1. INTRODUCTION

Cancer immunotherapy is conceptually based on the therapeutic stimulation of cancer immunosurveillance, a former theory which stated that tumor cells, mostly through the phenotypic alteration and the repertoire of tumor-associated neoantigens they often present, can be recognized and targeted by the immune system in the attempt to prevent disease progression. Nowadays, the dynamics of cancer and immune system cross-talk have been unequivocally unveiled to be extremely complex and has been renamed as cancer immunoediting. According to the three E’s theory, this process comprises three defined phases: elimination of cancer cells by the immune system; equilibrium between tumor growth and control by the immune system; escape of neoplastic cells from immunosurveillance. The first two phases in some way coincide with the concepts described by the former theory, but the second phase can last long, running asymptomatically, until it is overrun by the third one, which results in tumor growth and dissemination. In an immunocompetent host, this breakthrough results from a positive selection of tumor clones that are able to evade the inhibitory control of the immune system by downregulating/masking antigen epitopes or by increasing the immunosuppressive properties of the tumor microenvironment [1,2].

Modern approaches of cancer immunotherapy, designed to restore a robust degree of immune activity against tumor cells, encompass immune checkpoint blockade, adoptive cellular therapies, and cancer vaccines [1-5]. Among these therapeutic interventions, immune checkpoint inhibitors (ICKi) have substantially revolutionized the oncology field by prolonging the survival of patients affected by highly aggressive/advanced stage cancers, such as metastatic melanoma and non-small cell lung cancer (NSCLC). This approach is currently based on the use of monoclonal antibodies targeting inhibitory ICKs, such as CTLA4 (cytotoxic T lymphocyte antigen 4) and PD-1 (programmed cell death 1) or PD-L1 (programmed cell death 1 ligand) that regulate activated T lymphocytes function switching off the immune response. A number of preclinical and clinical studies have revealed that antibodies raised against the immune checkpoint molecules enhance the antitumor immunity through not completely identified mechanisms of action, which differ depending on the target and specificity of the monoclonal antibody used [1,3–5]. Despite the undoubtful clinical success of ICK blockade, the on-field experimentation has opened several tasks that are worth being addressed organ-involvement and cohorts of signs and symptoms; second, the clinical efficacy is limited to subgroups of responder patients or, in many other subjects, after an initial response drug resistance takes over. The latter event is generally due to the genetic and phenotypic heterogeneity of cancer cells and/or to tumor microenvironment remodeling during disease progression and dissemination [5–7]. Nonetheless, the overall efficacy of ICKi is affected by multiple factors, spanning from the expression and distribution of the target within the tumor microenvironment, the tumor mutational rate and alterations of antigen presentation [8–10]. In this context, a crucial role is played by components of the host microenvironment that infiltrate the tumor and exert immunosuppressive effects, thus counteracting ICKi activity (i.e., infiltration by T regulatory cells (Tregs), dendritic cells, immunosuppressive myeloid cells, cancer associated fibroblasts) [11].

Cancer cell immunogenicity is a key determinant for ICKi efficacy: malignancies which do not express tumor specific antigens are not potentially susceptible to this approach. This is somewhat strengthened by the evidence that an immunotherapy-non-responsive cancer can turn into an immunotherapy-responsive one upon enhancement of tumor immunogenicity [8–10,12].

Hence, a better knowledge of the “immunopeptidome” (the repertoire of peptides bound to and presented by MHC molecules) and how tumor-specific antigen repertoire change during tumor progression is expected to improve the current therapeutic strategies: thus, a challenging issue is the identification and characterization of MHC-1 presented peptides that modulate T-cell-based tumor response [12–14].

At the molecular level, the generation of effective T-cells that fight cancer require a functional and efficient machinery for the multi-step and multi-cellular mediated process, including antigen processing and presentation. The intracellular antigen processing pathway almost exclusively deals with the Ubiquitin Proteasome System (UPS) activity by which proteins are first ubiquitinated and then degraded. Indeed, the UPS carries out multiple functions in cells and in the onset and progression of different human pathologies spanning from neurodegeneration to cancer (see next sections) [15]. A major role in antigen processing is covered by a specialized and inducible form of proteasome (i.e., the multi-catalytic machine which degrades ubiquitinated proteins) named “immunoproteasome” [16]. Therefore, the reviewer of Tundo and co-workers focuses on immunoproteasome, its involvement in antigen generation, and on the therapeutic implications of its modulation to halt cancer progression. Finally, the potential crosstalk between proteasome modulators and immune checkpoint inhibitors is discussed.

