Immunogenic Cell Death: The Role of RAGE
Introduction
Millions of cells are always being removed in the human bodyin 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 evidence 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]. Anthracyclinesare are known to be active against many solid tumors. Their activity is very similar to that of topoisomerase II inhibition, which takes place due to its intercalation of anthracycline between adjacent DNA base pairs. However, there has been a hindrance to use anthracyclines because sometimes tumors become resistant to them and cause toxicity. Anthracycline tumor resistance is due to several mechanisms,one of which includes P-glycoprotein mediated resistance, where hydroxyl free radicals are produced and can make anti tumor effects and toxicity effects, especially to myocardial tissues which are easily damaged by free radicals.The effects of anthacyclines include acute myelosuppression, cumulative dose related cardio toxicity, nausea, vomiting, and inflammation of the mucous membrane of the mouth(stomatitis). Anthracyclines also cause effects such as cardiomyopathy,which is irreversible and often leads to congestive heart failure [27].A study conducted by Volkova et al in 2011 indicated that cardiotoxicity caused by chemotherapy can either be of type I or type II. Type I cardiotoxicity can be directly attributed by ICD and is irreversible, while type II is caused by cardiomyocyte dysfunction which is reversible. However, understanding the cardiotoxic etiology from anthracyclines has since led to developing strategies to deal with these cardiac damages in case they do end up occurringin some patients [33].
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. HSP70 is integrated into artificial bilayer openings of ion conductance pathways and it translocates into the plasma membrane after stress. It is released within membranous structures from intact cells[28]. Anthracyclines also induce immunogenic cell death by HSP90 and calreticulin to the surface of tumor cells. These three endoplasmic reticulum (ER) chaperones translocate to the cytosol where they eventually reach the cell surface mostly during ER stress, which may be induced by several factors including irradiation by UV, microbial stimuli, and drugs. Once in the extracellular space, they take on immunogenic characteristics to determine whether or not a cancer cell is targeted for cell death. This mechanism is an important target avenue for cancer cell elimination by the immune system. An underlying autoimmune condition such as multiple sclerosis and rheumatoid arthritis can sometimes be exacerbated by the release of these chaperones. Therefore, much caution and monitoring is required on the role of extracellular chaperones as potential cancer immunotherapies, especially if the patient has an autoimmune disease [29].
HSP70, HPS90, and calreticulin 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), which can bind to several pattern recognition receptors such as Toll-like receptor 2 and 4 (TLR2 and TLR4), and receptor for advanced glycosylation end products (RAGE). HMGB1 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 purigenic 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 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 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 the tumormicroenvironmentfacilitating tumor-antigen engulfment by DCs, and presenting the antigen to T-lymphocytes. These processes result in an immune response mediated by interferon (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 inside of the ER during ER stress from chemotherapy transfers itself into the plasma membrane’s outer surface (known as ecto-CRT) before onset of apoptotic cell death [7]. Ecto-CRT appears to be a potent signal of engulfment because itdestroys dendritic cellsfrom tumors. However, the CRT exposure 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].
Autophagy appears to be obligatory for stressed cells to be considered immunogenic. Autophagy-deficient tumor cells release 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 decrease immunogenic cell death. The opposite is the case for autophagy-competent tumor cells. This phenomenon can be corrected by the inhibition of extracellular ATP degrading enzymes. The inhibition of such enzymes causes an increase in ATP in tumor cells, which allow for the recruitment of dendritic cells [8]. Therefore, autophagy is essential for ICD so that ATP is released. Furthermore, it is important to note that increased concentrations of extracellular ATP providepotential 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].
In vitro, ICD has been seen in tumor cells by launching a response from the immune system and thereby protecting the body from tumor cells. For ICD occurring in vivo, it also elicits a local immune response with the recruitment of cognate and innate immune system effector cells into the tumor bed, which results in tumor growth inhibition. Scientists have considered various preclinical models and have provided evidence for 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 receptor, RAGE [10]. HMGB1 is a small protein that possesses three structural domains: 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 an 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, recombinant amphoterin from rat was purified and was shown to bind to RAGE in the same manner. 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].
