Inhibition of Proteasome LMP2 Activity Suppresses Chil3 Expression in Mouse Colon Adenocarcinoma Tissue and Restrains Tumor Growth.

Objectives: Proteasomes, multi-subunit proteases, are key actors of cellular protein catabolism and a number of regulatory processes. The detection of subtle proteasome functioning in tumors may contribute to our understanding of the mechanisms of cancer development. The current study aimed to identify the role of low molecular mass protein 2 (LMP2), a proteasome immune subunit, in the development of mouse colon 26 (C26) adenocarcinoma.

Methods: The functions of the LMP2 subunit in tumor development in Balb/c mice were studied using its irreversible inhibitor KZR-504. LMP2 activity was detected by the hydrolysis of the fluorogenic substrate Ac-Pro-Ala-Leu-AMC. Western blotting and Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) were used. We applied fluorescent tests for cell proliferation and apoptosis. M2 macrophages were obtained by polarization of mouse bone marrow-derived macrophages using the corresponding cytokines.

Results: KZR-504 showed high specificity only for the LMP2 subunit and had no negative effect on C26 cells in culture. However, KZR-504 suppressed the formation of tumor conglomerates (by 74%, p < 0.001) after C26 cell transplantation in vivo, inhibited the expression of chitinase-3-like protein 3 (Chil3) gene (by 90%, p < 0.001), a key marker of immunosuppressive M2 macrophages, in the tumor microenvironment, and reduced the tumor weight compared to the control (by 48%, p < 0.01). KZR-504 also suppressed the expression of Chil3 (by 68%, p < 0.05) and arginase-1 (Arg1) (by 90%, p < 0.001), another marker gene, in M2 macrophages and violated M0-M2 macrophage polarization in culture.

Conclusion: We discovered earlier unknown functions of the proteasome LMP2 subunit to facilitate the formation of tumor conglomerates and maintain Chil3 and Arg1 expression in immunosuppressive M2 macrophages. Our work demonstrates that the proteasome LMP2 subunit can be a target for antitumor treatment.