2. HATS OFF TO PROTEASOME VARIABILITY

Since the UPS catalyzes the degradation of the majority of intracellular proteins and its organization is tuned on the cellular metabolic demands, the maintenance of an adequate activity is essential for cell homeostasis as much as an appropriate plasticity, in terms of structural and functional organization, is required for cells to adapt to the stimuli they experience [15]. Canonical 26S proteasome, the central machine of the system, is a multifunctional holo-enzyme, composed by the 20S proteasome core particle (CP), which houses the proteolytic activity (the chymotryptic-like (β5 subunit); the trypsin-like (β2 subunit) and caspase-like (β1 subunit), capped by the 19S regulatory subunit (RP, also known as PA700), which carries out the ATP-dependent recognition, unfolding and translocation into the 20S of the poly-ubiquitinated substrate [4, 6, 20]. Thus, the proteasome machine is a highly dynamic complex, whose structural and conformational composition, substrate specificity is regulated at multiple steps, encompassing transcriptional regulation, kinetics of assembly, post-synthetic modifications and the interaction with a number of proteasome interacting proteins (PIPs) which act as regulatory factors [17–21]. Focusing on the proteasome structural composition, two main elements of proteasome plasticity and variability are represented by: 1) the interchangeability between constitutive and inducible catalytic subunits of the 20S; (2) the presence of different regulatory particles which can associate to just one or both the 20S free-ends. This allows to generate different subtypes of proteasome that can co-exist in a single cell and whose ratios may change among tissues. The metabolic and pathological stimuli that allow these canonical and non-canonical particles to form have been partially described, but to unequivocally address the interconnected, sometimes overlapping, or specific biological functions they carry out in vivo is a challenging task [18,22,23]. Noteworthy, in vertebrates, proteasome has gained considerable tissue-specificity, as indicated by the existence of alternative forms of proteasome: immunoproteasome, also known as inducible 20S (i20S), thymoproteasome and spermatoproteasome, in which the constitutive catalytic subunits of the 20S are replaced by inducible/tissue-specific homologs [18,24–26]. Immunoproteasome and thymoproteasome serve critical role in immunity, whereas spermatoproteasome is a testis-specific and chronologically-defined form of proteasome, exclusively identified in spermatocytes, spermatids and sperm. It is characterized by the presence of a specific α4 subunit (α4s) (PMSA8 gene) that replaces the constitutive α4: the incorporation of this subunit into a newly formed 20S is mutually exclusive with wild-type α4, and seems not to alter the 20S constitutive catalytic specificity [25,26]. Nevertheless, α4s incorporation seems to promote the association of the 20S with an alternative regulatory particle, named PA200, a nuclear-specific proteasome activator expressed in all mammalian tissues, but particularly abundant in the testis, where it plays a crucial role in spermatogenesis [27–31]. Remarkably, an increase of PSMA8 expression has been reported in different tumors, such as large B-cell lymphoma, thymomas and testicular germ cell tumors, even though its pathophysiological meaning and its relevance as novel therapeutic target have not been investigated yet [25,32,33]. As above mentioned, the reversible binding of activators (i.e., 19s and PA200) to either one or both the 20S α-subunit rings is another important level of proteasome organization, that contributes to the overall heterogeneity of proteasomes [15]. Besides the 19S, the best characterized regulatory particle is PA28 (i.e., 11S regulatory particle), which preferentially binds the immunoproteasome, forming the PA28/i20S complex as further discussed later (see Section 2.2) [16,34]. The binding of activators increases the proteolytic activity of the catalytic core, promoting the α-gate opening, influencing the substrate specificity of the complex and, more importantly, affecting the repertoire of cleavage products [15]. Hybrid proteasomes (i.e., 19S–20S 11S, 19S–20S-PA200) have also been identified. Their biological function remain obscure, but their identification underlies how the cells edit the proteasome repertoire in relation to their specific needs [35–37].

