Introduction
Millions of cells are always being removed in the human body in order to regulate biological pathways and keep cells healthy through a particular mechanism called cell death or apoptosis. Through apoptosis, such homeostatic death of cells has been defined as a tolerogenic or a non-immunogenic event for many years [1]. However, during the past few years, scientists have discovered the occurrence of a different concept of cell death – immunogenic cell death (ICD), where the death of a cell stimulates a response from the immune system against antigens of a dead cell, and this mechanism of cell death may play a particularly important role in cancer cells [2]. The efficacy of immunogenic cell death in cancer therapy has been tested in mouse and rat models. Elaborate experiments that have taken into account differences in species and strains have been performed in this context, proving that immune responses to tumors are able to define the efficiency of anti-cancer treatment [3].
In particular, human tumor cells that are exposed to anthracycline, a cancer drug, appear to be significantly immunogenic and it has therefore been successfully used as an effective treatment for cancer patients [4]. Using anthracycline for chemotherapy stimulates an immune response with the help of dendritic cells (DCs) in order to present an antigen from dying cells of tumors, which leads to increased recruitment of cytotoxic T-lymphocytes. Anthracyclines have been shown to induce immunogenic cell death by translocating HSP70, HSP90 and calreticulin to the surface of the tumor cells. These serve as signals for dendritic cells to engulf tumor cells. The cells undergoing immunogenic cell death induced by anthracyclines also release the late apoptotic marker high mobility group box1 (HMGB1) that can bind to several pattern recognition receptors such as Toll- like receptor 2 and 4 (TLR2 and TLR4), and receptor for advanced glysosylation end products (RAGE). HMGB release facilitates antigen presentation and further uptake of tumor cells by dendritic cells [5]. The secretion of ATP is also a characteristic feature of immunogenic cell death. ATP interacts with the purinogenic receptor P2rx7 on the surface of dendritic cells. Such mechanisms have been demonstrated in the laboratory with mice and humans, such as in clinical responses to chemotherapy with the loss of alleles of Toll-like receptor 4 and the purigenic receptor, P2rx7 [4].
Immunogenic cell death (ICD) is a result of autophagy and pre-mortem endoplasmic reticulum (ER) stress, which exposes calreticulin (CRT) on the plasma membrane’s outer leaflet at its pre-apoptotic stage with adenosine triphosphate (ATP) [6]. Moreover, during ICD, dying cells also release the nuclear protein high-mobility group box 1 (HMGB1), during the permeabilization of membranes during secondary necrosis. Accordingly, CRT binds to CD91 on the purigenic receptor, P2RX7, and HMGB1 binds to TLR4; these bindings stimulate dendritic cell (DC) recruitment into tumors and perform tumor-antigen engulfment by DCs, presenting the antigen to T-lymphocytes. These processes result in an immune response mediated by interferon γ (IFN- γ) and cytotoxic CD8+ T lymphocytes (CTLs), which lead to the eradication of chemotherapy-resistant tumor cells [6].
A number of studies have considered and analyzed the question of why certain cytotoxic chemotherapeutics result in CRT exposure with ATP’s secretion and HMGB1 release, while others may not [7]. In this regard, it is important to understand that ICD requires the appearance of two types of stress: autophagy and ER stress. CRT, which is in the ER lumen in its largest fraction during ER stress resulted from chemotherapy, transfers itself into the plasma membrane’s outer surface (known as ecto-CRT) before any sign of apoptotic cell death [7]. Ecto-CRT appears to be a potent signal of engulfment, which engulfs the DCs’ portion of dying cells of tumors. However, the CRT exposure blockade in tumors also appears to negatively affect the efficiency of chemotherapy in in vitro studies; thus, the ER stress-dependent signal of the immune system appears to be an essential aspect for anti-cancer responses [7].
