Marrone, Laura (2023) Insights into the mechanisms of alternative macrophage polarization to circumvent cancer immunotherapy resistance. [Tesi di dottorato]

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Abstract

Background: Tumor-associated-macrophages (TAMs) play a pivotal role in promoting tumor progression and therapy resistance; their targeting has recently emerged as a promising strategy for cancer defeat. Current approaches focus on reprogramming TAMs from the M2 pro-tumoral to the M1 anti-tumoral phenotype to kill cancer cells. Unfortunately, in-depth knowledge of TAMs and a signature that can reliably identify them still needs to be improved. We identified a splicing isoform of the FKBP5 gene, FKBP51s, exploited by cancer cells to suppress undesired immunity and highly expressed in circulating monocytes of cancer patients resistant to immunotherapy. Aim of this study is to decipher the role of FKBP51s in TAM biology to identify new potential therapeutic targets to their reprogramming. Methods: Macrophage polarization was assessed by qPCR, immunoblot, and flow cytometry both in a THP-1 cell line and in primary monocytes to obtain classically activated M1-macrophages, alternatively polarized M2a-macrophages, and the deactivated M2c-macrophages. Small interfering RNAs were transfected to specifically target FKBP51s expression and the silencing effect on the modulation of key macrophage polarization genes and cytokines was studied by qPCR, immunoblot, ELISA and flow cytometry. Migration and invasion were investigated by the transwell assay and immunosuppression by co-culturing macrophages with T cells. Proximity ligation assay assessed FKBP51s localization to the ER. Ribosome profiling served to assess the association of FKBP51s with polysomes. Agilent Seahorse measured oxygen consumption rate and extracellular acidification rate of macrophages. Results: Alternative-macrophage polarization showed typical features of M2 and M1, such as STAT1 downregulation in favor of STAT3/6, and a shift towards arginase 1 metabolism and scavenger receptors expression. Interestingly, FKBP51s levels strongly increased in M2-macrophages, thus suggesting that FKBP5 alternative splicing occurs in TAMs. FKBP51s silencing restored STAT1 activity, increased the secretion of pro-inflammatory cytokines, while IL-10 production was impaired. FKBP51s silencing strongly impacted on TAMs migration, invasiveness, and T-cells proliferation. Immunoblot of fractioned lysates showed that FKBP51s is mainly expressed in the cytoplasm of M2 and it is strongly associated with the cytoplasmic side of the ER. Polysome profiling showed that FKPB51s, but not the canonical form, is associated with polysomes, thus suggesting it could play a role in safeguarding the folding of the nascent protein at the ribosomal tunnel exit. Finally, silencing of FKBP51s also impaired OXPHOS and restored glycolysis in M2 macrophages. Conclusions: Results from this study suggest a relevant role for FKBP51s in the co-translational control of protein modification and folding, promoting a shift towards the expression of a different set of protein to accomplish the pro-tumoral activities of TAMs. Results from this thesis work highlight FKBP51s as a new potential therapeutic target to reprogram TAMs towards an anti-tumoral macrophage phenotype, thus overcoming the immune suppressive tumor microenvironment.

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