Rise of the natural red pigment ‘prodigiosin’ as an immunomodulator in cancer

Cancer is a heterogeneous disease with multifaceted drug resistance mechanisms (e.g., tumour microenvironment [TME], tumour heterogeneity, and immune evasion). Natural products are interesting repository of bioactive molecules, especially those with anticancer activities. Prodigiosin, a red pigment produced by Serratia marcescens, possesses inherent anticancer characteristics, showing interesting antitumour activities in different cancers (e.g., breast, gastric) with low or without harmful effects on normal cells. The present review discusses the potential role of prodigiosin in modulating and reprogramming the metabolism of the various immune cells in the TME, such as T and B lymphocytes, tumour-associated macrophages (TAMs), natural killer (NK) cells, and tumour-associated dendritic cells (TADCs), and myeloid-derived suppressor cells (MDSCs) which in turn might introduce as an immunomodulator in cancer therapy.


Introduction
Cancer is the leading cause of death in 135 countries according to the World Health Organisation (WHO) global health estimates in 2019 [1]. The Global Burden of Cancer (GLOBOCAN) also reported ~ 19 million new cancer cases in 2020 that are anticipated to increase by 47% (~ 28 million) in 2040 [2]. Understanding tumour biology has facilitated the development of targeted therapies; however, tumours display multidrug resistance (MDR) as a significant clinical burden due to heterogeneity [3][4][5][6]. Natural bioactive compounds from various sources (e.g., plants, microbes) have emerged as immunomodulators in diseases, such as diabetes, cardiovascular diseases (CVDs), inflammation, and cancer [7][8][9].
Research deems the use of natural compounds as 'immunomodulators' alongside the advanced understanding of the complex interactions between cancer and the immune system [10]. Immunomodulators boost the immune defences against threats (e.g., infections) or quench the abnormal immune response in immunerelated disorders [11]. Natural compounds are proven to affect immune cells and to enhance anticancer immune responses in vitro and in patients. For example, berries-which contain multiple chemopreventive compounds-enhance the function of natural killer (NK) cells and decrease the number of infiltrating neutrophils in colorectal cancer (CRC) [12][13][14]. Epigallocatechin gallate (EGCG), resveratrol, all-trans retinoic acid (ATRA), curcumin, polysaccharide K (PSK), β-glucans, and carotenoids are also immunomodulators (e.g., elevate NK cells and inhibit myeloid-derived suppressor cells [MDSCs]) [15][16][17][18][19]. Notably, bacteria-based cancer immunotherapy has lured attention thanks to its distinctive and ample components, mechanisms, and benefits to stimulate the host immunity against cancer [20].
Prodigiosin is a secondary metabolite anticancer red pigment that belongs to the "prodiginines" family, and is produced by the Gram-negative bacteria Serratia marcescens ( Fig. 1) [21]. It inhibits the mammalian target of rapamycin (mTOR) pathway and angiogenesis, and induces cycle arrest and apoptosis in cancer cells with minimal or without observed cytotoxicity on healthy cells [22]. Inherent toxicity is one of the major issues with immunosuppressants that prompted researchers to use combined regimens, especially in oncology. Prodigiosin offers interesting possibilities for a combinatorial applications, acting synergistically with cyclosporin A and additively with rapamycin, confirming its distinctiveness and the potential for further development as immunosuppressants [23][24][25]. Of note, prodigiosin analogues have demonstrated a good safety profile without genotoxicity in clinical trials for the treatment of chronic lymphocytic leukaemia (CLL) [26]. Although prodigiosin is a well-established anticancer molecule (Table 1), its immunomodulating and metabolic reprogramming activities were not studied─data are only available for a related compound, prodigiosin 25-C [27][28][29]. Therefore, the current review discusses a compendium of possible immunomodulating and metabolic reprogramming activities of prodigiosin on specific immune cells and their cytokines in cancer. The present review also provides a comprehensive list of target biomarkers for prodigiosin in the tumour microenvironment (TME) (Fig. 2) [30][31][32][33][34][35][36][37][38][39].