As previously stated, autophagy appears to be obligatory for stressed cells to be considered immunogenic. Autophagy-deficient tumor cells released less ATP as compared to their autophagy-competent counterparts, but Inhibiting autophagy influences neither HMGB1 release nor CRT exposure.. Autophagy is required for ATP secretion following chemotherapy treatment and immunogenic cell death. The decreased release of ATP causes autophagy-deficient tumor cells to stop the recruitment of dendritic cells and thus decreased immunogenic cell death. The opposite is the case for autophagy-competent timor cells. This phenomenon can be corrected by the inhibition of extracellular ATP degrading enzymes. The inhibition of these enzymes cause an increase in ATP in tumor cells, which allow the recruitment of endritic cells [8]. Autophagy is essential for ICD so that ATP is released. Furthermore, it is important to note that increased concentrations of extracellular ATP provide potential to improve the efficiency of anti-neoplastic chemotherapies in conditions when autophagy is unavailable or disabled [8].
Considering the aforementioned factors, the importance of immunogenic cell death lies within its potential to improve and enhance current cancer treatments [9]. Chemotherapeutics can induce significant combinations of the death and stress of tumor cells that would be immunogenic; moreover, the majority of such procedures have significantly high success rates to improve the status of cancer patients. In other words, the death of tumor cells in patients are nowadays becoming a considerably effective form of treatment, being able to stimulate an immune response aimed specifically to remove tumor cells, which results in further control and eradication of cancer cells [9].
ICD needs to fulfill two conditions in order to be effective. First, tumor cells that are associated with ICD in vitro, injected without any adjuvant into an experimental system, must launch a response from the immune system in order to protect the organism from these tumor cells of the same type that are currently alive and/or healthy in the body [9] Second, for ICD occurring in vivo, it should also elicit a local immune response with the recruitment of cognate and innate immune system effector cells into the tumor bed, resulting in tumor growth inhibition. Scientists have considered various preclinical models and have provided evidence for both these phenomena occurring in different immune system alterations [9].
During cell death, biomarkers are involved in the stimulation of the immune system, such as the HMGB1 protein along with its one of its main receptors, receptor for advanced glycation end products (RAGE) [10]. HMGB1 is a small protein that possesses three structural domains. These are two tandem HMG boxes and an acidic C-terminal tail separated by a short linker. This C-terminal tail of HMGB1 mediates its interaction with RAGE [11] HMGB1 was discovered as a ligand for RAGE during a search for the potential binding partners of RAGE in detergent extract of bovine lung acetone powder applied to sepharose and heparin columns by a step elution in which eluent fractions were checked for RAGE binding activity. The eluent fractions were purified and sequenced and was found to be HMGB1 or amphoterin. To further confirm this interaction, recombinantly expressed amphoterin from rat was purified and was shown to bind to RAGE in the same maner. This binding was proved to be highly specific [12]. In particular, HMGB1 represents a nuclear protein that has high association with chromatin playing a vital role in transcription processes and cell regulation. Previous studies related to HMGB1 have reported that it can only be released during necrotic cell death; however, the latest research has proved that the release is also possible during late stages of apoptosis. Afterwards, HMGB1 becomes closely attached to the chromatin, also being released in the form of HMGB1-nuclesome complexes. HMGB1 functions as a danger associated molecular pattern (DAMP), which binds to TLR4 to elicit signaling events that contribute to the effects of HMGB1. The efficacy of HMGB1 has been reported in conditions when nucleosomes, DNA or lipopolysaccharides (LPS) are bound to it. Its major mechanism of functioning lies within binding to particular receptors on antigens or dendritic cells such as TLR4 or RAGE in association with LPS [10]. HMGB1binding to RAGE leads to the activation of the IKK complex (inhibitor of NF-B (IB) kinase complex that phosphorylates IB. This phosphorylation induces the release of NF-B and its translocation to the nucleus. Thus, the transcription of pro-inflammatory genes (such as interleukin-1 (IL-1), IL-6 and tumour-necrosis factor) is induced (Fig 1).
As a result, phagocytized particles are intracellularly organized to present them at the surface of cells, which stimulates the cytotoxic response of T-lymphocytes, reacting to tumors [9]. Recent studies have proved that the DAMPs released through stressed cells with apoptosis and necrosis appears to be essential for a patient’s response after successful chemotherapy. In this regard, it has also been reported that knockdown or neutralization of TLR4 or HMGB1 is associated with significant reduction of an immune reaction both in vivo and in vitro, along with a weak outcome for therapy. Still, it remains uncertain whether HMGB1’s serum levels have a significant predictive or prognostic association with cancer [11].
