Abstract
The exposure of calreticulin (CRT) on the surface of stressed and dying cancer cells facilitates their uptake by dendritic cells and the subsequent presentation of tumor-associated antigens to T lymphocytes, hence stimulating an anticancer immune response. The chemotherapeutic agent mitoxantrone (MTX) can stimulate the peripheral relocation of CRT in both human and yeast cells, suggesting that the CRT exposure pathway is phylogenetically conserved. Here, we show that pheromones can act as physiological inducers of CRT exposure in yeast cells, thereby facilitating the formation of mating conjugates, and that a large-spectrum inhibitor of G protein-coupled receptors (which resemble the yeast pheromone receptor) prevents CRT exposure in human cancer cells exposed to MTX. An RNA interference screen as well as transcriptome analyses revealed that chemokines, in particular human CXCL8 (best known as interleukin-8) and its mouse ortholog Cxcl2, are involved in the immunogenic translocation of CRT to the outer leaflet of the plasma membrane. MTX stimulated the production of CXCL8 by human cancer cells in vitro and that of Cxcl2 by murine tumors in vivo. The knockdown of CXCL8/Cxcl2 receptors (CXCR1/Cxcr1 and Cxcr2) reduced MTX-induced CRT exposure in both human and murine cancer cells, as well as the capacity of the latter-on exposure to MTX-to elicit an anticancer immune response in vivo. Conversely, the addition of exogenous Cxcl2 increased the immunogenicity of dying cells in a CRT-dependent manner. Altogether, these results identify autocrine and paracrine chemokine signaling circuitries that modulate CRT exposure and the immunogenicity of cell death.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
Abbreviations
- BCG:
-
bacillus Calmette-Guérin
- CFU:
-
colony-forming unit
- CRT:
-
calreticulin
- DC:
-
dendritic cell
- eIF2α:
-
eukaryotic initiation factor 2α
- ELISA:
-
enzyme-linked immunosorbent assay
- ER:
-
endoplasmic reticulum
- FITC:
-
fluorescein isothiocianate
- GFP:
-
green fluorescent protein
- GPCR:
-
G protein-coupled receptor
- ICD:
-
immunogenic cell death
- IL-8:
-
interleukin-8
- mTOR:
-
mammalian target of rapamycin
- MTX:
-
mitoxantrone
- PDIA3:
-
protein disulfide isomerase family A, member 3
- PI:
-
propidium iodide
- PERK:
-
PKR-like ER kinase
- PI3K:
-
phosphoinositide-3-kinase
- r:
-
recombinant
- PTX:
-
pertussis toxin
- RFP:
-
red fluorescent protein
- SC:
-
synthetic complete
- siRNA:
-
small-interfering RNA
- SNARE:
-
SNAP receptor
- WT:
-
wild-type
- YEPD:
-
yeast extract peptone dextrose
References
Galluzzi L, Vitale I, Abrams JM, Alnemri ES, Baehrecke EH, Blagosklonny MV et al. Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012. Cell Death Differ 2012; 19: 107–120.
Beneteau M, Zunino B, Jacquin MA, Meynet O, Chiche J, Pradelli LA et al. Combination of glycolysis inhibition with chemotherapy results in an antitumor immune response. Proc Natl Acad Sci USA 2012; 109: 20071–20076.
Michaud M, Martins I, Sukkurwala AQ, Adjemian S, Ma Y, Pellegatti P et al. Autophagy-dependent anticancer immune responses induced by chemotherapeutic agents in mice. Science 2011; 334: 1573–1577.
Yang Y, Li XJ, Chen Z, Zhu XX, Wang J, Zhang LB et al. Wogonin-induced calreticulin/annexin A1 exposure dictates the immunogenicity of cancer cells in a PERK/AKT-dependent manner. PLoS One 2012; 7: e50811.
Kroemer G, Galluzzi L, Kepp O, Zitvogel L . Immunogenic cell death in cancer therapy. Annu Rev Immunol 2013; 31: 51–72.
Ma Y, Adjemian S, Mattarollo SR, Yamazaki T, Aymeric L, Yang H et al. Anticancer chemotherapy-induced intratumoral recruitment and differentiation of antigen-presenting cells. Immunity 2013; 38: 729–741.
Denkert C, Loibl S, Noske A, Roller M, Muller BM, Komor M et al. Tumor-associated lymphocytes as an independent predictor of response to neoadjuvant chemotherapy in breast cancer. J Clin Oncol 2010; 28: 105–113.
Fridman WH, Pages F, Sautes-Fridman C, Galon J . The immune contexture in human tumours: impact on clinical outcome. Nat Rev Cancer 2012; 12: 298–306.
