How Macrophages Become Transcriptionally Dysregulated: A Hidden Impact of Antitumor Therapy

Tumor-associated macrophages (TAMs) are the essential components of the tumor microenvironment. TAMs originate from blood monocytes and undergo pro- or anti-inflammatory polarization during their life span within the tumor. The balance between macrophage functional populations and the efficacy of their antitumor activities rely on the transcription factors such as STAT1, NF-κB, IRF, and others. These molecular tools are of primary importance, as they contribute to the tumor adaptations and resistance to radio- and chemotherapy and can become important biomarkers for theranostics. Herein, we describe the major transcriptional mechanisms specific for TAM, as well as how radio- and chemotherapy can impact gene transcription and functionality of macrophages, and what are the consequences of the TAM-tumor cooperation.


Introduction
Tumor-associated macrophages (TAMs) are essential components of the tumor microenvironment, along with other immune cells, fibroblasts, and neovasculature [1,2]. Macrophages, including TAMs, interact with the surrounding milieu and exhibit functional diversity with the specific release of pro-and anti-inflammatory cytokines and growth factors [3][4][5][6][7][8]. TAMs represent a mixed cell population, which includes pro-and antiinflammatory activated macrophages and newly infiltrated macrophages and monocytes that migrate from the surrounding tissues or through the blood vessel wall, respectively, and undergo further alterations in the tumor microenvironment. Of interest, phenotypically and functionally, TAMs are more similar to tissue-resident cells, such as alveolar macrophages (lungs), Kupffer cells (liver), microglia (the central nervous system), and others, depending on the tumor location, but not to blood-derived monocytes.
Additionally, the M1/M2 ratio and the exact cytokine secretion profile of macrophages vary with respect to cancer stage and tumor microenvironment [53,54]. With the disease progression, the number of M2-like TAMs gradually increases in the tumor microenvironment due to its immunosuppressive activities and abnormal blood vessel fenestration, as it is shown for lung carcinoma, cutaneous melanoma, colorectal, prostate, and ovarian cancer [55][56][57][58][59]. Moreover, the transition from acute to chronic inflammation, which occurs at the late stages, reduces antitumor immunoreactivity and increases growth factor production with subsequent tumor growth and metastasis [60,61].
The pro-or anti-oncogenic function of macrophages is precisely controlled by the specific TFs, which can be used as additional diagnostic and immunotherapeutic tools ( Figure 1) [13,62,63].
While therapeutic responses are modulated by TAMs, chemo-and radiotherapy also show direct and indirect effects on macrophage survival and activity. The direct action is associated with the off-target cytotoxicity and pro-or anti-inflammatory activation of macrophages. The majority of TAMs are polarized to M2-like phenotype and can release numerous tumor-promoting factors before the treatment. Radio-or chemotherapy can prevent these undesired events reducing M2 macrophage numbers and attracting the bloodderived monocytes with the higher anti-tumor potential. The monocyte infiltration and proinflammatory signaling are supported via pro-inflammatory cytokines and chemokines (ex., CCL2) from the tumor microenvironment (Figures 1 and 2) increased blood vessel wall permeability and signaling from dead and damaged cancer cells [87,162].
The indirect mechanisms are caused by the incoming signals from (a) the damaged, dying, and, at the later time points, resistant cancer cells and (b) the altered tumor microenvironment. Upon chemical and irradiation exposure, surviving cancer cells produce pro-tumor cytokines as IL-17, stromal cell-derived factor-1 (SDF-1), CCL2, and colonystimulating factor 1 (CSF-1), which limit the therapeutic benefits [44, [163][164][165]. At the same time, cancer cell damage and ROS generation induce proinflammatory macrophage activities, as phagocytosis, antigen presentation, and production of proinflammatory factors such as inducible nitric oxide synthase (iNOS) and TNF-α, and lymphocyte chemoattraction [85,124,125,[166][167][168]. With that, the therapeutic impact on TAMs remains controversial with possible pro-or anti-inflammatory polarization and hardly predictable outcomes.
As macrophage polarization and cytokine production rely on the cell transcriptional machinery, we aim to discuss the major TFs involved in cytokine network regulation-NF-κB, STAT-family, IRF-family, and p53,-in the context of the tumor microenvironment and chemo-and radiotherapy (Figures 1 and 2, Table 3).
NF-κB plays a critical role not only in macrophage polarization but also in their metabolism and surveillance in the tumor microenvironment upon chemotherapy. Such effects of chemotherapy are mediated via caspase-8-dependent apoptosis, which is selectively activated in monocytes and tumor-associated phagocytes upon trabectedin treatment and in M2-like TAMs upon platinum-containing drug exposure) [85,[181][182][183][184]. Upon chemotherapy, NF-κB acts as an important proinflammatory inductor and regulator of macrophage viability.