In addition, it has also been shown that HMGB1 not only launches the protective reactions of T-lymphocytes, but can also stimulate tumor invasiveness and neo-angiogenesis [14]. In –vitro experiments have proved that suppression of HMGB1 and RAGE by antisense S-oligodeoxynucleotide imparts tumor proliferation and migration. The same has been pbserved in animals where the suppression of RAGE-HMGB1 interaction induces the activation of Mitogen activated protein kinase pathway and NF-kB pathway and thus inhibits tumor growth. The downstream signaling components of HMGB1 needs to be elucidated in order to counteract this effect. HMGB1 and RAGE’s overexpression is vital for tumor metastasis, according to observations in colon and gastric cancer. However, RAGE’s serum levels have been reported to decline significantly in some forms of cancer. Therefore, there is a double role of RAGE, as it is involved in the DAMP cellular signal transmission as a surface receptor, and may also correlate with cellular high expression of RAGE [15].
Further sections of this research paper consider important biomarkers as an inevitable part of the cell death process, with the growth of tumors and immunogenicity represented in various forms of cancer to identify their role and importance in the prediction and prognosis of responses to different forms of chemotherapy during different phases of cancer treatment.
Chemotherapy is considered to be an immunosuppressive treatment due to the depletion of NK-cells and T-lymphocytes as they become functionally inactive [16]. As a result, the immune system of patients after chemotherapy is severely impaired, which often leads to the growth of residual tumors and infectious complications. In most cases, taking chemotherapeutic drugs and/or receiving radiotherapy treatment include apoptosis induction, which mediates the cytotoxic effects of the treatment. Generally, such methods have been considered to be non-immunogenic and non-inflammatory. Still, current research has discovered that the “danger signals” from cell death resulted from chemotherapy could potentially be antigen-specific T-lymphocyte immunity, which is TLR4/HMGB1-dependent [16].
Studies have also shown that chemo-radiation can result in local HMGB1 up-regulation in patients with esophageal squamous cell carcinoma (ESCC), as HMGB1’s high expression results in better overall survival of patients [13]. Additionally, respective in vitro studies have reported that there are significant variations in the production of HMGB1 after chemo-radiation procedures with ESCC cell lines; moreover, almost the same numbers of stressed cells have had no influence on this finding. Still, these studies have reported that the HMGB1-induced immune responses after chemo-radiation procedures may significantly influence the status of ESCC patients [17].
Cancer patients with tumors in the form of gastric, lung, rectal and esophageal cancers are treated with chemo-radiotherapy and the outcomes of treatment depend on each patient. For example, the same treatment may result positively for highly responsive patients but may provide weak outcomes for poorly responsive ones. Furthermore, the benefits of such treatment appear to be doubtful, taking into consideration significantly negative side effects of chemotherapy. However, in situations when immunogenic cell death mechanisms have been detected after chemotherapy, such treatment can have two possible applications. The first could be prognostic, having the ability to predict the chemotherapy’s success with the help of respective measurements of HMGB1 and CRT. The second application would be that this could enhance therapeutic benefits with the help of combining ICD with therapies that are aimed to activate the immune system of patients [18].
Rationale/Research Strategy
In addition to the aforementioned factors and research findings related to treating cancer with the help of ICD, according to the respective data from the American Cancer Society, between 2011 and 2013, the actual types of treatment of colon cancer has not changed significantly [19]. Even taking into consideration that the number of previously mentioned theoretic and practical studies reported the positive use of ICD in treating multiple types of cancer, during that period, the most widespread method of treatment for colon cancer was surgery and chemotherapy, depending on the status of the cancer and the patient [19].
In particular, during the carcinoma in situ stage of cancer, the growth of abnormal cells is eliminated by a colonoscopy or polypectomy. Segments of the colon resection are performed in cases of significant sizes of tumors. During the local stage of cancer, surgical resection is used for tumor removal.