3. IMMUNOPROTEASOME AS A SPECIALIZED APPARATUS OF THE SELF-TARGET DESIGNATION

In the early nineties, proteasome was discovered as the crucial player for the class I MHC-restricted antigen processing pathway and two proteasome genes, namely PSMB9 (LMP2) and PSMB8 (LMP7), which encode for two alternative subunits of the 20S, β1 and β5 respectively, were identified in close proximity of the transporter associated with antigen processing (TAP) gene in the MHC class II genomic region [38,39]. Concurrently, it was shown that synthesis and incorporation of these subunits into the 20S was driven by interferon-γ (IFN-γ) [40–43] . Thus, immunoproteasome, also known as inducible proteasome, is a specialized form of 20S with a prominent role in immunity. Immunoproteasome preferentially and cooperatively incorporates three immune subunits β1i, β2i (MECL-1) and β5i to replace the constitutive catalytic subunits into the β-ring of the 20S during its biogenesis pathway. The preferential assembly of inducible subunits is likely due to the higher affinity of β5i than β5c for the proteasome maturation protein (POMP) which mediates the β-ring formation [15,44,45]. The i20S assembly is four times faster than c20S, clearly reflecting the need of a rapid and transient response upon exposure to a pro-inflammatory stimulus. In fact, IFN-γ induces, via JAK/STAT signaling, the transcription of immune catalytic subunits, MHC-I and TAP genes, thus enhancing the entire class I antigen presentation machinery [18,18,38,40,46]. Immunoproteasome is constitutively expressed at basal level in hematopoietic cells and has a shorter half-life than c20S (average 27 hrs for immunoproteasome and 133 hrs for constitutive proteasome). Such a rapid turnover has the purpose to efficiently adapt to the environmental changes [47,48]. It has been shown that during the course of viral, bacterial and fungal infections, the immunoproteasome replaces up to 90% of the c20S pool [49,50]. As a matter of fact, besides the pioneering contribution of IFN-γ, immunoproteasome was shown to be further transcriptionally induced by a plethora of inflammatory stimuli, such as, IFN-α and β, tumor necrosis factor-α (TNF-α), lipopolysaccharide (LPS), but also redox unbalance [51–54]. It is important to recall that the different forms of proteasome particles can co-exist inside the cells as well as the different peptide antigens generated: Anyway, in dependence of the different stimuli the cells are exposed to, the relative abundance of antigens generated by each specific sub-populations can be adapted [55].

A side-by-side comparison of the three different substrate binding pockets of the c20S and i20S points out the enzymological differences of the two complexes [56]. In general, the i20S is characterized by increased chymotrypsin-like and trypsin-like activities, but a lower caspase activity [57]. In details, the caspase-like subunit β1 accommodates peptides with an acidic residue in P1 position, whereas β1i binds to peptides with a hydrophobic residue in the same position, exerting a branched-chain amino acidic-preferring activity.

The folded trimeric complex formed by a given peptide and the MHC-I ligand cleft is exposed to the cell surface for presentation to the immune cells. The requisite for tight peptide – class I binding are essentially two: 1) the length of 8-9 amino acids, and 2) an anchor of basic or hydrophobic residues located at C-terminus or within the peptide sequence [24]. MHC-I does not accept C-termini with acidic anchor residues, thus, the substitution of β1c with β1i produces antigenic peptides with a hydrophobic C-termini that can efficiently bind to MHC-1 molecules. Additionally, also the structural properties of β5i contribute to generate peptides with a preferred C-terminal anchor amino acids for MHC-I molecules. In fact, the β5i S1 pocket accommodates larger hydrophobic amino acids chains than β5c (which presents, instead, a “small neutral amino acids-preferring activity”) and is characterized by a more hydrophilic environment around the catalytic threonine favoring the chymotryptic-like catalytic properties of the inducible subunit. Despite β5 and β1 subunits, the active sites of β2c and β2i seem to be structurally identical, rendering more enigmatic this substitution, even though several studies reported an increase of trypsin-like activity of i20S with respect to c20S [24,56].

