Metabolic regulation of Cathepsin B in tumor macrophages drives their pro-metastatic function

Tumor macrophages possess tumor-promoting functions, but the mechanism regulating such functions is poorly understood. Providing new insight into such mechanism, Shi et al. in this issue of Cancer Cell identify how metabolic regulation of Cathepsin B and its O-GlcNAcylation by lysosomal O-GlcNAc transferase (OGT) in macrophages drives pro-metastatic function. Tumor macrophages possess tumor-promoting functions, but the mechanism regulating such functions is poorly understood. Providing new insight into such mechanism, Shi et al. in this issue of Cancer Cell identify how metabolic regulation of Cathepsin B and its O-GlcNAcylation by lysosomal O-GlcNAc transferase (OGT) in macrophages drives pro-metastatic function. Macrophages perform diverse functions ranging from triggering inflammation, phagocytosis, and microbicidal/tumoricidal activity to immunosuppression and tissue remodeling. Among the mechanisms that regulate the phenotypic and functional diversity of macrophages, metabolic regulation of these cells is emerging as a key player (Murray et al., 2015Murray P.J. Rathmell J. Pearce E. SnapShot: Immunometabolism.Cell Metab. 2015; 22: 190-190.e1https://doi.org/10.1016/j.cmet.2015.06.014Abstract Full Text PDF PubMed Scopus (68) Google Scholar). While distinct modes of glucose and amino acid metabolism were reported for IFNγ+LPS(M1)- and IL-4(M2)-polarized macrophages in vitro (Biswas and Mantovani, 2012Biswas S. Mantovani A. Orchestration of metabolism by macrophages.Cell Metab. 2012; 15: 432-437https://doi.org/10.1016/j.cmet.2011.11.013Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar), how metabolism guides macrophage function in vivo remains poorly understood. Tumor-associated macrophages (TAMs) infiltrate many solid tumors where they affect many aspects of cancer progression (Engblom et al., 2016Engblom C. Pfirschke C. Pittet M.J. The role of myeloid cells in cancer therapies.Nat. Rev. Cancer. 2016; 16: 447-462https://doi.org/10.1038/nrc.2016.54Crossref PubMed Scopus (462) Google Scholar). However, the metabolic changes in TAMs and how they regulate their pro- or anti-tumor functions are not well characterized. In this issue, Shi et al. report how glucose metabolism-driven hexosamine biosynthetic pathway (HBP) in TAMs regulates their role in cancer metastasis and chemoresistance (Shi et al., 2022Shi Q.S. Liu Q.W. Shi Y. Huang W. Wang X. Li Z. Chai Y. Wang H. Hu X. Li N. et al.Increased glucose metabolism in TAMs fuels O-GlcNAcylation of lysosomal Cathepsin B to promote cancer metastasis and chemoresistance.Cancer Cell. 2022; 40: 1207-1222Abstract Full Text Full Text PDF Scopus (35) Google Scholar). Using a murine B16 melanoma model, the authors characterized various tumor infiltrating myeloid cells and found that M2-like TAMs show an upregulation of the glucose transporter (gene Slc2a1 and protein GLUT1) and increased glucose uptake, as compared with M1-like TAMs, splenic, peritoneal macrophages, and blood monocytes. The same was true for M2-like TAMs from a Lewis lung carcinoma (LLC) model. M2-like TAMs upregulated several genes encoding enzymes related to the glucose metabolism and the HBP that are responsible for the production of UDP-N-acetylglucosamine (UDP-GlcNAc), a substrate for protein O-linked N-acetylglucosamine modification (O-GlcNAcylation; Figure 1A). O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) are key enzymes that catalyze and remove O-GlcNAc, respectively. Accordingly, M2-like TAMs showed increased OGT and O-GlcNAcylation. Blocking glucose uptake in vitro and in vivo inhibited O-GlcNAcylation in M2-like TAMs, indicating that glucose uptake drives HBP and O-GlcNAcylation. TAMs show changes in glucose metabolism in several cancer types (Arts et al., 2016Arts R.J.W. Plantinga T.S. Tuit S. Ulas T. Heinhuis B. Tesselaar M. Sloot Y. Adema G.J. Joosten L.A.B. Smit J.W.A. et al.Transcriptional and metabolic reprogramming induce an inflammatory phenotype in non-medullary thyroid carcinoma-induced macrophages.OncoImmunology. 2016; 5e1229725https://doi.org/10.1080/2162402x.2016.1229725Crossref PubMed Google Scholar; Zhang et al., 2021Zhang Q. Wang J. Yadav D.K. Bai X. Liang T. Glucose Metabolism: The Metabolic Signature of Tumor Associated Macrophage.Front. Immunol. 