Galluzzi L, Senovilla L, Zitvogel L, Kroemer G . The secret ally: immunostimulation by anticancer drugs. Nat Rev Drug Discov 2012; 11: 215–233.
Senovilla L, Vacchelli E, Galon J, Adjemian S, Eggermont A, Fridman WH et al. Trial watch: prognostic and predictive value of the immune infiltrate in cancer. Oncoimmunology 2012; 1: 1323–1343.
Krysko DV, Garg AD, Kaczmarek A, Krysko O, Agostinis P, Vandenabeele P . Immunogenic cell death and DAMPs in cancer therapy. Nat Rev Cancer 2012; 12: 860–875.
Casares N, Pequignot MO, Tesniere A, Ghiringhelli F, Roux S, Chaput N et al. Caspase-dependent immunogenicity of doxorubicin-induced tumor cell death. J Exp Med 2005; 202: 1691–1701.
Martins I, Kepp O, Schlemmer F, Adjemian S, Tailler M, Shen S et al. Restoration of the immunogenicity of cisplatin-induced cancer cell death by endoplasmic reticulum stress. Oncogene 2011; 30: 1147–1158.
Obeid M, Tesniere A, Ghiringhelli F, Fimia GM, Apetoh L, Perfettini JL et al. Calreticulin exposure dictates the immunogenicity of cancer cell death. Nat Med 2007; 13: 54–61.
Gardai SJ, McPhillips KA, Frasch SC, Janssen WJ, Starefeldt A, Murphy-Ullrich JE et al. Cell-surface calreticulin initiates clearance of viable or apoptotic cells through trans-activation of LRP on the phagocyte. Cell 2005; 123: 321–334.
Panaretakis T, Kepp O, Brockmeier U, Tesniere A, Bjorklund AC, Chapman DC et al. Mechanisms of pre-apoptotic calreticulin exposure in immunogenic cell death. EMBO J 2009; 28: 578–590.
Garg AD, Krysko DV, Verfaillie T, Kaczmarek A, Ferreira GB, Marysael T et al. A novel pathway combining calreticulin exposure and ATP secretion in immunogenic cancer cell death. EMBO J 2012; 31: 1062–1079.
Menger L, Vacchelli E, Adjemian S, Martins I, Ma Y, Shen S et al. Cardiac glycosides exert anticancer effects by inducing immunogenic cell death. Sci Transl Med 2012; 4: 143ra199.
Garg AD, Krysko DV, Vandenabeele P, Agostinis P . The emergence of phox-ER stress induced immunogenic apoptosis. Oncoimmunology 2012; 1: 786–788.
Raghavan M, Wijeyesakere SJ, Peters LR, Del Cid N . Calreticulin in the immune system: ins and outs. Trends Immunol 2013; 34: 13–21.
Nakamura M, Moriya M, Baba T, Michikawa Y, Yamanobe T, Arai K et al. An endoplasmic reticulum protein, calreticulin, is transported into the acrosome of rat sperm. Exp Cell Res 1993; 205: 101–110.
Tutuncu L, Stein P, Ord TS, Jorgez CJ, Williams CJ . Calreticulin on the mouse egg surface mediates transmembrane signaling linked to cell cycle resumption. Dev Biol 2004; 270: 246–260.
Nakamura M, Oshio S, Tamura A, Okinaga S, Arai K . Antisera to calreticulin inhibits sperm motility in mice. Biochem Biophys Res Commun 1992; 186: 984–990.
Park BJ, Lee DG, Yu JR, Jung SK, Choi K, Lee J et al. Calreticulin, a calcium-binding molecular chaperone, is required for stress response and fertility in Caenorhabditis elegans. Mol Biol Cell 2001; 12: 2835–2845.
Madeo F, Durchschlag M, Kepp O, Panaretakis T, Zitvogel L, Frohlich KU et al. Phylogenetic conservation of the preapoptotic calreticulin exposure pathway from yeast to mammals. Cell Cycle 2009; 8: 639–642.
Ferreira V, Valck C, Sanchez G, Gingras A, Tzima S, Molina MC et al. The classical activation pathway of the human complement system is specifically inhibited by calreticulin from Trypanosoma cruzi. J Immunol 2004; 172: 3042–3050.
Girard-Misguich F, Sachse M, Santi-Rocca J, Guillen N . The endoplasmic reticulum chaperone calreticulin is recruited to the uropod during capping of surface receptors in Entamoeba histolytica. Mol Biochem Parasitol 2008; 157: 236–240.
Vacchelli E, Galluzzi L, Fridman WH, Galon J, Sautes-Fridman C, Tartour E et al. Trial watch: chemotherapy with immunogenic cell death inducers. Oncoimmunology 2012; 1: 179–188.