The possible role of prodigiosin as an 'immunomodulator' in cancer
Prodigiosin might improve the efficacy of immunotherapy by regulating multiple immune cells (e.g., T cells) and other proteins in the TME (e.g., programmed death ligand-1 [PD-L1]) [83][84][85][86][87]. Genetic mutations that occur during DNA replication and increased genetic instability in tumours, create neoantigens that evoke an immune response [88]. Failure of immune surveillance facilitates tumour growth and progression despite the expression of immunogenic target expression [89].

The role of prodigiosin on immune-associated molecules PD-L1
Immune checkpoint inhibitors provide durable clinical response and have become an important anticancer strategy versus standard-of-care (SOC) [90]. Targeting PD-1/ PD-L1 by antibodies is minimally effective in several cancers, including renal cell carcinoma (RCC) and non-small cell lung cancer (NSCLC) [90,91]. However, research has reported that failure of immune checkpoint inhibition is attributed to an increased mTOR activity [92]. Inhibition of mTORC1 decreased PD-L1 levels in NSCLC cell lines [93]; although, such inhibition increased PD-L1 levels in other tumour models. For example, everolimus upregulated PD-L1 expression in RCC cell lines and in xenografted tumour tissues [94]. These results indicate that PD-L1 expression levels following mTOR inhibition vary based on tumour types. Hence, using prodigiosin as an effective mTOR inhibitor in different cancer types in vitro and in vivo might explain why mTOR inhibition effects PD-L1 expression levels differently.

Heat shock protein 90 (HSP90)
Heat shock proteins are stress hallmarks that are abnormally regulated in cancer to prevent cell degradation and death and preserve the protein structure in a stressful environment [86]. They are essential for the immune system regulatory function in healthy cells; although, cancer cells are drug-resistant due to elevated expression levels of HSP90 [95]. For example, combining bortezomib with HSP90 inhibition improved survival and delayed disease progression in mouse models, and suppressed tumour growth in multiple myeloma (MM) cell culture [96,97]. Moreover, anti-HSP90 treatment improved T-cell killing in melanoma cell lines, and significantly sustained responses with a better safety profile in relapsed/refractory MM (RRMM) patients [96,[98][99][100][101]. Recently, a combination of prodigiosin and the HSP inhibitor, PU-H71, decreased the levels of HSP90α in MDA-MB-231 cells [102]. Among other HSPs, HSP90 stimulates T regs and T helper 1 (Th1) and Th2 cells that support other cells in the immune system. Inhibition of HSP90 using prodigiosin may have the potential to modify T regs and enhance tumour therapy [59].

T lymphocytes
Despite their antigen-directed cancer cytotoxicity, overstimulation of T cells (i.e., T cell exhaustion) causes T cell senescence with defects in effector functions and proliferation, preventing tumour control [121][122][123]. Persistent antigen exposure helps tumours evade the immune surveillance and causes T cell dysfunction, where dysfunctional T cells have multiple inhibitory receptors such as PD-1 [124]. Prodigiosin selectively suppressed the proliferation and immune functions of T cells but not B-cells in vitro and in vivo [125]. However, data are insufficient to confirm whether prodigiosin directly or indirectly inhibited the immune functions of T cells. Prodigiosin also suppressed IL-2Rα expression in the IL-2/IL-2R signalling to block T-cell activation, inhibiting graft versus host disease (GvHD) and delayed the progression of autoimmune diabetes without toxicity in mice [126]. Prodigiosin 25-C (a related compound) directly attacked the activated CD8 + T cells by inhibiting the acidification of intracellular organelles needed for cytotoxic T lymphocytes (CTLs) functions [127]. Prodigiosin represents an effective molecule in an immunosuppressive TME characterised by dysfunctional T cells, and might be an important molecule for immunologic studies on T cells [126]. Studying the extent of T-cell inhibition after treatment with prodigiosin is noteworthy, because deficient T-cell inhibition causes autoimmune diseases, whereas cancers arise due to excessive T-cell inhibition [128].