2021; 12702580https://doi.org/10.3389/fimmu.2021.702580Crossref Scopus (24) Google Scholar). However, the mode of glucose metabolism varies according to the TAM phenotype and cancer type. How and what differences in the tumor microenvironment (TME) decide which mode of glucose metabolism a TAM would preferentially adopt are not known. Shi et al. showed that IL-4 upregulated key components of the glucose-HBP (e.g., Slc2a1, OGT, Glucose-6-phosphate, and Glucosamine-6-phosphate) and O-GlcNAcylation in bone marrow-derived macrophages (BMDM; Figure 1B), but the mechanism by which IL-4 induced OGT and HBP remains unclear (Shi et al., 2022Shi Q.S. Liu Q.W. Shi Y. Huang W. Wang X. Li Z. Chai Y. Wang H. Hu X. Li N. et al.Increased glucose metabolism in TAMs fuels O-GlcNAcylation of lysosomal Cathepsin B to promote cancer metastasis and chemoresistance.Cancer Cell. 2022; 40: 1207-1222Abstract Full Text Full Text PDF Scopus (35) Google Scholar). UDP-GlcNAc synthesis pathway was shown to play a critical role in IL-4-induced M2 polarization of macrophages (Jha et al., 2015Jha A. Huang S.C. Sergushichev A. Lampropoulou V. Ivanova Y. Loginicheva E. Chmielewski K. Stewart K. Ashall J. Everts B. et al.Network Integration of Parallel Metabolic and Transcriptional Data Reveals Metabolic Modules that Regulate Macrophage Polarization.Immunity. 2015; 42: 419-430https://doi.org/10.1016/j.immuni.2015.02.005Abstract Full Text Full Text PDF PubMed Scopus (1182) Google Scholar). Increased HBP flux and O-GlcNAc level was shown to contribute to the M2 polarization of TAMs in a colon cancer model, under hyperglycemia (Rodrigues Mantuano et al., 2020Rodrigues Mantuano N. Stanczak M.A. Oliveira I.d.A. Kirchhammer N. Filardy A.A. Monaco G. Santos R.C. Fonseca A.C. Fontes M. Bastos C.d.S. et al.Hyperglycemia Enhances Cancer Immune Evasion by Inducing Alternative Macrophage Polarization through Increased O-GlcNAcylation.Cancer immunology research. 2020; 8: 1262-1272https://doi.org/10.1158/2326-6066.cir-19-0904Crossref PubMed Scopus (0) Google Scholar). However, HBP had no direct role on M2 macrophage polarization in the present study. This divergence could be due to the impact of hyperglycemia. Lactate is another important TME-associated factor that triggers mTOR-dependent glycolysis in TAMs (Arts et al., 2016Arts R.J.W. Plantinga T.S. Tuit S. Ulas T. Heinhuis B. Tesselaar M. Sloot Y. Adema G.J. Joosten L.A.B. Smit J.W.A. et al.Transcriptional and metabolic reprogramming induce an inflammatory phenotype in non-medullary thyroid carcinoma-induced macrophages.OncoImmunology. 2016; 5e1229725https://doi.org/10.1080/2162402x.2016.1229725Crossref PubMed Google Scholar). Since mTOR can cross-regulate O-GlcNAcylation, lactate-induced activation of the mTOR pathway may potentially regulate O-GlcNAcylation in TAMs, though this needs to be tested. To understand how heightened O-GlcNAcylation might impact the function of M2-like TAMs, the authors first identified target proteins that are likely to undergo O-GlcNAcylation. Binding studies revealed lysosomal Cathepsin B as a binding partner of lysosomal OGT. Using IL-4-polarized BMDMs, Shi et al. showed that OGT catalyzes Serine 210 O-GlcNAcylation of Cathepsin B, resulting in increased maturation and secretion of active Cathepsin B. This process is glucose dependent since inhibiting glucose affected S210 O-GlcNAcylated Cathepsin B and matured Cathepsin B levels in both IL-4-polarized BMDMs and TAMs from B16 melanoma. M2-like TAMs showed increased mature form of Cathepsin B compared with M1-like TAMs, and OGT deficiency abrogated matured Cathepsin B levels in these TAMs. Collectively, these results demonstrate a "metabolic regulation" of Cathepsin B in TAMs. Importantly, this provides a mechanistic link connecting upstream metabolic changes (in HBP) in TAMs to downstream Cathepsin B-mediated functions, as described below. Since Cathepsins promote tumor metastasis (Gocheva et al., 2010Gocheva V. Wang H.W. Gadea B.B. Shree T. Hunter K.E. Garfall A.L. Berman T. Joyce J.A. IL-4 induces cathepsin protease activity in tumor-associated macrophages to promote cancer growth and invasion.Genes Dev. 2010; 24: 241-255https://doi.org/10.1101/gad.1874010Crossref PubMed Scopus (544) Google Scholar), the authors investigated whether Cathepsin B production by TAMs