Additional forms of proteasome bearing a mix of standard and inducible subunits were identified. These intermediate proteasomes, which represent one-third to one-half of the overall proteasome content in different tissues, such as liver, colon and kidney, contains these triads of subunits, β1/β2/β5i or β1i/β2/β5i. Remarkably, a recently discovered mechanism of antigen generation through which proteasome increases the repertoire of antigens for presentation to immune system is the “proteasome-catalyzed peptide splicing”: spliced peptides, which are made by two not contiguous fragments of parental proteins, are produced efficiently both by immunoproteasome and constitutive particles [58–61]. The existence of these mechanisms along with the co-presence of different proteasome populations, beyond constitutive proteasome, involved in antigen-peptide generations broadens the repertoire of antigens produced by a cell [48,62,63]. Whether the incorporation of immune subunits triggers qualitative or quantitative effects on peptide repertoire generation is still not resolved, since different studies report somewhat controversial results. In fact, a number of studies highlighted the positive role of immunoproteasome mainly against viral and bacterial antigens, whereas some studies reported that immunoproteasome expression can abrogate the presentation of some tumor epitopes [62–69].

Anyway, a defect in antigen presentation was found in the triple inducible subunits ko mice and this alteration is now reported to be qualitatively and quantitatively much broader than that previously described in any of the β1i, β2i or β5i single subunit ko mice and to be still far greater than the sum of the defects these single ko animal were reported to bear [48,70,71]. Moreover, analysis of MHC class I bound peptides shows that the antigen repertoire of ko mice differs from that of wt mice, reinforcing the hypothesis that immunoproteasomes generate peptides that apparently cannot be produced by constitutive proteasomes [64,72,73]. On the other hand, other studies suggest that immunoproteasomes affect the quantity rather than the quality of the given generated peptides, influencing also in this case the immune response [68,74,75]. Thus, some antigens are exclusively produced by the immunoproteasome or the constitutive one, while others can be processed by both or some others can be preferentially processed by intermediate-type proteasomes [76,77]. Of note, this distinction between quantity and quality effect of antigen repertoire in dependence of the expression rate of immune-constitutive or mixed proteasome is not simply semantic. In fact, it is of basic significance not only to better understand the enzymatic properties of the different proteasome populations but also to better define the MHC class I dependent CD8+ T-cell response in the context of specific physio pathological conditions [78].

4. IMMUNOPROTEASOME: AN EMERGING TARGET IN CANCER

Alterations of different genes belonging to the UPS are a hallmark of cancer. UPS dysregulation may occur at multiple levels, spanning from genetic modifications (i.e., mutations, amplifications, deletions), transcriptional network alterations (i.e., p53; NRF-1 and NRF-2), epigenetic and post-translational modifications [15,79].

A number of studies underline the i20S role in cancer progression, strengthening its therapeutic potentiality. The tumor microenvironment is profoundly different from that of healthy tissues; this is also due to the presence of tumor infiltrating lymphocytes (TILs) that release IFN-γ and other inflammatory cytokines. Interestingly, immunoproteasome seems to possess both pro- and anti-tumorigenic properties, which are associated with the modulation of cytokine expression and tumor associated peptide presentation, respectively [77].