B lymphocytes
Regardless of the available consensus about the immunosuppressive role of prodigiosin on T-cell proliferation, little is known about its effects on B cells [129,130]. B cells constitute ~ 25% of all cells in some cancers and 40% of tumour-infiltrating lymphocytes (TILs) in breast cancer patients [131][132][133]. B cells destruct tumours by increasing T cell responses and support tumour growth by favouring immunosuppression via complement activation or immune complex formation [134]. Prodigiosin inhibited polyclonal B-cell proliferation and immortalisation in human peripheral blood lymphocytes (PBLs) and Epstein Barr virus (EBV) [130]. The differential response of prodigiosin on T and B cells might be attributed to the source of the cells used in the experiments. For instance, human cells demonstrate selective inhibition of T-cell proliferation compared to mouse cells [23,129,130,135]. Moreover, B cells are heterogeneous and diverse, and might increase T-cell anticancer activities or facilitate carcinogenesis through angiogenesis, inflammation, and immunosuppression [134].
Prodigiosin might modulate the immune response of TAMs by inhibiting TNF-α, IL-2, and interferon-gamma (IFN-γ), reducing TAMs-mediated immunosuppression. Cuevas et al., recently showed that prodigiosin modulated the immune response and stabilised atherosclerotic lesions by inhibiting circulating TNF-α, IL-2, and IFN-γ in vivo (Fig. 3) [158]. Activation of M1 macrophages via IFN-γ is essential in immune function and contributes to tissue damage by proinflammatory cytokines [136]. For example, IFN-γ switched the immunosuppressive TAMs into immunostimulatory cells, potentiating the efficacy of antitumour immunotherapies by generating effector T cells in ovarian cancer [137]. Nevertheless, IFN-γ also conditioned protumourigenic effects in solid tumours and induced lung colonisation and enhanced expression of class I major histocompatibility complex (MHC I)related antigens [159,160]. Prodigiosin also inhibited the onset and progression of autoimmune diabetes in nonobese diabetic mice, and reduced IL-2, IFN-γ, and TNF-α mRNA levels in prodigiosin-treated group without side effects [161]. Nonetheless, prodigiosin did not inhibit the secretion of IL-2 in vitro but inhibited the mitogenic signalling from IL-2, suggesting an unusual mechanism of action [135]. It is important to consider the negative effect of prodigiosin on IL-2, because it is among the most potent inducers of antitumour activity in preclinical studies [162].
Solid tumours are characterised by suppressed antitumour immunity due to high PGE2 levels that reduce apoptosis, and increase tumour growth, invasion, metastasis, and angiogenesis [163][164][165][166]. There is a TAMs-PGE2 reciprocal relationship where TAMs secrete PGE2 that directly inhibits CD4 + and CD8 + T cells' effector function, while PGE2 regulates macrophage polarisation into M2 TAMs [166][167][168]. Activation of the COX-2/PGE2 pathway also stimulates PD-L1 expression via TAMs to inhibit the immune response and promote immune tolerance by modulating T-cell activity and facilitating cancer immune escape [155,169]. Accordingly, it is wise to consider that prodigiosin-related mTOR inhibition may interfere indirectly with the COX-2/PGE2 pathway by decreasing PD-L1 levels (Fig. 4) [93]. However, mTOR inhibition simultaneously upregulates PD-L1 expression in some circumstances such as in xenografted tumour tissues and in RCC cell lines [94].

Tumour-associated dendritic cells (TADCs)
Similar to TAMs, prodigiosin might modulate TADCs immune functions via PGE2. Although DCs initiate T-cell anticancer immune response, malignant tumours possess other types of DCs with reduced migration and accumulation in lymphoid organs that lead to immunosuppressive T cells [170]. High PGE2 levels shift the immunostimulatory DCs into immunosuppressive cells to reduce the proliferation of anticancer T cells by upregulating PD-L1 [170]. Prostaglandin E2 inhibits MHC II expression and upregulates IL-10 via EP2 and EP4 receptors, suppressing DCs' antigen presentation mediated via the COX-2/EP3 signalling [171,172]. The immunomodulatory actions of prodigiosin on TAMs discussed earlier denote that it may affect DCs in the TME. Prodigiosin might reverse TAM-mediated attenuation of tumouricidal and tumour antigen-presenting behaviours occurring to DCs due to the established metabolic crosstalk [142].

