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UBR5 promotes tumor immune evasion through enhancing IFN-γ-induced PDL1 transcription in triple negative breast cancer.

ABSTRACT: Background: The up-regulation of PD-L1 is recognized as an adaption of cancer cells to evade immune surveillance and attack. However, the intrinsic mechanisms of the induction of PD-L1 by interferon-γ (IFN-γ) in tumor microenvironment remain incompletely characterized. Ubiquitin ligase E3 component N-recognition protein 5 (UBR5) has a critical role in tumorigenesis of triple negative breast cancer (TNBC) by triggering specific immune responses to the tumor. Dual targeting of UBR5 and PD-L1 exhibited superior therapeutic benefits in a preclinical TNBC model in short term. Methods: The regulation of UBR5 to PD-L1 upon IFN-γ stimulation was evaluated through in UBR5 deficiency, reconstitution or overexpression cell line models by quantitative PCR, immunohistochemistry and RNA-seq. The effects of PD-L1 regulation by UBR5 and double blockade of both genes were evaluated in mouse TNBC model. Luciferase reporter assay, chromatin immunoprecipitation-qPCR and bioinformatics analysis were performed to explore the transcription factors involved in the regulation of UBR5 to PD-L1. Results: E3 ubiquitin ligase UBR5 plays a key role in IFN-γ-induced PDL1 transcription in TNBC in an E3 ubiquitination activity-independent manner. RNA-seq-based transcriptomic analyses reveal that UBR5 globally affects the genes in the IFN-γ-induced signaling pathway. Through its poly adenylate binding (PABC) domain, UBR5 enhances the transactivation of PDL1 by upregulating protein kinase RNA-activated (PKR), and PKR's downstream factors including signal transducers and activators of transcription 1 (STAT1) and interferon regulatory factor 1 (IRF1). Restoration of PD-L1 expression in UBR5-deficient tumor cells recoups their malignancy in vivo, whereas CRISPR/Cas9-mediated simultaneous abrogation of UBR5 and PD-L1 expression yields synergistic therapeutic benefits than either blockade alone, with a strong impact on the tumor microenvironment. Conclusions: This study identifies a novel regulator of PDL1 transcription, elucidates the underlying molecular mechanisms and provides a strong rationale for combination cancer immunotherapies targeting UBR5 and PD-L1.

SUBMITTER: Wu B

PROVIDER: S-EPMC9274738 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:HORMA domain‑containing protein 1 (HORMAD1), is normally expressed only in the germline, but is frequently re‑activated in human triple‑negative breast cancer (TNBC); however, its function in TNBC is largely unknown. In the present study, the expression and biological significance of HORMAD1 in human TNBC was evaluated. Bioinformatics analysis and reverse transcription‑quantitative PCR were used to evaluate HORMAD1 expression in datasets and cell lines. HORMAD1 protein expression was detected in TNBC samples using immunohistochemical assays, and the effect of HORMAD1 on cell proliferation was determined using Cell Counting Kit‑8, plate colony formation and standard growth curve assays. Cell cycle, reactive oxygen species (ROS) and apoptosis analyses were conducted using flow cytometry. The activity of caspases was measured using caspase activity assay kit. The levels of key apoptosis regulators and autophagy markers were detected by western blot analysis. TNBC cell survival and apoptosis were not influenced by small interfering RNA targeting HORMAD1 alone; however, HORMAD1 knockdown enhanced autophagy and docetaxel (Doc)‑induced apoptosis, compared with the control group. Furthermore, higher ROS levels and caspase‑3, ‑8 and ‑9 activity were detected in MDA‑MB‑436 TNBC cells with HORMAD1 knockdown upon exposure to Doc. The levels of the induced DNA damage marker γH2AX were also higher, while those of the DNA repair protein RAD51 were lower in TNBC cells with HORMAD1 knockdown compared with the controls. Furthermore, the expression of the autophagy marker P62 was enhanced in MDA‑MB‑231 cells in response to HORMAD1 overexpression. Notably, Doc‑induced apoptosis was similarly increased by both HORMAD1 overexpression and treatment with the autophagy inhibitor, 3‑methyladenine (3MA); however, the Doc‑induced increase in autophagy was not inhibited by 3MA. The present data indicated that HORMAD1 was involved in autophagy and that the inhibition of autophagy can partially enhance the induction of apoptosis by Doc. The role of HORMAD1 in the DNA damage tolerance of tumor cells may be the main reason for Doc resistance; hence, HORMAD1 could be an important therapeutic target in TNBC.

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UBR5 promotes tumor immune evasion through enhancing IFN-γ-induced PDL1 transcription in triple negative breast cancer.

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