in their study was responsible for promoting tumor cell invasion. Conditioned medium (CM) from M2-like TAMs induced higher tumor cell invasion in vitro than M1-like TAMs, which was abrogated by OGT deficiency but replenished by recombinant Cathepsin B. OGT and glucose dependency of Cathepsin B-mediated tumor cell invasion was also validated in IL-4-polarized BMDMs. Co-expression of OGT and wild-type Cathepsin B, but not S210-mutated Cathepsin B, induced tumor cell invasion. These results confirm that glucose-dependent, OGT-mediated, O-GlcNAcylation of Cathepsin B in M2-like TAMs drive their ability to induce tumor cell invasion. Growth of B16 melanoma and LLC in myeloid-specific OGT knockout mice markedly inhibited lung metastasis and prolonged survival compared with wild-type counterparts (Figure 1C). Mechanistically, co-localization of OGT and Cathepsin B was observed in TAM lysosomes, and OGT deficiency in TAMs decreased S210 O-GlcNAcylation of Cathepsin B as well as its intracellular and secreted mature forms in tumor interstitial fluids. The authors showed chemotherapy such as Oxaliplatin or Paclitaxel led to increased levels of OGT-mediated S210 O-GlcNAcylation of Cathepsin B and secretion of matured Cathepsin B in BMDMs. In vitro, CM from IL-4-polarized BMDMs and M2-like TAMs blunted the effect of chemotherapy on cancer cells in an OGT- and Cathepsin B-dependent manner. In vivo, myeloid-specific OGT deficiency in tumor-bearing mice (B16, LLC, and MC38) increased the chemotherapeutic effects of these drugs: inhibiting tumor growth, lung metastasis (shown only for B16 and LLC), and prolonging survival. Indeed, in a cohort of colorectal cancer (CRC) patients that received oxaliplatin-based, neoadjuvant chemotherapy, higher proportions of OGThi TAMs and Cathepsin B levels in the TME correlated with higher tumor regression grade (TRG) and shorter overall survival (Figure 1C). Together, these results support the role of OGT-mediated O-GlcNAcylation of Cathepsin B in TAMs to support chemoresistance. Metabolic re-programming of TAMs in different cancer models suggest that the mechanisms by which TME drives metabolic changes in these cells and how such metabolic changes regulate TAM functions are diverse and poorly understood. A case-by-case study across different tumor types and TAM subsets is therefore necessary before translational approaches to "metabolically" target TAMs can be designed. As TAMs are the highest producers of Cathepsin B in the TME, the use of O-GlcNAcylation inhibitors could provide a two-pronged attack by (1) curbing TAMs' pro-metastatic and chemoresistance activity and (2) exerting direct inhibitory effect on cancer cells, given the importance of O-GlcNAcylation in cancer. The feasibility of this approach must be weighed against the toxicity of these inhibitors and their off-target effects since O-GlcNAcylation impacts several aspects of immune cell function (Chang et al., 2020Chang Y.H. Weng C.L. Lin K.I. O-GlcNAcylation and its role in the immune system.J. Biomed. Sci. 2020; 27: 57https://doi.org/10.1186/s12929-020-00648-9Crossref PubMed Scopus (60) Google Scholar). Further, the fact that higher proportions of OGThi TAMs correlate with shorter overall survival in chemotherapy-treated CRC patients raises the question whether this metabolic signature could be predictive of response to chemotherapy or immunotherapy, though the latter needs future investigation. Finally, though metabolic regulation of Cathepsin B was defined in M2-like TAMs, its in vivo relevance in other TAM subpopulations is not clear. Future high-dimensional analysis of metabolic signatures at a single-cell and spatial level in human cancer tissues might help clarify this issue. The authors declare no competing interests. Increased glucose metabolism in TAMs fuels O-GlcNAcylation of lysosomal Cathepsin B to promote cancer metastasis and chemoresistanceShi et al.Cancer CellSeptember 8, 2022In BriefShi et al. show that M2-like tumor-associated macrophages (TAMs) bear the highest glucose uptake ability. Cathepsin B O-GlcNAcylation mediated by lysosomal OGT is the major effector downstream of glucose uptake by TAMs promoting tumor metastasis and chemoresistance. OGT expression in TAMs correlates with response to chemotherapy in individuals with colon cancer. Full-Text PDF Open Archive