HMGB1 binding to RAGE leads to the activation of the IKK [inhibitor of NF-κB (IκB)] kinase complex that phosphorylates IκBα, which takes place when inactive NF-KB residues in the cell’scytoplasm are bound to its inhibitor proteins. Inflammatory cytokines activate the IKB kinase complex, which is composed of IKK1, IKK2, and NF-Kb essential modulator (NEMO); the HMBG1 binding to RAGE phosphorylation is therefore through the activated IKK [30]. Thisphosphorylation induces the release of NF-κB and its translocation to the nucleus.Thus, the transcription of pro-inflammatory genes (such as interleukin-1 (IL-1), IL-6 and tumor-necrosis factor) is induced, as shown in figure 1.
1Fig.1: HMGB1-RAGE pathway.
1Lotze, 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
As a result, phagocytized particles are intracellularly organized for presentation at the cell surface, which stimulates the cytotoxic response of T-lymphocytes, as response to tumors [9]. Recent studies haveshown 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 demonstrated that suppression of HMGB1 and RAGE by antisense S-oligodeoxynucleotide imparts tumor proliferation and migration. The same has been observed 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 need to be elucidated in order to counteract this effect. HMGB1 and RAGE’s over expression is vital for tumor metastasis, according toevidence obtained 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), asHMGB1’s high expression results in better overall survival of patients [13]. Additionally, 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 of gastric, lung, rectal and esophageal cancers are treated with chemo-radiotherapy and the outcomes of treatment are patient-specific. For example, the same treatment may yield positive outcome for some patients but only elicit weak responses forothers Incases where immunogenic cell death mechanisms have been detected after chemotherapy, treatments might have a prognostic value, having the ability to predict the chemotherapy’s success with the help of respective measurements of HMGB1 and CRT. Another application would be that ICD may enhance therapeutic benefits by combining ICD with therapies that activate the immune system of patients [18].
As mentioned, calreticulin exposure dictates the immunogenicity of cancer cell death. ICD leads to changes in the cell surface composition occurring in temporal sequences, leading to the stimulation and presentation of tumor antigens [31].Currently, ICD is being studied as a potential benefit for the treatment of colon cancer [22]. 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, in colon cancer, 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 emerge in 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), which is an effective drug to improve patients’ survival and reduce the recurrence of colon 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,some cancer patients with aggressive tumors have had no benefited from these drugs[21].
According to recent research, it has been found that apoptosis can be immunogenic under certain conditions. However, immune effector cells during the pre-apoptotic stage can attack pre-apoptotic tumor cells. DNA damage elements produce signals that lead to tumor cell immunogenicity, such as the p53 activation. ER elements such as eukaryotic initiation factor 2A phosphorylation produce similar signals as well. Tumor cells responding to chemotherapy therefore release immune-stimulatory factors that stimulate innate immune effector cells. It is therefore anticipated that the mechanisms governing the immunogenicity of cell death will have a major impact in the design of anticancer therapies [34].
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 howICDcould improve the situation with cancer treatments as well as the survivability of cancer patients.
Benefits of ICD for treatment of colon cancer.The most valuable factor and characteristic of ICD for treating colon cancer is that it restores calreticulin (CRT) because it becomes exposed on the cell surface before cells manifest any apoptosis signs. ICD also boosts ATP secretion and stimulates HMGB1 release [22].The death of cancer cells depend on the initial stimulus. The initiating stimulus can be of two types: immunogenic stimulus or non-immunogenic stimulus. ICD leads to cell surface changes and the release of soluble mediators, which take place in a particular sequence. Tumor antigens are presented to T cells, which are then stimulated by dendritic cell receptors. Dendritic cellsthemselves are regulated by signals produced by cell surface changes. ICD therefore constitutes a very important pathway that leads to the activation of the immune system against cancer cells, which is a major factor to the success of cancer therapies.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. These proteins have the ability to translocate calreticulin (CRT)[32],and such composition and structure allows the phosphorylation of EIF2A to the surface of the cell from the ER lumen martins regardless of overt ER stress [22]. CT26 cells, which are a colon carcinoma cell line, have been treated with etoposide (chemotherapy drug), as it is used for a variety of malignant tumors. This drug is a topoisomerase II inhibitor that, alone show CRT exposure, but combined with other inhibitors, such as salubrinal and calyculin A, results in CRT exposure [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].