Tumor cells can evade recognition by cytotoxic T lymphocytes (CTLs or CD8+ T cells) through downregulation of MHC-I at the cell surface or, additionally, by reducing immunoproteasome subunits expression [77,80]. In fact, non-small cell lung cancer, that undergoes the epithelial–mesenchymal transition, shows a reduced immunoproteasome subunits expression: this leads to a dramatic drop of the heterogeneity of antigen peptide repertoire produced by tumor cells and to poor clinical outcomes [81]. Thus, it has been proposed that a decrease of immunoproteasome expression might represent a mechanism of immune escape in tumor cells which show a mesenchymal phenotype, since this downregulation is associated with a decline in the amount and diversity of MHC-1 presented peptides [81]. In accordance with these data, transforming growth factor-β (TGF-β) induced epithelial-mesenchymal transition leads to a decrease of the immunoproteasome content [81]. Moreover, in the early stage of NSCLC, low expression of i20S is linked to an increased risk of recurrence and metastasis onset [81]. At the molecular level, one proposed mechanism which links carcinogenesis with immunoproteasome deficiency is the differential expression of β5i subunit. Two main β5i variants have been described, which are both induced by IFN-γ: LMP7E2 and LMP7E1. LMP7E2, usually expressed in normal cells and in certain cancer types, is regularly incorporated into the mature i20S. However, many cancer cell lines express only the LMP7E1 isoform that does not interact with the 20S assembly chaperone POMP and, thus, cannot be integrated into the mature i20S, leading to a deficiency of functional immunoproteasome [82]. Moreover, a polymorphism at amino acid 49 of LMP7 (K49 instead of Q49), localized at the pre-sequence of β5i, reduces the rate of proteasome assembly and is associated with a higher risk of developing colon carcinoma [83]. On the other hand, overexpression of immunoproteasome subunits, due to an increase of I-FN-γ production by TILs, correlates with a better prognosis in different tumors, such as melanoma and breast cancer [77,79]. Immunoproteasome expression is not only triggered by paracrine production of pro-inflammatory cytokines (such as IFN-γ) by immune cells, but it is constitutively elevated in hematological malignancies [84,85]. In myeloid leukemia cells, the i20S increase was associated with a higher survival rates [86]. Interestingly, the upregulation of immunoproteasome by IFN-γ overcomes resistance to the proteasome inhibitor bortezomib and sensitizes hematological malignant cells (such as multiple myeloma and leukemia) to a selective immunoproteasome inhibitor ONX0914 [87]. This opens up the perspective of developing therapeutic approaches based on selective inhibition of immunoproteasome subunits, different from those targeting the constitutive ones. Moreover, some evidence indicates that immunoproteasome alterations can impact on the onset of inflammation-driven carcinogenesis: indeed, β5i inhibition prevents colitis associated with colon carcinoma [88]. Thus, the emerging complex picture indicates that the altered expression of immunoproteasome subunits (mainly LMP2 and LMP7) is common in various tumors, but the extent of the expression and its biological significance vary in dependence of cancer type and grading [50,88]. As matter of fact, the immunopeptidome changes in the context of tumor microenvironment and in dependence of the relative abundance of constitutive or inducible proteasome. For example, a number of cancer antigens derived from members of “melanoma antigen gene” protein family (MAGE), whose expression is restricted to reproductive tissues, but are aberrantly expressed also in a wide-variety of cancer types, such as MAGA3_(114-122) and MAGEC_(42-50) and MAGEA2_(338-344), are produced by immunoproteasome but not by the constitutive one [59,89]. Since the identification and characterization of neoantigens is of clinical relevance, modern strategies which combine genomic, proteomic and immunopeptidomic approaches are powerful way to discover novel presented antigens and tumor associated antigens, paving the way to novel therapeutic potential [90,91]. As mentioned in the previous section, intermediate proteasomes broaden the repertoire of MHC-I antigenic peptides and, intriguingly, are involved in the production of unique tumor antigens. In fact, it has been reported that some peptides derived from proteins belonging to the melanoma antigen gene (MAGE) family are generated by intermediate forms. Specifically, β1i-β2-β5i intermediate produces MAGE-A10 (254-262) peptide, whereas β1-β2-β5i intermediates generate MAGE-C2 (191-200) and MAGE-A3 (271-279) peptides. On the other hand, other antigenic peptides, such as MAGE-A3 (114-122) and MAGE-C2 (42-50), are produced with equal efficiency by the i20S and intermediate proteasomes [48,92,93]. Moreover, intermediate proteasomes were detected in a number of tumor cells, including lung carcinoma, myeloma, osteosarcoma and melanoma [22,35,48,92]. Despite this evidence, the role of these forms of proteasome in cancer onset and development is poorly known.