MDSCs
Tumours maintain an immunosuppressive TME through high levels of heterogeneous immature myeloid cells, referred to as MDSCs [179]. It is mandatory to target myeloid populations that stop anticancer immunity or activate stimulatory cells that promote antitumour immunity. In addition to ILs and VEGF, PGE2-rich tumoural exosomes induce MDSCs activation and migration and promote MDSCs-dependent tumour growth [179][180][181][182]. Prostaglandin E2 controls MDSCs differentiation and increases their levels, enhancing the stemness of cervical cancer cells in vitro and in vivo (Fig. 4). For example, MDSCs express the four PGE2 receptors (i.e., EP1-4) in tumour-bearing mice [183,184].
COX-2 inhibitors reduce MDSCs levels and delay the burden of primary carcinoma tumour, because PGE2induced COX-2 activates the secretion of endogenous MDSCs-related PGE2 (Fig. 4) [185]. Production of PGE2 in lung and ovarian cancers is correlated with COX-2 expression, promoting recruitment and retention of MDSCs [185,186]. COX-2 inhibitors reduce PGE2 production resulting in decreased levels of MDSC-attracting C-C Motif Chemokine Ligand 2 (CCL2) in vivo, suggesting that blocking COX-2 impedes the development and accumulation of MDSCs [187]. In-silico molecular docking analysis revealed that prodigiosin inhibited COX-2 effectively and could be assessed as an antiinflammatory compound in further research [188]. Inflammation is characterised by high levels of PGE2 through COX-2. Moreover, overexpression of COX-2 (also a downstream target of mTORC1) promoted proliferation and growth of several cancers. Downregulation of COX-2 exerts a protective effect against hyperactivated mTORC1-mediated tumourigenesis caused by loss of tuberous sclerosis complex (TSC) in TSC-null cell [189]. Likewise, the ability of prodigiosin to inhibit the mTOR pathway might prevent COX-2-mediated tumourigenesis via increased PGE2 production (Fig. 4).
Production of PGE2 by MDSCs increases PD-L1 expression in ovarian cancer through the mTOR signalling pathway (Fig. 4). Bone marrow (BM) cells cultured with bladder cancer cells showed significant PD-L1 expression in monocytic MDSCs [183]. Tumour-infiltrating PD-L1 + cells also showed high expression levels of both COX-2 and PGE2 synthase 1 (mPGES1) in tumourbearing mice. Inhibition of mPGES1/COX-2 by prodigiosin may reduce the expression of MDSCs-related PD-L1, arguing that reprogramming PGE2 metabolism enhances tumour sensitivity to immunotherapy.
NK cells The mTOR/PI3K pathway is sensitive to a high number of extracellular signals and is a key regulator of cellular growth, proliferation, and metabolism. Cancer is characterised by an aberrant mTOR signalling that supports tumour proliferation, survival, metabolic programming, and drug resistance [195]. The mTOR signalling pathway enhances glycolysis and mitochondrial function to regulate the metabolism of NK cells. Inhibition of mTOR by rapamycin reduced both IL-2/ IL-12-stimulated glycolysis and IL-2-stimulated levels in mouse NK cells and human NK cell glycolysis, respectively (Fig. 5) [196,197]. For instance, higher glucose transporter 1 (GLUT1) levels that absorb glucose exist following the upregulation of both CD71 and CD98 [198,199]. The anti-mTOR activity of prodigiosin in Table 1 highlight that it might have a pivotal role in the metabolic reprogramming of NK cells [59].
TAMs Macrophages with different polarisation states are also different in glycometabolism. Anaerobic glycolysis provides instant energy to help proinflammatory M1 macrophages eliminate pathogens, whereas mitochondrial oxidative phosphorylation (OXPHOS) generates energy for antiinflammatory M2 macrophages [200]. Therefore, mitochondrial dysfunction impedes M2 repolarisation that inhibits regulatory immune signals, and facilitates tumour angiogenesis, migration, and metastasis [166,201].
T lymphocytes Naïve T cells preserve a resting state using OXPHOS in contrast to activated T cells that grow Fig. 4 The inhibitory effect of prodigiosin on mTOR and COX-2/PGE-2 pathways that further sensitise tumour cells to drugs. COX-2, cyclooxygenase-2; CSCs, cancer stem cells; EMT, epithelial-mesenchymal transition; PGE-2, prostaglandin E2; MDSCs, myeloid-derived suppressor cells; mTOR, mammalian target of rapamycin; TAM, tumour-associated macrophages via glucose and lipid metabolism. Proliferating cells exhibit higher aerobic glycolysis rate, referred to as the Warburg effect where the principal driver of aerobic glycolysis is 'mitochondrial dysfunction' [204,205]. Imbalance of ROS production due to mitochondrial dysfunction destroys cell membranes and DNA, disrupts cell proliferation, induces apoptosis, and inhibits autophagy [206][207][208][209][210]. Prodigiosin fosters the protecting antioxidative function of nuclear factor erythroid 2-related factor 2 (Nrf2) and scavenges ROS in hepatocellular carcinoma (HCC) cells [211,212]. However, it upregulates ROS levels and suppressed proliferation and autophagy in leukaemia cell line. These data suggest that prodigiosin might interfere with be the metabolic reprogramming of T cells via ROS, considering its ROS stimulatory and scavenging roles (Fig. 3).
The mTOR pathway is crucial in upregulating GLUT1 expression in naïve T cells to promote glucose absorption and to improve the immune response [213][214][215]. It also helps Th2 cells' differentiation via the OXPHOS-aerobic glycolysis metabolic transition. Mammalian target of rapamycin complex 1 (mTORC1) is essential for Th1 cells while mammalian target of rapamycin complex 2 (mTORC2) regulates OXPHOS and glycolysis in Th2 cells [216][217][218]. Moreover, the mTOR pathway regulates the production and memory differentiation of CD8 + T cells [219][220][221][222][223]. Consistent with the inhibition of the PI3K/Akt/mTOR pathway by prodigiosin in cancer, it might be involved in the metabolic reprogramming of Th cells and CD8 + T cells [42,48,71,72]. However, these data should be used cautiously because the ability of prodigiosin to suppress the immune functions of T cells has not been confirmed yet.
TADCs Metabolic reprogramming (i.e., decreased OXPHOS and an increased glycolysis) is essential for activation and functions of DCs [224]. Stimulated LPS helps DCs regulate the mTOR signal, stabilise HIF1-α, and increase inducible nitric oxide synthase (iNOS) expression [225,226]. Since prodigiosin inhibits the mTOR pathway and reduces iNOS expression by inhibiting LPS-triggered inflammatory responses, it might prevent the manipulation of the metabolic processes that affect the activation and functions of DCs (Fig. 5) [72,227]. However, the metabolic environment where DCs compete with neighbouring cells for nutrition is difficult to simulate and measure both in vitro and in vivo [203].

