The Role of Immunoproteasomes in Tumor-Immune Cell Interactions in Melanoma and Colon Cancer

Immunoproteasomes are constitutively expressed in the cells of the immune system such as antigen-presenting cells (APCs) and T lymphocytes (McCarthy and Weinberg 2015). The altered catalytic activity of immunoproteasome subunits influences the quantity and quality of peptides presented by MHC class I molecules (Basler et al. 2013). Immunoproteasomes have a reduced caspase-like activity and enhanced chymotrypsin-like activity leading to more efficient generation of specific epitopes (Driscoll et al. 1993). According to current model, the immunoproteasome assembly is efficiently achieved by competitive integration of catalytic β subunits. The high affinity of immunosubunits to assembling proteasome complexes facilitates the replacement of constitutive proteasomes by immunoproteasomes during infection even in non-immune cells. A strong interaction of LMP7 and POMP (a chaperone that selectively binds to precursor subunits of the proteasome) promotes the immunoproteasome assembly and increases the proteasome amount in infected cells during infections (Heink et al. 2005), which is an essential step for clearance of intercellular pathogens by mammalian cells.

One of the main functions of the immunoproteasome is an efficient antigen processing for presentation on MHC class I molecules. Peptides generated by immunoproteasome show a higher binding affinity for the MHC class I complex (Aki et al. 1994). The lack of immunoproteasomes in mice reduces the CD8⁺ T-cell-mediated immune responses during influence virus, hepatitis B virus and lymphocytic choriomeningitis virus (LCMV) infections (Chen et al. 2001; Moebius et al. 2010; Robek et al. 2007). In addition, immunoproteasomes seem to be essential for survival of LCMV-specific CD8⁺ T cells in infected mice (Moebius et al. 2010). Furthermore, it was shown that mice with genetic deletion of immunoproteasomes are more susceptible to infections with intracellular bacteria and protozoan parasites. The combined deficiency of all three immunoproteasome subunits in mice abrogated the development of an effective host resistance against the human protozoan parasite Trypanosoma cruzi. Immunoproteasome-deficient mice exhibited significantly lower magnitude and quality of T. cruzi-specific CD8⁺ T-cell responses (Ersching et al. 2016). Moreover, during infection of mice with the intracellular bacterium Brucella abortus, a lower MHC I surface expression, an impairment of granzyme B and IFN-γ expression, as well as a reduced cytotoxic activity of CD8⁺ T lymphocytes was observed in the absence of immunoproteasomes (Guimaraes et al. 2018). In general, the immunoproteasome-mediated generation of peptides with higher binding affinity crucially impacts the availability and repertoire of epitopes for antigen presentation. Cells completely lacking immunoproteasomes have a restricted repertoire of presented peptides and a strong reduction of MHC class I surface expression compared to wild-type (WT) cells. Of note, the animals lacking immunoproteasomes reject skin transplants or splenic cells from WT mice, suggesting that cells in WT and immunoproteasome-deficient mice present a markedly different set of peptides (Kincaid et al. 2012; Toes et al. 2001). Recently, novel functions of immunoproteasomes in modulating a complex network of pro-inflammatory signaling pathways in APCs that are linked to autoimmunity, gut and neuro-inflammation, as well as to T helper (Th) cell differentiation and cytokine production have been proposed (Basler et al. 2013). Moreover, lack of immunoproteasomes is associated with decreased cellular ability to degrade oxidized proteins, showing so far unknown role for this protease complex in the cells (Ebstein et al. 2013; Seifert et al. 2010). Thus, in addition to immune functions, the immunoproteasome provides also protection against the accumulation of oxidatively damaged cellular proteins (Pickering et al. 2010).