5. IMMUNOPROTEASOME AND IMMUNE CHECKPOINT INHIBITORS: A GLANCE TO THE FUTURE?

As mentioned in the previous sections, the production of tumor-associated antigenic peptides recognized by CTLs is a process that starts in the cytoplasm with the degradation of cellular proteins mainly by the immunoproteasome [94,95]. Peptide antigens produced by cancer cells are commonly classified into two main groups, namely with high and low specificity. Antigens with high tumor specificity are encoded by viral genes (expressed only in infected cells), mutated genes (generated by the intrinsic instability of cancer cells and hereafter referred to as neoantigens) and cancer germinal genes (expressed as a result of genome-wide demethylation occurring in germinal cells) [55,96]. Moreover, it is known that tumorigenesis is strictly related to genetic diversity and to high mutational burden of cancer cells, which increase the possibility of production of neoantigens [79,91,97,98]. This high mutational heterogeneity and neoantigens frequency positively correlate with the response to ICKi therapy. In fact, ICKi are particularly effective against cancers that show a high burden of mutations and that are characterized by DNA mismatch repair deficiency, such as colorectal cancer and NSCLC [99–101]. Thus, neoantigens have been proposed to be a prognostic marker for positive clinical outcomes [79,91,97,98]. As a matter of fact, one of the main reasons of acquired resistance to ICKi therapy seems to be the loss of neoantigens recognized by circulating T-cells, suggesting that tumors are “able” to eliminate some mutations during the acquisition of a resistant phenotype [101]. Remarkably, despite the approval of ICK therapy for cancers characterized by a high mutational burden, a very recent study fails to support the concept that the high mutational burden is a positive biomarker for ICKi treatment in all solid tumors. In fact, a high mutational burden seems to behave as predictive marker e of response to ICK-based therapies only when the CD8⁺ infiltration level correlates with neoantigen load (such as melanoma, lung, bladder cancers and colon cancer). On the other hand, for tumors in which no relationship between CD8⁺ levels and neoantigens load is reported (such as glioma), the high mutational burden failed to predict a positive response to therapy [102]. Thus, it clearly emerges that additional tumor type-specific studies should be performed to unveil the role of this biomarker in ICKi response [103,104].

In light of the plethora of antigenic peptides produced by the proteasome pathway, it is not surprising that alterations of proteasome activity and composition could be linked to antigen processing and ICKi response. Of note, the local production of IFN-γ within the tumor microenvironment by infiltrating T lymphocytes positively correlates with clinical outcomes to ICKi therapy and cancer vaccination in tumors like metastatic melanoma [105,106]. In fact, the upregulation and secretion of chemotactic cytokines (such as IFN-γ and TNF-α) increase the recruitment of additional immune cells and alter the tumor microenvironment, stimulating the inhibition of immune exclusion of cancer signatures [107]. Interestingly, some studies show that the primary response to anti-CTLA4 antibodies required high level exposure of MHC-1 on the surface of cancer cells at baseline; on the other hand, the response to anti PD-1 antibodies is linked to a pre-existing IFN-γ transcriptome signature [108]. In a recent study, the transcriptome of baseline and on-therapy tumor biopsies from a cohort of 101 patients with advanced melanoma, included within the CheckMate 038 study (NCT01621490) and treated with nivolumab alone or in combination with ipilimumab, has been analyzed [109]. These data, together with in vitro studies, suggest that the immune activation, which follows the administration of ICKi, is associated to the expression of IFN-γ response genes, mediated by the increase of T-cell infiltration. Among different sets of genes induced by IFN-γ, the most relevant in mediating the response at the therapy are those involved in antigen presenting machinery [110]. Thus, a combination therapy of ICKi with agents that independently trigger the intra-tumoral production of IFN-γ could become meaningful  [110–112]. Consistently with the role of IFN-γ signature in driving ICKi response, it has been proposed that upregulation of immunoproteasome subunits in tumor cells might be also involved in this process [105,109]. In fact, the local production of I-FN-γ induced by ICKi, which are routinely used in the treatment of advanced/metastatic melanoma, leads to the modulation of proteasome composition, thus inducing the generation of antigenic peptides [105,106]. Consistent with this observation, the expression of immunoproteasome subunits β1i and β5i was associated with a better prognosis in the case of tumors with a high mutational burden (i.e., melanoma and NSCLC) and positively correlated with the response to ICKi and with the survival rate of patients [81,105,113]. Thus, it has been proposed that, at least in some tumors, the expression level of β1i and β5i subunits might represent a predictive marker of response to ICKi [105,114]. As a matter of fact, the overexpression of these subunits is linked to longer survival and improved response to ICKi therapy in melanoma patients and the mechanism proposed underlying this connection is an enhanced reactivity of TILs toward melanoma cells, as a consequence of an altered repertoire of presented antigens [105]. Importantly, it has been also reported that immunoproteasome subunit overexpression occurs sometimes regardless of IFN-γ and T-cell infiltration, suggesting that these subunits should be independent prognostic biomarker with respect to IFN-γ level and the rate of T-cell infiltration in the context of tumor microenvironment [105]. Thus, this last observation open up to a possible and yet poorly investigated scenario concerning the role of immunoproteasome in mediating ICKi response independently on IFN-γ pathway. Therefore, even though many crucial points deserve to be clarified, it clearly emerges that immunoproteasome expression seems to be an important predictive marker in colorectal, melanoma and NSCLC for which ICKi therapy has proven to be effective. Thus, an intriguing therapeutic strategy that should be explored in the next future is the combination of ICKi and drugs that directly modulate immunoproteasome activity and/or induce immunoproteasome expression in order to increase its pool inside the cells.