Tumour mutation burden (TMB)
Analysis of ~ 5000 mutations from ~ 7000 cancers highlighted that tumour mutation burden (TMB) status in cancer cells-the number of somatic mutations/ megabase of the genome encoding tumours-successfully predicted the efficacy of immune checkpoint blockade [229]. Studies demonstrated that PI3K/mTOR pathway mutations are correlated with TMB status in NSCLC and nasopharyngeal carcinoma [230,231]. Treatment with an mTOR inhibitor (i.e., everolimus) led to tumour shrinkage and disease stabilisation in patients with NSCLC [230]. Prodigiosin may have an interesting role on TMB by inhibiting PI3K/mTOR pathway and P53 (Table 1) [50].

Conclusion
The current review demonstrated the compelling immunomodulatory and metabolic reprogramming activities of prodigiosin on TME-related immune cells (Fig. 5). Particularly, the crosstalk between the immune cells, the involvement of the mTOR pathway, and expression of PGE-2 and COX-2, dictate the potential of prodigiosin as an immunomodulator in the TME. Using prodigiosin to inhibit the PI3K/mTOR pathway may clarify its hypothesised effects on TMB, especially because the TMB status associated with PI3K/mTOR pathway gene mutations is still unclear.

Future perspectives
Further research may confirm whether prodigiosin has a compensatory mechanism that overcomes cancer resistance, and whether it renders B cells pro or antitumourigenic. More data are required to examine the controversial role of prodigiosin in blocking T-cell activation by suppressing IL-2Rα expression in the IL-2/IL-2R signalling. It is also important to focus on prodigiosin encapsulation in nanoparticles because limited research demonstrated that it might be an excellent alternative in cancer treatment. Based on the crosstalk between immune cells, using prodigiosin in cancer immunotherapy elicits outstanding inquiries such as: • Could prodigiosin be used as a tool to identify the point at which immune tolerance occurs? • What is the dominant role (immunostimulatory/ immunosuppressive) of prodigiosin on every immune cell in the TME? • How could prodigiosin modulate NK-DC crosstalk and strengthen both DC-and NK-cell-mediated immune response?