ICD requires ATP stimulation to restore its concentrations Particularly the CD39’s pharmacologigal inhibition [19]. During anthracycline reactions, some amounts of ATP are secreted by CT26 cells that lack vital components necessary for autophagy, including Benclin 1, Atg5 and/or Atg7 [23].
Nevertheless, this can be fixed with the help of ARL67156 co-administration, in other words, the administration of extracellular nucleotidase inhibitors.
These results indicate that these inhibitors are able to constitute conditions necessary 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, traditionally, autophagy’s pharmacological activation does not fulfill the requirements for cancer cells immunogenicity. In conditions where anti-cancer agents are combined with molecules that perform the up-regulation of autophagic flux, RCD may convert to ICD with high levels of proximity [23]. Further research is however needed to better understand these mechanisms.
Production of Type I IFN and subsequent stimulation during ICD, and its promotion has been defined and introduced only recently; therefore, the requirement of type I IFN signaling plays a KEY 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 isn’t a better treatment route. 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 ICD. In colorectal cancer patients, the levels of HMGB1 in serum following radiation therapy are increased. Chemo-radiation 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 vary significantly depending on thetype of tumor, which may also control tumor progression. Therefore, some malignant cells release some levels of HMGB1 to an extent which 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 has been proven to be compromised just like 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 types of cancers, further research is still needed in order to enhance the current ICD mechanisms.
RAGE:HMGB1 interaction as evidence for treating colon cancer
In a study by Fahmueller et al [25], ICD and RAGE implications were shown specifically for the treatment of colon cancer [25]. These scientists chose 49 patients with colorectal cancer who also had liver metastases. The patients received treatment in the form of radio-embolization (RE) for two years. Additionally, all patients had received surgical resection along with adjuvant chemotherapy and all had liver metastases [25]. During the treatment procedure of RE therapy and after it, patients did not receive any additional cytotoxic or cytostatic therapy [25].
Fahmueller et al dded CYFRA 21-1, CA 19-9 and CEA tests for the patients in order to take into consideration nucleosome activity in the liver. These analyses included C-reactive protein (CRP), lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), choline, lipase and amylase esterase and alkaline phosphatase (AP) due to their prognostic abilities in univariate analyses [25].
Fahmueller et al collected blood samples 24 hours before REand 48 hours after receiving RE. They found that the concentration of HMGB1 was evaluated for each patient according to a sandwich ELISA method. The RAGE levels of the patients were also evaluated by sandwich ELISA. HMGB1 serum concentrations were increased significantly in 24 hours after RE returning back to pre-therapeutic levels 48 hours later. On the contrary, the levels of RAGE weredecreased in both 24 and 48 hours after RE, with activity of DNase remaining nearly at stable levels [25].
Before receiving RE, there was a correlation between HMGB1 and CEA, but not with nucleosomes, activity of DNAse, or RAGE. In 24 hours after receiving RE, there was a strong correlation of HMGB1 and the nucleosomes of cell death biomarkers. In addition, post-RE correlated with CEA and CYFRA 21-1, but did not correlate with DNAse or RAGE. The same correlations have been registered for HMGB1 with CEA and with nucleosomes 48 hours after RE [25].
RE responses after 3 months led to only 14 patients showingeither some levels of remission or no changes in their disease. The rest of them were reported as deceased or developed progressive disease either in liver or in other organs. [25].
Analyzing these ICD markers, the HMGB1 levels before therapy have been reported as considerably higher for non-responsive patients in comparison to responsive patients. The levels of DNAse and RAGE, however, have not been reported to significantly change in this patient pool. However, the radiologic response of the patients to therapy significantly correlated with their time of survival. Moreover, it has been reported that high levels of serum HMGB1 were closely related to low survivability of the patients with the rest of the parameters showing no significant association [25]. Thus, it can be concluded that HMGB1 has been proved a very important marker in the prognosis of colorectal cancer. Moreover, Fahmueller et al has stated that HMGB1 levels along with CRP in the liver at 24 hours post-RE was the most effective predicting model.
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