6. CONCLUSION

Cancer cells are often more dependent on a proper integrity and functionality of UPS than non-malignant cells due to the rapid proliferation rate, increased metabolic activity and continuous exposure to a variety of extrinsic stress perturbations (such as nutrient deprivation, hypoxia and acidosis) under which cancer cells live. All these conditions lead to a decrease of protein quality control and make UPS a suitable target for cancer therapy [219,220]. Accordingly, a number of proteasome based-strategies have been proposed, spanning from i) inhibition of proteasome proteolytic activity, ii) modulation of the abundance of proteasome regulatory particles (i.e 19S or PA28) and of their interaction with 20S; iii) modulation of the activity of enzymes involved in proteasome subunit post-translational modifications and iv) interference with transport of natural low molecular-weight proteasome activators (e.g., spermine) [18,117–121]. Additionally, a series of strategies targeting ubiquitination cascade have been studied. One of the most intriguing and novel therapeutic approach involves the use of PROTAC or Proteolysis Targeting Chimeric Molecules, which are hetero-bifunctional molecules that recruit specific target proteins to E3 ligase, thus inducing the increase of target ubiquitination and degradation. This strategy has been already applied to the degradation of a number of selected targets[122–124]. However, even though promising, it is still at its infancy for application to immunotherapy. On the other hand, the most deeply investigated strategy consists in the use of broad specificity inhibitors of 20S activity, like bortezomib, carfilzomib and ixazomib that inhibit proteolysis of all the proteasome forms present in different cells [15,18]. A second approach encompasses the identification of specific inhibitors targeting inducible, tissue-specific forms of proteasome, mainly i20S, which is involved in the production of antigenic peptides. Though this mechanism is well known, how such inhibitors might either decrease or stimulate cancer cell recognition by T cells is debated (see Section 4) [18]. As a matter of fact, a challenging but poorly investigated issue is the precise in vivo impact of broad-specificity 20S proteasome inhibitors commonly used in clinical practice on antigen presentation by cancer cells [55] and it should deserve more attention. A key question for improving the efficacy and safety profile of immunotherapy includes the identification of the most appropriate strategy to optimize the antigenic peptide repertoire of the tumor, required for an efficient immune response [55]. Notably, some recent data suggest that enhanced immunoproteasome activity might play an important role in the response of melanoma to ICKi [105]. It seems to indicate that, at least in some tumors, the more efficient strategy could be to “enhance” immunoproteasome expression and activity. Thus, the choice of the best strategy whether to inhibit or activate immunoproteasome should take into consideration the biological features of the specific tumour that has to be treated. Even though additional in vitro and in vivo investigation needs to be performed, the current evidence indicates that selective modulation of proteasome activity might have a role in improving the outcome of ICKi or of other immunotherapeutic approaches.

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This entry is adapted from the peer-reviewed paper 10.3390/cancers13194852