Fig. 2E). These leased into the cytosol upon extracellular signaling by either of two results establish that calcineurin modulates TRAIL–DR endocy- ER calcium channels: inositol triphosphate receptors (IP3R) or tosis, thus regulating DR signaling, via the activation of Dyn1. RyRs (35). We tested the involvement of each of these by in- We confirmed that calcineurin inhibition acts at early stages in cubating cells with Xestospongin C (XesC) or Ryanodine (Ry), DR signaling by analyzing the effects of CsA treatment on cas- specific inhibitors of IP3R or RyR, respectively. Preincubation of pase-8 processing. Both the rate of onset and the extent of MDA-MB-231 cells with up to 20 μM XesC, which potently in- TRAIL-induced caspase-8 cleavage were markedly increased in hibits IP3R (Fig. S7A), did not affect TRAIL-induced apoptosis CsA-treated cells, compared with control cells (Fig. S6D). More- (Fig. 3B). In contrast, treatment with Ry, known to inhibit RyR at over, the increased sensitivity of MDA-MB-231 cells to TRAIL- higher concentrations (>10 μM), resulted in a dose-dependent induced apoptosis upon inhibition of calcineurin was especially increase in sensitivity to TRAIL-induced cell death (Fig. 3C), and apparent when measuring caspase-3/7 activation (Fig. 2F). We a corresponding TRAIL-dependent increase in caspase-3/7 acti- also sought direct evidence that TRAIL and calcineurin inhibitors vation (Fig. 3D). As predicted, TRAIL–DR endocytosis was also were indeed acting through Dyn1 by modulating its phosphory- inhibited upon blocking RyR (Fig. 3E), whereas TfnR uptake lation. To this end, MDA-MB-231 cells were incubated with remained unaltered (Fig. 3F). Moreover, Dyn1KO A549 cells TRAIL for different time periods, and changes in Dyn1 phos- reconstituted with the constitutively active Dyn1S774A/S778A phorylation levels were measured using a phospho-S774–specific mutant, but not WT, were insensitive to RyR inhibition (Fig. S7B), antibody. Our results show a time-dependent decrease in Dyn1 confirming that RyR activity is upstream of Dyn1 activation. phosphorylation upon TRAIL incubation, relative to total Dyn1 (Fig. 2G). Importantly, TRAIL-mediated dephosphorylation of TRAIL-Induced Caspase-8 Activation Promotes RyR-Dependent Dyn1 was completely dependent on calcineurin, as CsA abolished Calcium Spikes. We next sought direct evidence for TRAIL- + Dyn1 dephosphorylation mediated by TRAIL (Fig. 2G). From stimulated ER Ca2 release using MDA-MB-231 cells trans- these results, we conclude that TRAIL-induced activation of Dyn1 fected with a genetically encoded calcium sensor, GCaMP6f (36). is regulated by calcineurin activation. Cells imaged after incubation with TRAIL exhibited periodic and + Dyn1 is overexpressed in several cancers, including leukemia, transient spikes of elevated Ca2 (Fig. 4 A and B and Fig. S8), lung, and colon adenocarcinomas (33, 34). Therefore, we tested which were completely blocked when RyR was inhibited by high the impact of Dyn1 overexpression on TRAIL-induced apoptosis concentrations of Ry (Fig. 4B). Calcium signaling downstream of in TRAIL-sensitive MDA-MB-231 cells. Transient overexpression TRAIL–DR activation has not previously been reported, raising

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Fig. 4. TRAIL induces RyR-dependent calcium spike formation. (A) Total internal reflection fluorescence microscopy (TIRFM) time-lapse images of MDA-MB- 231 expressing GCaMP6f after TRAIL incubation (100 ng/mL) in the presence or absence of Ry. (Scale bars: 5 μm.) (B) Quantification of Ca2+ oscillations in control, TRAIL, and TRAIL+Ry-treated cells. Boxes illustrate the time course where the Ca2+ oscillations induced by TRAIL shown in A occurred. (C) Quanti- + fication of Ca2 oscillations in TRAIL + caspase-8 inhibitor (Z-IETD-FMK, 20 μM) -treated cells. (D) TRAIL and TfnR uptake in control cells (Ctrl) and cells pretreated with caspase-8 inhibitor 30 min before the uptake assay. Internalization rates were calculated relative to surface bound TRAIL or TfnR, re- spectively. All of the results are mean values ± SD (n = 3). Two-tailed Student’s t tests were used to assess statistical significance. *P < 0.05, **P < 0.005.