In cases where cancer has not reached the lymph nodes, corresponding surgical resection of the colon’s segment that contains the tumor may be the only treatment needed. If the cancer is suspected to infect the colon again, chemotherapy and/or radiotherapy could also be applied. Otherwise, once the cancer reaches the lymph nodes, all the aforementioned measures are performed in order to eliminate the cancer; the difference is that the infected lymph nodes are also removed with the tumor. As for chemotherapy, it is based on fluorouracil (5-FU), an effective drug to improve both the patients’ survival and reduce the recurrence of cancer [20]. In cases of considerably high tumor growth rates, radiotherapy could also be applied.
Often, surgery is aimed to remove colon blockage in order to prevent further complications during cancer metastasis. Still, during this stage, surgery is not recommended for any patient. Instead, radiotherapy, chemotherapy and/or biologically targeted procedures should be given to prolong the patient’s survival along with providing symptom relief. Recently, three monoclonal antibody therapies have been approved by the US Food and Drug Administration (FDA) in order to provide treatment for metastatic colon cancer. For example, Avastin (bevacizumab) have been used to block the blood flow to the specified tumor. Ebitux (cetuximab) and Vectibix (panitumumab) have been used to eliminate the effects of hormone-like factors that influence colon cancer. Nevertheless, in cases where the cancerous tumor appears to be aggressive, some patients have had no benefit from treating their cancer with these drugs [21].
It is clear that the previously widespread methods of treating colon cancer have been treated with the help of surgeries and chemotherapies and/or radiation treatments that also could not guarantee complete recovery from cancer. Thus, the following sections in this paper will consider how ICD could improve the situation with cancer treatments as well as the survivability of cancer patients.
Benefits of ICD in treating colon cancer. The most valuable factor and characteristic of ICD for treating colon cancer is that it restores calreticulin (CRT) exposure, boosts ATP secretion, and stimulates HMGB1 release [22]. There are a variety of strategies designed specifically to restore the exposure of CRT despite the negative impacts of chemotherapy; one is the co-administration of EIF2A phosphatase inhibitors with protein phosphatase 1 (PP1) and its regulatory subunit 15A. Such composition and structure allows the phosphorylation of EIF2A regardless of overt ER stress [22]. CT26 cells, which are a colon carcinoma cell line, have been treated with the help of etoposide (chemotherapy drug), as it is used for a variety of malignant tumors. This drug is a topoisomerase II inhibitor that does not tend to show any traits of CRT exposure, but with the presence of other inhibitors, such as salubrinal and calyculin A, CRT exposure is present [22].
Such co-administration promotes the phosphorylation of EIF2A which has high potential in recovering regulated cell death immunogenicity resulted from anti-cancer agents which do not traditionally stimulate CRT exposure, but result in an increased secretion of ATP and HMGB1 release. Moreover, it has been shown that the inability to stimulate CRT’s translocation to the plasma membrane’s outer leaflet can be corrected with the help of administering recombinant and exogenous CRT. Moreover, in vitro, its absorption on malignant cells attached to cells that are not immunogenic has been reported as completely restoring the ability of these cells to provide an effective treatment [22].
As for the ATP stimulation needed for ICD, there is one widely reported strategy to restore its concentrations; in particular, it is CD39’s pharmacological inhibition [19]. Therefore, CT26 cells that lack vital components necessary for autophagy, including Beclin 1, Atg5 and/or Atg7, tend to secrete some amounts of ATP during their reaction to anthracyclines [23]. Nevertheless, this can be fixed with the help of ARL67156 co-administration, in other words, the administration of extracellular nucleotidase inhibitors. Consequently, such results show that these inhibitors are able to constitute a set of conditions to further stimulate regulated cell death (RCD) immunogenicity, especially in situations that are hardly related to increasing secretion of ATP. What is more important is that autophagy’s pharmacological activation does not traditionally fulfill the requirements for cancer cells to turn immunogenic. Still, in conditions where anti-cancer agents are combined with molecules that perform the up-regulation of autophagic flux, RCD may convert into ICD with high levels of proximity [23]. Nevertheless, thorough research is still needed to better understand these mechanisms.