NF-κB and Radiotherapy
Radiotherapy can shift the balance between NF-κB subunits depending on the irradiation dose (Figure 2). Low radiation doses increase the nuclear translocation of p50-p50 homodimer and inhibit p65 translocation, thereby reducing IL1B expression and proinflammatory macrophage activity [173]. Moderate doses (5-10 Gy) preferentially stimulate p65-p50 transcriptional activity in macrophage thereby reprogramming them into M1 phenotype with increased TNF-α, IL-6, IL-8 and reduced EGF [68]. High irradiation dose utilizes p50 subunit for M2 polarization and maintenance of the immunosuppressive microenvironment following radiotherapy [153]. NF-κB mediates pro-survival signaling in macrophages exposed to 10 Gy and higher cumulative doses [148]. This mechanism partially protects macrophages from irradiation. The preferred strategy for NF-κB implementation in radiotherapy is to target p50/p65 and to consider the doses of irradiation.

STATs and Chemotherapy
Chemotherapy has a strong impact on macrophage STAT activities resulting in TAM abundance and phenotypic alterations. Among the routinely applied anticancer drugs, cisplatin and carboplatin increase STAT3 and STAT6 activity and M2-like phenotype of the TAMs [85]. Doxorubicin treatment alone stimulates STAT6 and is also associated with anti-inflammatory effects. At the same time, combined therapy with doxorubicin and cyclophosphamide or EGFR inhibitor lapatinib implies M1-associated STAT1 to stimu-late and prolong macrophage antitumor activity [87]. Imatinib and paclitaxel inhibit the STAT6 pathway and M2-like cytokine production in macrophages showing, thus, proinflammatory potential [4,188]. Taken together, STAT TFs exhibit multidirectional effects in TAM functionality due to counterplay between the various STAT members, NF-κB, and other TFs.

STATs and Radiotherapy
Various radiotherapeutic strategies have a differential and, sometimes, non-specific impact on the members of the STAT TF family [172]. The generalized effect of X-ray and γ-photon radiotherapy is STAT3 activation, which is observed in TAMs in response to all clinical doses of radiation. Irradiation promotes IL-6 production by the tumor microenvironment, which results in STAT3 phosphorylation and subsequent anti-inflammatory CCL2, CCL4, VEGF, and TGF-β cytokine production [149,158,160,189]. It is worth noting that STAT3 signaling also promotes cell survival after irradiation exposure via induction of anti-apoptotic proteins (survivin and Bcl-2), and this effect is more profound for M2-like TAMs [158,160,190]. The low radiation doses show a bidirectional impact on the anti-inflammatory TFs, comprised of STAT3 stimulation, as mentioned earlier, and STAT6 suppression with high IL-5 and 13 and low TGF-β cytokine profile [150,156]. Considering that NF-κB is also suppressed upon low-dose radiotherapy, macrophages may finally acquire anti-inflammatory characteristics, although this has to be further studied. Intermediate radiation doses stimulate the transcriptional activities of STAT1, STAT3, and STAT6 [172]. The immunomodulatory effect of such treatment is the most difficult to control, as it simultaneously triggers intracellular pro-and anti-inflammatory signaling pathways. The functional outcome likely depends on individual cell characteristics (time after infiltration of the tumor), the other TFs impacted by radiotherapy (for example, NF-κB) and tumor microenvironment [191]. Finally, high-dose radiation activates STAT6 and therefore has the most pronounced anti-inflammatory effect [149,156].

