UBE2I depletion regulated tumor-associated macrophage polarization into M1 type through reprogramming glycolysis and increases immunotherapy efficacy of anti-PD-L1 in ovarian cancer

Abstract

There is ample evidence that ubiquitin-conjugating enzyme E2I (UBE2I) is involved in progression of diverse cancers. However, the influence of UBE2I on ovarian cancer (OC) has been poorly reported. This study tries to discover the mechanisms and functions of UBE2I in OC. Relative mRNA expression of UBE2I, CD86, iNOS, MHC II and programmed death ligand 1 (PD-L1) was detected through qRT-PCR. We identified UBE2I, Vimentin, E-Cadherin, N-Cadherin and Ki67 protein expression levels in tumor tissues through immunohistochemistry staining. Protein levels of UBE2I, cleaved caspase-3, cleaved PARP, E-cadherin, N-cadherin and Vimentin were detected through western blot. Cell viability, invasion, and migration were examined by means of cell counting kit-8 (CCK-8), transwell, and wound healing assays. Immunofluorescence was used to detect colocalization between UBE2I and CD68. We assessed expression levels of IFN-γ and TNF-α via flow cytometry and ELISA. We used the TUNEL assay to assess tumor cell apoptosis. Glycolysis was assessed through the consumption of glucose, ATP production, production of lactate, and extracellular acidification rate. For establishing a xenograft model, OC cells were subcutaneously injected into mice. UBE2I expression was boosted in OC cells and tissues, which was negatively associated with OC patients’ prognosis. Silencing of UBE2I suppressed OC cell proliferation, invasion, EMT (epithelial-to-mesenchymal transition) and migration. UBE2I inhibition promoted macrophages toward the M1 phenotype and macrophage viability. After deletion of UBE2I in vivo, mice tumor growth and EMT were suppressed, and apoptosis of tumor cells was increased. Meantime, an increasing proportion of CD86+ TAMs (tumor-associated macrophages) was observed after the deletion of UBE2I. Besides, increases in consumption of glucose, lactate production, ATP production and ECAR in THP-1 cells were observed by silencing of UBE2I; however, glycolysis inhibitor reversed UBE2I-mediated polarization of M1 macrophages in a dose-dependent fashion. Importantly, UBE2I-mediated M1 macrophages promoted PD-L1 expression. Furthermore, the combinatorial therapy of UBE2I inhibitor plus anti-PD-1 repressed tumor growth, reduced Ki67 expression, and promoted apoptosis in tumor cells, exhibiting higher efficiency than UBE2I inhibitor/anti-PD-L1 alone. UBE2I inhibition regulated polarization of M1 macrophages via glycolysis and improved anti-PD-L1 immunotherapy efficacy, paving a novel avenue to prevent OC development.