TIMELESS Upregulates PD-L1 Expression and Exerts an Immunosuppressive Role in Breast Cancer

Background: Upregulation of the PD-L1 (CD274) immune checkpoint ligand on the tumor surface facilitates tumor immune escape and limits the application of immunotherapy in various cancers, including breast cancer. However, the mechanisms underlying high PD-L1 levels in cancers are still poorly understood.

Methods: Bioinformatics analyses and in vivo and in vitro experiments were carried out to assess the association between CD8 T lymphocytes and TIMELESS (TIM) expression, and to discover the mechanisms of TIM, the transcription factor c-Myc, and PD-L1 in breast cancer cell lines.

Results: The circadian gene TIM enhanced PD-L1 transcription and facilitated the aggressiveness and progression of breast cancer through the intrinsic and extrinsic roles of PD-L1 overexpression. Bioinformatic analyses of our RNA sequencing data in TIM-knockdown breast cancer cells and public transcriptomic datasets showed that TIM might play an immunosuppressive role in breast cancer. We found that TIM expression was inversely associated with CD8 T lymphocyte infiltration in human breast cancer samples and subcutaneous tumor tissues. In vivo and in vitro experiments demonstrated that TIM knockdown increased CD8 T lymphocyte antitumor activity. Furthermore, our results showed that TIM interacts with c-Myc to enhance the transcriptional capability of PD-L1 and facilitates the aggressiveness and progression of breast cancer through the intrinsic and extrinsic roles of PD-L1 overexpression. Moreover, public database analysis suggested that high TIM levels were positively related to PD-L1 inhibitor therapeutic response.

Conclusions: Mechanistically, we first found that TIM could upregulate PD-L1 by interacting with c-Myc to enhance the transcriptional capability of c-Myc to PD-L1. Altogether, our findings not only provide a novel therapeutic strategy to treat breast cancer by targeting the oncogenic effect of TIM but also indicate that TIM is a promising biomarker for predicting the benefit of anti-PD-L1 immunotherapy.