Interferon Regulatory Factor (IRF)
The IRF family is represented by nine members ranging from IRF1 to IRF9. IRFs promote host defense against viral and microbial pathogens by regulating type I and II IFN-responsive genes. IRF TFs are also active in TAMs and are linked to the pro-and antiinflammatory cytokine production [192]. IRF3, 5, 7, and 8 are involved in proinflammatory macrophage polarization and control of chemokine (S100A8, S100A9S100a9, matrix metallopeptidase (MMP) 9 and 14, CXCL2, and CCL5) production [65,75,193]. IRF3, along with IRF4, can also exhibit an alternative activity inducing the expression of anti-inflammatory genes IL1RA, IL10, IFNB [76,194].

IRFs and Chemotherapy
IRF TFs strongly echo the STAT-mediated transcription in macrophages and it becomes challenging to evaluate exclusively IRFs contribution to macrophage function. The majority of data show that chemotherapy-induced IRF activation supports the tumoricidal activity of macrophages. For instance, IRF5 activation and, subsequently, macrophage proinflammatory functionalization are induced by PARP inhibitor olaparib [145]. However, some studies show that IRFs are involved in M2-like polarization following chemotherapy and can be associated with resistance [200,201].

IRFs and Radiotherapy
The IRF input into macrophage polarization upon radiotherapy remains poorly investigated. In fact, according to the authors' knowledge, alterations in IRF1, 2, 3, and 5 activity were reported in irradiated macrophages so far [72,73,202]. Low dose-irradiation induces ROS generation with subsequent ATM activation to stimulate IRF5 expression and M1like macrophage polarization with increased IL-6, TNF-α, and IFN-γ [145]. Furthermore, IRF5 cooperates with the NF-κB Rel-A subunit, regulates proinflammatory cytokine gene expression, which can result in M1 polarization. However, the IRF5-NF-κB interactions have not yet been studied in irradiated macrophages [71]. Moderate dose-radiotherapy stimulates IRF1 and 5 revealing the potential proinflammatory effects. High radiation doses reduce IRF2 and 5 levels increasing numbers of M2-like macrophages in the tumor [151]. At least for the high-dose irradiation IRF1 is suppressed in monocytes, while upregulated in macrophages, suggesting the different responses from infiltrated cells and TAMs [72,73].

P53
Despite the extensive studies on the tumor suppressor p53, its roles in immune cells, including TAMs, are not fully understood. It is known that p53 regulates inflammatory responses in the tumor microenvironment. In macrophages p53 is involved in cell survival and death, monocyte-to-macrophage differentiation, and M1/M2 polarization [52, 74,203,204]. P53 is up-regulated and activated following both pro-and antiinflammatory macrophage activation and promotes pro-apoptotic pathways predominantly in M1 cells [205,206].

P53 and Chemotherapy
P53 is involved in cell responses to cytotoxic, cytostatic, and targeted drugs, which impact macrophages. Doxorubicin, methotrexate 5-fluorouracil, and other chemotherapeutic compounds induce p53 activity in TAMs with subsequent increase in p53 target p21 and IL6 expression. The p53-driven proinflammatory polarization results in tumor sensitization to chemotherapy and is specific for monocyte/macrophage subsets [140]. Among the targeted drugs, nutlin-3, which inhibits p53/MDM2 interaction and stabilizes p53, MDM2 inhibitor APG-115, and anti-VEGF receptor 3 activate p53 and promote p53-NF-κB cooperation to stimulate antitumor macrophage reactivity [52,210,211,215]. P53 maintains the physiological levels of programmed cell death protein 1 (PD-1) ligand, restricts excessive extracellular vesicle formation, and subsequently limits T-cell exhaustion and immunosuppression. [216,217]. With that, p53 can become a multifaceted target for immunotherapy.