As for the type I IFN production stimulation during ICD, the possibility to promote it has been defined and introduced only recently; therefore, the requirement of type I IFN signaling also appears to play a role in ICD [24]. In particular, cancer cells react to different anthracyclines with TLR3-elicited signal transduction, paracrine type I IFN signaling, and the secretion of chemokine ligand 10. However, IFNAR1−/− and TLR3−/− exposure to anthracyclines are not able to act as a treatment. Nevertheless, the inability of these cells to perform ICD is also possible to correct with the co-administration of recombinant CXCL10 (motif chemokine 10) or recombinant type I IFNs. Therefore, with the help of these reactions, even non-immunogenic cells could turn immunogenic [24]. However, more clinical and experimental evidence for these reactions are also required to better understand this.
Radiation therapy increases the secretion of HMGB1 and thus induces immunogenic cell death. In colorectal cancer patients, the levels of HMGB1 in the serum following radiation therapy increased. Chemoradiation is known to increase antigen-specific responses [25]. Thus the interaction of HMGB1 and its receptors has a role in promoting immunogenic cell death following radiation therapy in colorectal cancer patients. This interaction can be coupled with radiation therapy for the treatment of cancer [18].
HMGB1’s expression levels significantly vary depending on the type of tumor, which also results into how tumors progress. Therefore, several malignant cells may release the levels of HMGB1 to an extent but may not be compatible with RAGE and TLR4 activation in immune cells [26]. What is more important is that the immunogenicity of such dead cells is proved to be compromised similarly to the cells that were depleted of HMGB1. Therefore, considering these factors, one can conclude that colon cancer cells that secrete ATP, provide CRT exposure along with type I IFNs production but with limited levels of HMGB1 release, will hardly provide any immune responses. Particularly, for colon cancer, as for other cancers, further research is still needed in order to enhance the current ICD mechanisms.
References
Galluzzi L, Vitale I, Abrams J et al. Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012. Cell Death Differ. 2011;19(1):107-120. doi:10.1038/cdd.2011.96.
Green D, Ferguson T, Zitvogel L, Kroemer G. Immunogenic and tolerogenic cell death. Nat Rev Immunol. 2009;9(5):353-363. doi:10.1038/nri2545.
Garg, D.A, Galluzzi, L, Apetoh, L et al. Molecular and Translational Classifications of DAMPs in Immunogenic Cell Death. Front Immunol. 2015. doi: 10.3389/fimmu.2015.00588.
Apetoh L, Ghiringhelli F, Tesniere A et al. Toll-like receptor 4–dependent contribution of the immune system to anticancer chemotherapy and radiotherapy. Nature Medicine. 2007;13(9):1050-1059. doi:10.1038/nm1622.
Fucikova, J, Kralikova, P, Flialova, A. Human Tumor Cells Killed by Anthracyclines Induce a Tumor-Specific Immune Response. Cancer Res. 2011. 71(14):4821-33. doi: 10.1158/0008-5472.CAN-11-0950.
Kroemer G, Galluzzi L, Kepp O, Zitvogel L. Immunogenic Cell Death in Cancer Therapy. Annual Review of Immunology. 2013;31(1):51-72. doi:10.1146/annurev-immunol-032712-100008.
6. Panaretakis T, Kepp O, Brockmeier U et al. Mechanisms of pre-apoptotic calreticulin exposure in immunogenic cell death. EMBO J. 2009;28(5):578-590. doi:10.1038/emboj.2009.1.
6. Michaud M, Martins I, Sukkurwala A et al. Autophagy-Dependent Anticancer Immune Responses Induced by Chemotherapeutic Agents in Mice. Science. 2011;334(6062):1573-1577. doi:10.1126/science.1208347.
7. Galluzzi L, Senovilla L, Zitvogel L, Kroemer G. The secret ally: immunostimulation by anticancer drugs. Nature Reviews Drug Discovery. 2012;11(3):215-233. doi:10.1038/nrd3626.
