Molecular mechanisms of ATP secretion during immunogenic cell death

Abstract

The immunogenic demise of cancer cells can be induced by various chemotherapeutics, such as anthracyclines and oxaliplatin, and provokes an immune response against tumor-associated antigens. Thus, immunogenic cell death (ICD)-inducing antineoplastic agents stimulate a tumor-specific immune response that determines the long-term success of therapy. The release of ATP from dying cells constitutes one of the three major hallmarks of ICD and occurs independently of the two others, namely, the pre-apoptotic exposure of calreticulin on the cell surface and the postmortem release of high-mobility group box 1 (HMBG1) into the extracellular space. Pre-mortem autophagy is known to be required for the ICD-associated secretion of ATP, implying that autophagy-deficient cancer cells fail to elicit therapy-relevant immune responses in vivo. However, the precise molecular mechanisms whereby ATP is actively secreted in the course of ICD remain elusive. Using a combination of pharmacological screens, silencing experiments and techniques to monitor the subcellular localization of ATP, we show here that, in response to ICD inducers, ATP redistributes from lysosomes to autolysosomes and is secreted by a mechanism that requires the lysosomal protein LAMP1, which translocates to the plasma membrane in a strictly caspase-dependent manner. The secretion of ATP additionally involves the caspase-dependent activation of Rho-associated, coiled-coil containing protein kinase 1 (ROCK1)-mediated, myosin II-dependent cellular blebbing, as well as the opening of pannexin 1 (PANX1) channels, which is also triggered by caspases. Of note, although autophagy and LAMP1 fail to influence PANX1 channel opening, PANX1 is required for the ICD-associated translocation of LAMP1 to the plasma membrane. Altogether, these findings suggest that caspase-and PANX1-dependent lysosomal exocytosis has an essential role in ATP release as triggered by immunogenic chemotherapy.

Contribution of RIP3 and MLKL to immunogenic cell death signaling in cancer chemotherapy

“…ATP release, occurring in the context of caspase-dependent ICD, has been linked to premortem autophagy. 17,36 However, Rip3 ¡/¡ and Mlkl ¡/¡ exhibited a normal autophagic response to MTX in vitro (Fig. S5D), suggesting that differences in autophagy cannot explain the reduced ATP release.…”

“…Although caspase inhibition fails to prevent chemotherapy-induced cell death (which then occurs in a non-apoptotic fashion), it does prevent ICD due to the suppression of calreticulin (CRT) exposure (which is an "eat-me" signal facilitating the transfer of tumor antigens into immature dendritic cells (DC)) 13,15 and the reduction of ATP release (which serves as a chemotactic signal for the attraction of immune cells into the tumor bed). 16,17 CT26 and MCA205 cells that have been lysed by freeze-thawing fail to immunize mice against cancer. 12 These two cell lines, when killed by chemotherapy in the context of caspase inhibition, undergo necrotic cell death, which is non-immunogenic as well.…”

Systemic autophagy in the therapeutic response to anthracycline-based chemotherapy

“…13,14 The release of ATP can be passive (as a result of plasma membrane permeabilization during primary or secondary necrosis) or active via lysosomal secretion. 15 This latter phenomenon is linked to autophagy, which is required for optimal release of ATP from dying cancer cells. 3 For this reason, autophagy-deficient cancer cells fail to release ATP in response to chemotherapy and thus are unable to recruit DCs into the tumor bed and to elicit an anti-tumor immune response.…”

Immunological metagene signatures derived from immunogenic cancer cell death associate with improved survival of patients with lung, breast or ovarian malignancies: A large-scale meta-analysis

“…[1][2][3]14,15 ICD is associated with danger signaling pathways mediating the extracellular emission of danger signals like surface calreticulin/heat-shock proteins, secreted ATP and secreted HMGB1. 14,[16][17][18][19][20][21][22][23][24] These danger signals help in activating the innate immune system 14,22 which further boosts the adaptive immunity leading to anti-tumor immunity. 14,25-27 A significant advantage of using ICD for biomarker discovery is that it, uniquely, simultaneously integrates several immune-related pathways such as danger signaling, effector T cell infiltration/ activity and others into a single paradigm.…”