P53 and Radiotherapy
As a pro-apoptotic protein, p53 sensitizes cells to radiation, and this effect is also present in irradiated macrophages. P53 becomes activated in macrophages during radiotherapy and is associated with TAM polarization and modulation of cell survival. However, the impact of different irradiation doses has not been studied. At least the intermediate (4-5 Gy) irradiation increases p53 protein levels and its transcriptional activity together with the antitumor (TNF-α, FasL) and tumorigenic activities (MMP-2, MMP-9) of macrophages [218,219]. P53 is directly linked to macrophage survival, as TP53-/-macrophages are highly radioresistant due to reduced caspase-8 expression [220]. Interestingly, p53 proapoptotic activity is stronger in M1, than in M2, macrophages, which correlates with the improved radioresistance of M2 cells [221]. Thus, p53 may regulate the balance of pro-and anti-inflammatory macrophages in the irradiated tumor microenvironment [221,222]. P53 plays an important role in macrophages simultaneously regulating cell functional activities and survival during chemo-and radiotherapy. These facts have to be considered when applying p53-targeted therapy.

Other Transcription Factors Affected by Radio-and Chemotherapy
Transcriptional networks essential for macrophage polarization include a broad range of TFs, which are also involved in therapeutic responses. When summarizing the existing data it can be observed that various treatments tend to impact M2-associated TFs, such as nuclear factor erythroid 2-related factor 2 (Nrf2), peroxisome proliferator-activated receptor gamma (PPAR-γ), cAMP response element-binding protein (CREB), and some others, which may be due to the immunosuppressive properties of the tumor microenvironment. However, a substantial number of transcriptional mechanisms have not yet been studied in the context of chemo-and radiotherapy.
Nrf2 shifts macrophages toward anti-inflammatory polarization altering cytokine, growth factor and cell adhesion molecule profiles [223][224][225]. Nrf2 deficiency in myeloid cells is associated with the enhanced metastatic profile of the tumor and tumorigenic immune activity [226,227]. The Nrf2 activation orchestrated by p21, which is observed after γ-irradiation, is dose-dependent, reduces oxidative stress in macrophages, and may, thus, protect the cells against γ-ray damage [228]. While the Nrf2 activities in chemotherapy have not yet been studied, Nrf2 controls local tissue inflammation and can potentially protect TAMs from drug-induced oxidative damage [229,230].
Peroxisome proliferator-activated receptors (PPARs) comprise another family of antiinflammatory TFs [231]. Macrophage-specific PPAR-γ impairs the chemotherapeutic efficiency [232]. During radiotherapy, PPAR-γ is involved in macrophage activation by irradiated cancer cells and immunogenic antitumor activity thereby reducing cancer progression and metastasis [233]. Thus, regulators of PPAR-γ may emerge as promising candidates in targeting both cancer cells and the tumor microenvironment [234].
Other candidate TFs, which can shape macrophage behavior, include cAMP response element-binding protein (CREB) and CCAAT-enhancer-binding proteins (C/EBPs) TF family [235][236][237][238]. The phosphorylated CREB inhibits NF-κB activation, thus limiting proinflammatory responses [239,240]. Furthermore, overexpression of CREB in committed macrophages provokes myeloproliferative diseases [241]. The C/EBP family consists of six members, from C/EBP-α to C/EBP-ζ, which may function in opposite ways. For instance, C/EBP-α and C/EBP-δ are one of the most important TFs that contribute to the M1 polarization, whereas C/EBPβ is associated with M2 macrophage polarization [11,242]. Furthermore, C/EBPβ activated by vitamin D3 or its derivatives can aggravate alternative macrophage polarization [225,243]. Interestingly, C/EBPβ is downstream of the rapamycin kinase (mTOR) pathway which is a target of immunosuppressive and anticancer drugs [244,245]. Therefore, mTOR targeting drugs should be considered in the framework of macrophage polarization and could represent a novel therapeutic approach. Highmobility group box protein 1 (HMGB1) is a chromatin-binding factor that promotes M2 polarization by activation of the receptor for advanced glycation end-products (RAGE) [246]. High expression of HMGB1 in TAMs has been shown to enhance lymphatic metastasis [247]. Current observations show opposite effects after radiotherapy; HMGB1 release can lead to immunosuppression and potentiate macrophages reprogramming towards M1 phenotype [166,248]. Chemotherapy stimulates HMGB1 production to promote antigenpresenting function of immune cells [249].
Many other factors that have an impact on TAM polarization have been not yet sufficiently investigated. For example, the Maf family consists of a wide number of TFs including V-maf musculoaponeurotic fibrosarcoma oncogene homolog B (Mafb) and c-Maf.
Both of them are highly expressed in TAMs and correlate with anti-inflammatory responses in both human and murine models [250][251][252]. At the same time, JunB governs functional behaviour of both pro and anti-inflammatory macrophages. JunB upregulates IL-1β in proinflammatory polarized macrophages while enhancing anti-inflammatory markers in M2-like cells [253]. MAP kinase-interacting kinase (Mnk2) promotes education of M2 phenotype through induction of anti-inflammatory marker translation by activation of eukaryotic translation initiation factor (eIF4E) [254]. All of them are promising research targets in the framework of resistance overcoming.