8. Bianchi M. HMGB1 loves company. Journal of Leukocyte Biology. 2009;86(3):573-576. doi:10.1189/jlb.1008585.
Hori, O, Brett, J, Slattery, T et al. The Receptor for Advanced Glycation End Products (RAGE) Is a Cellular Binding Site for Amphoterin. J Biol Chem. 1995 Oct 27;270(43):25752-61
9. Kepp O, Tesniere A, Schlemmer F et al. Immunogenic cell death modalities and their impact on cancer treatment. Apoptosis. 2009;14(4):364-375. doi:10.1007/s10495-008-0303-9.
Lotze, M. T, Tracey, K.J, High-mobility group box 1 protein (HMGB1): nuclear weapon in the immune arsenal. Nat Rev Immunol. 2005 Apr;5(4):331-42
10. Zitvogel L, Apetoh L, Ghiringhelli F, Kroemer G. Immunological aspects of cancer chemotherapy. Nat Rev Immunol. 2008;8(1):59-73. doi:10.1038/nri2216.
11. Sparvero L, Asafu-Adjei D, Kang R et al. RAGE (Receptor for Advanced Glycation Endproducts), RAGE Ligands, and their role in Cancer and Inflammation. Journal of Translational Medicine. 2009;7(1):17. doi:10.1186/1479-5876-7-17.
12. Igney F, Krammer P. DEATH AND ANTI-DEATH: TUMOUR RESISTANCE TO APOPTOSIS. Nature Reviews Cancer. 2002;2(4):277-288. doi:10.1038/nrc776.
13. Suzuki Y, Mimura K, Yoshimoto Y et al. Immunogenic Tumor Cell Death Induced by Chemoradiotherapy in Patients with Esophageal Squamous Cell Carcinoma. Cancer Research. 2012;72(16):3967-3976. doi:10.1158/0008-5472.can-12-0851.
14. Kono K, Mimura K, Kiessling R. Immunogenic tumor cell death induced by chemoradiotherapy: molecular mechanisms and a clinical translation. Cell Death Dis. 2013;4(6):e688. doi:10.1038/cddis.2013.207.
15. Colorectal Cancer Facts & Figures 2011-2013. 1st ed. Atlanta: American Cancer Society, Inc.; 2011. Available at: http://www.cancer.org/acs/groups/content/@epidemiologysurveilance/documents/document/acspc-028312.pdf. Accessed January 9, 2016.
16. Sargent D, Sobrero A, Grothey A et al. Evidence for Cure by Adjuvant Therapy in Colon Cancer: Observations Based on Individual Patient Data From 20,898 Patients on 18. Randomized Trials. Journal of Clinical Oncology. 2009;27(6):872-877. doi:10.1200/jco.2008.19.5362.
17. Wang G, Kelley R, GAPPNet. KRAS mutational analysis for colorectal cancer Application: Pharmacogenomic. PLoS Curr. 2010;2:RRN1175. doi:10.1371/currents.rrn1175.
18. Obeid M, Tesniere A, Ghiringhelli F et al. Calreticulin exposure dictates the immunogenicity of cancer cell death. Nature Medicine. 2006;13(1):54-61. doi:10.1038/nm1523.
19. Michaud M, Martins I, Sukkurwala A et al. Autophagy-Dependent Anticancer Immune Responses Induced by Chemotherapeutic Agents in Mice. Science. 2011;334(6062):1573-1577. doi:10.1126/science.1208347.
20. Sistigu A, Yamazaki T, Vacchelli E et al. Cancer cell–autonomous contribution of type I interferon signaling to the efficacy of chemotherapy. Nature Medicine. 2014;20(11):1301-1309. doi:10.1038/nm.3708.
Fahmueller,Y. N, Nagel, D, Hoffman, R.T, Immunogenic cell death biomarkers HMGB1, RAGE, and DNAse indicate response to radioembolization therapy and prognosis in colorectal cancer patients. Int J Cancer. 2013 May 15;132(10):2349-58. doi: 10.1002/ijc.27894.
21. Yamazaki T, Hannani D, Poirier-Colame V et al. Defective immunogenic cell death of HMGB1-deficient tumors: compensatory therapy with TLR4 agonists. Cell Death Differ. 2013;21(1):69-78. doi:10.1038/cdd.2013.72.