Immunoproteasome is crucially involved in mediating protective immunity against viral and bacterial antigens, but it is also implicated in the pathogenesis of several autoimmune diseases such as rheumatoid arthritis and multiple sclerosis (Basler et al. 2018; Muchamuel et al. 2009). We and others have shown that immunoproteasome activity in the inflamed intestine promotes the production of pro-inflammatory cytokines, such as IL-6, TNF-α, IL-17A and IL-23 (Basler et al. 2010; Schmidt et al. 2010; Visekruna et al. 2006). Because these cytokines are the integral part of the signaling network that synergistically activates NF-κB and STAT3 in colonic epithelial cells (De Simone et al. 2015), they are also crucially implicated in the onset of inflammation-associated carcinogenesis in the gut. IL-17A, IL-21, IL-22, TNF-α, IL-6 and IL-23 are also excessively produced in the early colonic lesions in patients with inflammatory bowel disease who have an increased risk for development of colon tumorigenesis (Karin 2009; Neurath 2014; West et al. 2015). Thus, the cytokine network abundantly secreted in the inflamed areas of gastrointestinal tract contributes to the induction of oncogenic transcription factors in colonocytes, which promotes cell survival and uncontrolled proliferation. Interestingly, the simultaneous neutralization of IL-17A and TNF-α abrogating NF-κB signaling pathways, or IL-22 and IL-6 inhibiting STAT3-mediated signaling impairs the mitogenic effects on colorectal cancer cells (De Simone et al. 2015). In mice, the deletion of IL-17A or IL-23 was sufficient to significantly reduce the number and size of tumors in experimental model of colitis-associated cancer (Grivennikov et al. 2012; Hyun et al. 2012). Importantly, the specific inhibition of the immunoproteasome subunit LMP7, but also the non-specific blockade of proteasomes by bortezomib, suppresses the expression of these inflammatory mediators and prevents the development of acute colitis in mice (Kalim et al. 2012; Schmidt et al. 2010). The immunoproteasome-specific inhibitor, ONX 0914 was effective in suppressing the onset of inflammation-driven cancer even when applied in a therapeutic setting when the tumor size was macroscopically visible in mice (Koerner et al. 2017; Vachharajani et al. 2017). In recent study, we addressed the role of immunoproteasomes in the azoxymethane (AOM)-dextran sodium sulfate (DSS) model of colitis-associated cancer (CAC) in mice with combined deficiency of all three immunoproteasome subunits, LMP2, LMP7 and MECL-1. Remarkably, immunoproteasome-deficient triple-knockout mice were not susceptible to the development of CAC, since no visible tumors were detected in these mice (Leister et al. 2021). In addition, in the lamina propria, a negligible expression of pro-inflammatory chemokines CXCL1, CXCL2 and CXCL3, as well as cytokines that contribute to the CAC progression such IL-6, TNF-α, IL-17A and IL-23 was observed. Of note, we also found an upregulation of immunoproteasome-regulated pro-tumorigenic chemokines in patients with ulcerative colitis, who have a high risk for development of colorectal cancer (Leister et al. 2021). These findings suggest that in humans a similar mechanisms may lead to the recruitment of neutrophils and other innate immune cells that promote the damage in the gut epithelial cells and contribute to the onset of tumorigenesis. Collectively, these data have identified the immunoproteasome as a key regulator of pro-tumorigenic signaling networks in the inflamed gut, leading to the onset of colorectal carcinoma. Interestingly, one study demonstrated that LMP7 inhibition was effective not only in a model of inflammation-driven cancer, but also in APC^Min/+ mice, a preclinical model that closely resembles familial adenomatous polyposis in humans, suggesting that immunoproteasomes might be also involved in promoting inflammation-independent carcinogenesis (Koerner et al. 2017). The components of the UPS such as immunoproteasomes might be directly involved in the regulation of proliferation and differentiation, as well as in pro-apoptotic signaling pathways in colorectal cancer cells (Voutsadakis 2008). An effective targeting strategy for colorectal cancer in future may be a specific blockade of immunoproteasomes with small compounds, which, in contrast to non-specific proteasome inhibitors, could reduce therapeutic side effects, as the cells expressing constitutive proteasomes are not targeted.

In conclusion, accumulating evidence strongly suggests that immunoproteasomes regulate the activity of infiltrated immune cells in the inflamed gut and promote the development of CAC. Thus, novel immunoproteasome-specific inhibitors should be tested in future clinical studies to optimize the treatment of rectal and colon adenocarcinomas.