Gardner BM, Walter P . Unfolded proteins are Ire1-activating ligands that directly induce the unfolded protein response. Science 2011; 333: 1891–1894.
Cole GM, Reed SI . Pheromone-induced phosphorylation of a G protein beta subunit in S. cerevisiae is associated with an adaptive response to mating pheromone. Cell 1991; 64: 703–716.
Hildebrandt JD, Sekura RD, Codina J, Iyengar R, Manclark CR, Birnbaumer L . Stimulation and inhibition of adenylyl cyclases mediated by distinct regulatory proteins. Nature 1983; 302: 706–709.
Kroemer G, Galluzzi L, Brenner C . Mitochondrial membrane permeabilization in cell death. Physiol Rev 2007; 87: 99–163.
Miyamoto S, Inoue H, Nakamura T, Yamada M, Sakamoto C, Urata Y et al. Coxsackievirus B3 is an oncolytic virus with immunostimulatory properties that is active against lung adenocarcinoma. Cancer Res 2012; 72: 2609–2621.
Paget C, Duret H, Ngiow SF, Kansara M, Thomas DM, Smyth MJ . Studying the role of the immune system on the antitumor activity of a Hedgehog inhibitor against murine osteosarcoma. Oncoimmunology 2012; 1: 1313–1322.
Zappasodi R, Pupa SM, Ghedini GC, Bongarzone I, Magni M, Cabras AD et al. Improved clinical outcome in indolent B-cell lymphoma patients vaccinated with autologous tumor cells experiencing immunogenic death. Cancer Res 2010; 70: 9062–9072.
Chao MP, Jaiswal S, Weissman-Tsukamoto R, Alizadeh AA, Gentles AJ, Volkmer J et al. Calreticulin is the dominant pro-phagocytic signal on multiple human cancers and is counterbalanced by CD47. Sci Transl Med 2010; 2: 63ra94.
Fucikova J, Kralikova P, Fialova A, Brtnicky T, Rob L, Bartunkova J et al. Human tumor cells killed by anthracyclines induce a tumor-specific immune response. Cancer Res 2011; 71: 4821–4833.
Haber JE . Mating-type genes and MAT switching in Saccharomyces cerevisiae. Genetics 2012; 191: 33–64.
Bauer MA, Carmona-Gutierrez D, Ruckenstuhl C, Reisenbichler A, Megalou EV, Eisenberg T et al. Spermidine promotes mating and fertilization efficiency in model organisms. Cell Cycle 2013; 12: 346–352.
Rajagopal S, Rajagopal K, Lefkowitz RJ . Teaching old receptors new tricks: biasing seven-transmembrane receptors. Nat Rev Drug Discov 2010; 9: 373–386.
Mishra P, Banerjee D, Ben-Baruch A . Chemokines at the crossroads of tumor-fibroblast interactions that promote malignancy. J Leukoc Biol 2011; 89: 31–39.
Verbeke H, Struyf S, Laureys G, Van Damme J . The expression and role of CXC chemokines in colorectal cancer. Cytokine Growth Factor Rev 2011; 22: 345–358.
Pazolli E, Alspach E, Milczarek A, Prior J, Piwnica-Worms D, Stewart SA . Chromatin remodeling underlies the senescence-associated secretory phenotype of tumor stromal fibroblasts that supports cancer progression. Cancer Res 2012; 72: 2251–2261.
Britschgi A, Andraos R, Brinkhaus H, Klebba I, Romanet V, Muller U et al. JAK2/STAT5 inhibition circumvents resistance to PI3K/mTOR blockade: a rationale for cotargeting these pathways in metastatic breast cancer. Cancer Cell 2012; 22: 796–811.
Piperi C, Samaras V, Levidou G, Kavantzas N, Boviatsis E, Petraki K et al. Prognostic significance of IL-8-STAT-3 pathway in astrocytomas: correlation with IL-6, VEGF and microvessel morphometry. Cytokine 2011; 55: 387–395.
Saintigny P, Massarelli E, Lin S, Ahn YH, Chen Y, Goswami S et al. CXCR2 expression in tumor cells is a poor prognostic factor and promotes invasion and metastasis in lung adenocarcinoma. Cancer Res 2013; 73: 571–582.
Hei TK, Zhou H, Chai Y, Ponnaiya B, Ivanov VN . Radiation induced non-targeted response: mechanism and potential clinical implications. Curr Mol Pharmacol 2011; 4: 96–105.
Frey B, Rubner Y, Wunderlich R, Weiss EM, Pockley AG, Fietkau R et al. Induction of abscopal anti-tumor immunity and immunogenic tumor cell death by ionizing irradiation—implications for cancer therapies. Curr Med Chem 2012; 19: 1751–1764.