Macrophage Transcription Factors in Antitumor Therapy
Numerous preclinical and clinical studies confirm the promising effects of multitargeted combinations based on radio-, chemo-, and immunotherapy in cancer medicine [255,256]. The most prominent treatment effects on monocyte/macrophage subsets are associated with the increased immune cell recruitment to the tumor site and promoted antigen presentation, which are now recognized as essential components of the tumor microenvironment [257].
TF-based macrophage regulation can be considered as one of the most potent and efficient strategies against tumor therapeutic resistance. As described previously, a substantial number of chemical agents are already available for TAM transcriptional reprogramming. For NF-κB-based approaches target deletion of IκB kinase β (IKKβ) or inhibition of downstream PI3K significantly improve antitumor activities [273,274]. TLR agonists can be used for stimulation of NF-κB-mediated transcription, as shown for alveolar macrophages and monocytes (paraquat), for lung, breast, and melanoma cancers (chloroquine), for melanoma and squamous cell carcinoma (imiquimod) [275][276][277]. Cancer-associated chronic inflammation may request the application of nonsteroidal-based anti-inflammatory drugs (NSAIDs), such as acetaminophen or sulindac, to suppress undesired NF-κB-promoted production of growth and metastatic factors. Furthermore, NF-κB inhibition can prevent cisplatinand carboplatin-induced M2-like phenotype in cervical and ovarian cancer [85]. Other possible additives for oncotherapy may implement salicylates (aspirin, sulfasalazine, triflusal), antioxidants (pyrrolidine dithiocarbamate, N-acetylcysteine, vitamin E, vitamin C), and peptides (SN50, nuclear localization signal peptide, NEMO-binding domain peptide, Toll/interleukin-1 receptor domain-containing adaptor protein) for non-specific NF-κB regulation [278][279][280].
STAT family-based immunotherapy can enhance the effects of γ-radiation via regulation of macrophage subset and cytokine balance in the tumor microenvironment [281]. For instance, tyrosine kinase inhibitors (sunitinib, sorafenib), WP1066, plant-derived imodin (PM37), and resveratrol inhibit STAT TF activity in macrophages and restricts M2 polarization, suggesting the improved antitumor reactivity against pancreatic adenocarcinoma, breast, and lung cancer [49, 160,191,258,[282][283][284]. Additionally, STATs, IRFs, NF-κB, and c-MYC mediated polarization can be regulated by microRNAs. MicroRNAs regulate gene expression through translation repression or mRNA degradation. It has been shown as a prospective macrophage-centered diagnostic and therapeutic strategy [22].
In conclusion, macrophage TFs are among the prospective diagnostic markers and therapeutic targets, which allow repolarize TAMs and impact tumor microenvironment preventing tumor resistance. In macrophage-based immunotherapy, various immunomodulators can be applied to optimize the chemo-or radiotherapeutic outcome and prolong the treatment benefits. Currently NF-κB, STAT, and IRF transcriptional machinery, which is implied in cancer and immune cell functionality, present the most substantial potential for cancer immunotherapy. Tumor status and disease type have to be considered for preferential pro-or anti-inflammatory alterations.

Conflicts of Interest:
The authors declare no conflict of interest.