Infiltration of cytotoxic T lymphocytes (CTLs) and Th1 cells into the microenvironment of solid tumor is a pivotal step in recognizing tumor-associated antigens and in preventing the tumor growth and progression (Ritter and Greten 2019). Further, immunoproteasomes are involved in shaping the repertoire of neo-antigens and the activation of antigen-specific CD8⁺ T-cell responses in the tumor microenvironment. Recent studies have revealed that the local production of IFN-γ and high expression of the immunoproteasome subunits LMP2, LMP7 and MECL-1 strongly correlate with the abundance of tumor-infiltrating lymphocytes, and with better survival of melanoma patients (Hugo et al. 2016; Kalaora et al. 2020; Leister et al. 2021). Of note, the overexpression of immunoproteasomes in human melanoma cell lines led to the generation of more immunogenic repertoire of tumor peptides and better lysis of tumor cells by co-cultured autologous tumor-infiltrating lymphocytes (Kalaora et al. 2020). These findings support the hypothesis that the high expression of immunoproteasomes in solid tumors might result in superior killing of tumor cells by CTLs due to alterations in antigen repertoire, and possibly by increased generation and recognition of neo-antigens. Importantly, the enhanced activity of immunoproteasomes correlated also with a better response of melanoma patients to immune-checkpoint inhibitor therapy such as anti-CTLA4 and anti-PD1 (Kalaora et al. 2020). Furthermore, in a murine model of melanoma, in which the melanoma cell line B16-F10 was able to express immunoproteasomes, while the tumor recipient mice were immunoproteasome-deficient, an increased tumor volume with impaired anti-tumor immunity was observed (Leister et al. 2021). These data demonstrate that immune cells, such as dendritic cells, Th1 cells and CTLs, which express high amount of immunoproteasomes in the tumor microenvironment, are essential for an effective immunity against melanoma, and probably also against other solid tumors such as lung cancer (Spits and Neefjes 2016). Immunoproteasomes are known to regulate the production of IL-12 by APCs, which ultimately leads to enhanced infiltration of lymphocytes in the tumor tissue and a better differentiation of tumor-specific Th1 cells and CTLs in tumor-draining lymph nodes (Fig. 2). Notably, by inoculating melanoma cells into WT animals and mice lacking immunoproteasomes, we observed that immunoproteasomes were strongly induced in tumor cells derived from WT mice, but not in that from immunoproteasome-deficient animals (Leister et al. 2021). These results demonstrate that Th1 lymphocytes and CTLs, surrounding the tumors and producing large amounts of IFN-γ, force cancer cells to enhance their immunoproteasome expression and activity. This significantly affects the repertoire of tumor antigens and might lead to more efficient presentation of neo-antigens derived from driver mutations (Fig. 2). Consequently, this would lead to a better elicitation of an antigen-specific immune response, and even to improved response to checkpoint inhibitor therapy. Thus, simultaneous activation of the immunoproteasome activity in solid tumors and in immune cells of the tumor microenvironment might result in improved cancer immunotherapies against various solid cancers of different origin. In accordance, one study in patients with non-small cell lung cancer (NSCLC) revealed that the immunoproteasome expression in tumor cells influences the responsiveness to immunotherapy (Tripathi et al. 2016). Likely, due to enhanced activity of immunoproteasomes in tumor cells, a more diverse tumor antigen pool is efficiently recognized by CTLs, resulting in a better outcome of disease. Accordingly, the deficiency of immunoproteasome expression was associated with a poor outcome in patients with NSCLC. Thus, the high immunoproteasome amount in solid tumors, such as melanoma and NSCLC, is linked to an increased diversity of tumor antigens, as well as to better responsiveness to immune-checkpoint therapy, and most importantly, also to better overall survival of patients.

Collectively, tumors have developed many strategies to avoid immune surveillance and to minimize the production of neo-antigens. Hence, a better understanding and a deeper knowledge of multiple interaction between tumor and immune cells in the tumor microenvironment is needed to develop therapeutic strategies for facilitating generation of more immunogenic tumor antigens. This might be achieved by enhancing the activity of immunoproteasomes in both, tumor and immune cells.