Galluzzi L, Vacchelli E, Eggermont A, Fridman WH, Galon J, Sautes-Fridman C et al. Trial watch: experimental Toll-like receptor agonists for cancer therapy. Oncoimmunology 2012; 1: 699–716.
Vacchelli E, Galluzzi L, Eggermont A, Fridman WH, Galon J, Sautes-Fridman C et al. Trial watch: FDA-approved Toll-like receptor agonists for cancer therapy. Oncoimmunology 2012; 1: 894–907.
Biot C, Rentsch CA, Gsponer JR, Birkhauser FD, Jusforgues-Saklani H, Lemaitre F et al. Preexisting BCG-specific T cells improve intravesical immunotherapy for bladder cancer. Sci Transl Med 2012; 4: 137ra172.
Thalmann GN, Sermier A, Rentsch C, Mohrle K, Cecchini MG, Studer UE . Urinary Interleukin-8 and 18 predict the response of superficial bladder cancer to intravesical therapy with bacillus Calmette-Guerin. J Urol 2000; 164: 2129–2133.
DiPaola RS, Lattime EC . Bacillus Calmette-Guerin mechanism of action: the role of immunity, apoptosis, necrosis and autophagy. J Urol 2007; 178: 1840–1841.
Sheff MA, Thorn KS . Optimized cassettes for fluorescent protein tagging in Saccharomyces cerevisiae. Yeast 2004; 21: 661–670.
Galluzzi L, Morselli E, Vitale I, Kepp O, Senovilla L, Criollo A et al. miR-181a and miR-630 regulate cisplatin-induced cancer cell death. Cancer Res 2010; 70: 1793–1803.
Galluzzi L, Vitale I, Senovilla L, Olaussen KA, Pinna G, Eisenberg T et al. Prognostic impact of vitamin B6 metabolism in lung cancer. Cell Rep 2012; 2: 257–269.
Senovilla L, Vitale I, Martins I, Tailler M, Pailleret C, Michaud M et al. An immunosurveillance mechanism controls cancer cell ploidy. Science 2012; 337: 1678–1684.
Galluzzi L, Aaronson SA, Abrams J, Alnemri ES, Andrews DW, Baehrecke EH et al. Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes. Cell Death Differ 2009; 16: 1093–1107.
Kepp O, Galluzzi L, Lipinski M, Yuan J, Kroemer G . Cell death assays for drug discovery. Nat Rev Drug Discov 2011; 10: 221–237.
Criollo A, Galluzzi L, Maiuri MC, Tasdemir E, Lavandero S, Kroemer G . Mitochondrial control of cell death induced by hyperosmotic stress. Apoptosis 2007; 12: 3–18.
Acknowledgements
GK is supported by the European Commission (ArtForce); Agence National de la Recherche (ANR); Ligue Nationale contre le Cancer (Equipe labellisée); Fondation pour la Recherche Médicale (FRM); Institut National du Cancer (INCa); LabEx Immuno-Oncologie; Fondation de France; Fondation Bettencourt-Schueller; AXA Chair for Longevity Research; Cancéropôle Ile-de-France and Paris Alliance of Cancer Research Institutes (PACRI). AQS is supported by Fondation ARC pour la recherche sur le cancer. IM is supported by la Ligue Nationale contre le Cancer. We thank SIRIC SOCRATE for their critical support. We are grateful to the Austrian Science Fund FWF (Austria) for grants P23490-B12 and P24381-B20 to FM.
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Competing interests
The authors declare no conflict of interest.
Additional information
Edited by M Piacentini
Supplementary Information accompanies this paper on Cell Death and Differentiation website
Rights and permissions
About this article
Cite this article
Sukkurwala, A., Martins, I., Wang, Y. et al. Immunogenic calreticulin exposure occurs through a phylogenetically conserved stress pathway involving the chemokine CXCL8. Cell Death Differ 21, 59–68 (2014). https://doi.org/10.1038/cdd.2013.73
Received:
Revised:
Accepted:
Published:
Issue date:
DOI: https://doi.org/10.1038/cdd.2013.73
Keywords
This article is cited by
-
Stress-induced inflammation evoked by immunogenic cell death is blunted by the IRE1α kinase inhibitor KIRA6 through HSP60 targeting
Cell Death & Differentiation (2022)
-
Immunogenic necroptosis in the anti-tumor photodynamic action of BAM-SiPc, a silicon(IV) phthalocyanine-based photosensitizer
Cancer Immunology, Immunotherapy (2021)
-
Natural compound inducers of immunogenic cell death
Archives of Pharmacal Research (2019)
-
Dying cells actively regulate adaptive immune responses
Nature Reviews Immunology (2017)
-
Chemokines in the cancer microenvironment and their relevance in cancer immunotherapy
Nature Reviews Immunology (2017)