the question of the mechanism for coupling DRs to RyR. In- regulate CCP dynamics via recruitment of dynamin (8), although terestingly, another TNF superfamily member, TNF-α,potentiates isoform-specificity and effects on signaling have not been studied. terminal afferent responses through a RyR-mediated calcium- Interestingly, the α-adaptin subunit of AP2 is a substrate for release mechanism (37). TNF-α–induced caspase-8 activation in- caspase-8–dependent cleavage, resulting in removal of the + duces RyR S-nitrosylation, resulting in Ca2 release (38). Thus, we C-terminal appendage domain (21). We previously showed that tested for the specific involvement of caspase-8 on TRAIL- Dyn1 is activated in cells expressing a similar truncation muta- 2+ induced Ca -signaling. We observed a significant decrease in tion of α-adaptin, altering the regulation of CME (15). Thus, this – TRAIL-stimulated calcium spike formation in caspase-8 inhibited cleavage, which occurs at later times after exposure to TRAIL cells, with most cells showing much fewer and less-intense calcium (21), may be a mechanism for sustained regulation of CME that elevations, compared with TRAIL-treated cells (Fig. 4 B and C + is likely less cargo-selective. and Fig. S9A). Consistent with the block in Ca2 release, inhibition of caspase-8—or its siRNA-mediated knockdown (Fig. S9B)—also inhibited TRAIL–DR endocytosis, without significantly affecting the uptake of TfnR (Fig. 4D). Thus, activation of caspase-8 can either trigger or negatively regulate apoptotic signaling, presumably reflecting a threshold effect that controls apoptosis. Conclusions and Perspectives Endocytosis is a major regulator of cellular signaling (2, 39). Herein we present evidence for the direct, reciprocal regulation of CME by signaling receptors. Our studies uncover an early feedback loop relying on TRAIL–DR-mediated activation of initiator caspases to promote TRAIL–DR endocytosis. We show + that TRAIL-activated DRs trigger RyR-dependent Ca2 release from ER stores, induce calcineurin-dependent dephosphorylation, and thereby activation of Dyn1, leading to cargo-selective uptake of DRs and the attenuation of their apoptotic signaling (Fig. 5). – Although DRs are the first surface-signaling receptor shown Fig. 5. Proposed model for Dyn1-dependent endocytosis of TRAIL DR complexes to suppress apoptotic signaling. TRAIL-induced DR activation to regulate their own CME and signaling, through a dynamin- leads to caspase-8 cleavage/activation, which in turn activates RyR-mediated – + isoform specific mechanism, we predict that other surface sig- Ca2 release from ER stores and calcineurin activation to dephosphorylate naling receptors participate in this bidirectional crosstalk between and activate Dyn1. The selective regulation of TRAIL–DR endocytosis sup- signaling and CME. Indeed, others have shown that GPCRs can presses TRAIL-mediated apoptosis in cancer cells.

Reis et al. PNAS Early Edition | 5of6 Downloaded by guest on September 23, 2021 The overexpression and activation of Dyn1 in cancer cells siRNA transfections were performed with previously established siRNA might be an adaptation that enhances their survival, migration, sequences using RNAiMAX (Life Technologies), as described in SI Materials and other properties that contribute to cancer cell aggressive- and Methods. ness. In support of this possibility, Dyn1—but not Dyn2—is Dyn1 KO A549 and H1299 cells were generated using a CRISPR-Cas9n overexpressed in many lung tumor-derived lung cancer cells double-nicking strategy. Cell viability assays were performed by using the relative to normal bronchial epithelial cells (Fig. S10A). More- CCK-8 Counting Kit (Dojindo), according to the manufacturer’s instructions. over, lower survival rates in lung cancer patients are linked to Caspase-3/7 activation was assessed using the Apo-ONE Homogeneous high levels of Dyn1, but not Dyn2 expression, especially among Caspase-3/7 assay (Promega). smokers (Fig. S10B). Thus, Dyn1-mediated modulation of endo- Endocytosis of Transferrin receptors or N-terminal FLAG-tag TRAIL was cytic trafficking emerges as an important physiological regulator of measured using the anti-TfnR (HTR-D65) or anti-FLAG mouse mAbs. TfnR and DR-mediated signals, further implicating the regulation of CME TRAIL receptor cell surface expression were measured in parallel by in- by dynamins as a mechanism exploited by cancer cells to escape cubating MDA-MB-231 or A549 cells with the respective ligands at 4 °C for apoptotic cell death. More generally, given that Dyn1 can also be 30 min. Internalized ligand was expressed as the percentage of the total activated downstream of oncogenic Akt signaling (15), Dyn1 surface-bound ligand at 4 °C (i.e., without the acid wash step), measured becomes a potential nexus for reciprocal regulation of cell-surface in parallel. receptor signaling and CME in cancer cells. Further exploration of Calcium imaging was performed using MDA-MB-231 transiently express- the differential endocytic adaptations exploited by cancer cells ing GCaMP6f or A549 cells preincubated with Fura2. may provide new targeting strategies to combat this disease. ACKNOWLEDGMENTS. We thank S. Srinivasan, W. Burford, A. Mohanakrishnan, and D. Reed for technical support; Boning Gao for the tumor and lung cell Dyn1 Materials and Methods and Dyn2 expression data; all the members of the S.L.S. laboratory for valuable Detailed methods are provided in SI Materials and Methods. In brief, cells discussions and feedback; R. H. Cool, R. Setroikromo, and Wim J. Quax for pro- lines used in this study, including MDA-MB-231 breast adenocarcinoma, viding us with human recombinant TRAIL–death receptor ligands; and Daniel H1299 nonsmall-cell lung cancer cells, A549 lung carcinoma, HT1080 fibrosar- Ryskamp and Ilya Bezprozvanny for help with Ca2+ imaging in A549 cells. coma, MV3 melanoma cells, HCT116 colon adenocarcinoma cells, MCF-7 breast pGP-CMV-GCaMP6f was a gift from Douglas Kim (Addgene plasmid #40755). This adenocarcinoma, and Scc61 squamous cell carcinoma cells were cultured as work was funded by NIH Grants GM73165 and GM42455, and Cancer Prevention described in SI Materials and Methods. Research Institute of Texas Grant RP150573 (to S.L.S.).

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