A series of studies have dissected the putative molecular mechanisms underlying the ICB resistance in TNBC. High expression of immune checkpoint genes has been proved to be one of the immune evasion mechanisms. In this study, we discovered HuR as a novel regulator of immune checkpoint PD-L1 mRNA in TNBC cells, which have a limited response to ICB targeting PD-1/PD-L1. Furthermore, we identified here, for the first time, niclosamide inhibits both HuR nucleo-cytoplasmic translocation and PD-L1 glycosylation that niclosamide can improve the anti-PD-1 immunotherapy efficacy in syngeneic murine breast and lung cancer models. Thus, our findings provide a new approach to improve anti-PD-1 immunotherapy in breast and lung tumors by repurposing niclosamide.

HuR has been reported to play an important role in cancer immune evasion by regulating TGF-β and cytokines [39, 40]. However, considering the complicated intrinsic mechanisms of ICB resistance in TNBC, the molecular mechanism driven by HuR has yet to be fully determined. Recently, Liu et al. described that HuR up-regulates PD-L1 expression on cancer cell surface via controlling CMTM6 [25]. However, given the CLIP-seq data results of HuR binding sites in identified AREs in CD274 3′-UTR regulated by Tristetraprolin (TTP) [41, 42], their results cannot preclude the possibility that HuR was directly involved in PD-L1 regulation. In the current study, we identified HuR as a novel modulator of PD-L1 by directly binding to and stabilizing its mRNA. Additionally, we observed the function of HuR in impeding IL-2 secretion by T cells, which is crucial for T cell activation [43, 44]. These data enriched our understanding toward the roles of HuR in cancer immune evasion and PD-L1 regulation, suggesting a way for enhanced antitumor immunity by targeting HuR.

Drug repurposing has emerged as a promising strategy for developing anti-cancer drug, given the high cost and lengthy timeline of developing a new drug. Among those approved drugs, niclosamide has been revealed to exert a synergistic effect with numerous cancer drugs in human cancer cells. Besides the reported multi-mechanisms of niclosamide in Wnt/β-catenin signaling, NF-κB, mTORC1, STAT, or Notch pathways [28, 34], we report here a previously not yet recognized function of niclosamide: inhibiting HuR nucleocytoplasmic translocation, which was recently reported by our group in another study [45]. Interestingly, niclosamide shows different effects on HuR translocation at high or low concentrations, which may be attributed to the nature of HuR as a cell-stress responding protein, but the detailed underlying mechanism is still not fully understood. Yet, considering that niclosamide is not a HuR-specific inhibitor, this result indicates that niclosamide may have a broad inhibitive effect on other proteins that possesses nucleo-cytoplasmic shuttling activities. A previous study in NSCLC reported that niclosamide down-regulates PD-L1 expression by inhibiting p-STAT3 [46]. Considering the strong correlation between cytoplasmic HuR and HG PD-L1, we conclude that niclosamide reduces PD-L1 protein via inhibiting cytoplasmic HuR function. These findings not only uncover a new molecular mechanism by which niclosamide improves antitumor immunity, but also identify HuR as a novel post-transcriptional regulator of PD-L1. More in-depth analysis is warranted to better understand the multi-faceted roles of niclosamide in HuR-mediated PD-L1 regulation.

Besides the consistent downregulation of PD-L1 levels with niclosamide, we unexpectedly observed that niclosamide also has a potent effect on PD-L1 glycosylation profiling in various cancer cell lines. PD-L1 protein undergoes extensive post-translational modifications including phosphorylation, ubiquitination, acetylation, and glycosylation [47]. N-linked glycosylation is one of the most abundant post-translational modifications and contributes to various biological functions, including protein stability, ligand-receptor interaction, and subcellular localization [48]. The mechanisms of PD-L1 N-linked glycosylation via B3GNT3 [49], STT3 [50], and Sigma1 [51, 52] are studied in different cancer cells, and we didn’t detect changes of these proteins upon niclosamide treatment. However, we observed changes in several ER proteins following niclosamide treatment, indicating that ER stress may be a potential cause of PD-L1 glycosylation dysregulation. Considering the strong heterogeneity among cell signaling, clinical characteristics, and therapeutic responses in different cancer types, the niclosamide-induced PD-L1 glycosylation disorder is still worth elucidating.

Our experiments in murine breast and lung cancer models show that the combination of niclosamide and anti-PD-1 antibody had a synergetic effect compared with monotherapies. These results are consistent with the report from Fu et al. that inhibiting PD-L1 expression by niclosamide enhances PD-1/PD-L1 ICB. Our results also verified the strategy proposed by Li, et al. that targeting glycosylated PD-L1 to eradicate cancer cells, suggesting that niclosamide shows promise in improve immunotherapy efficacy through several pathways. Benefiting from the well-studied pharmacokinetics and pharmacodynamics and biological safety of niclosamide, repurposing of this drug can be attractive as the process is less risky, more cost-effective, and can be quickly moved into clinical testing. Currently, there are over 6 clinical trials testing niclosamide for its anti-tumor activity in various cancer populations (clinicaltrials.gov). With our findings of its new mechanism of action on HuR-PD-L1 signaling, it is worth redesigning the clinical trials to select patients with high levels of cytoplasmic HuR and PD-L1, who may have a better response to ICB immunotherapy. Other drugs with similar effect on HuR may also be explored for cancer immunotherapy.

Although this study sheds light on the roles of HuR in ICB resistance and the potential therapeutic value of niclosamide for TNBC patients, it has several limitations. Firstly, as a multifunctional drug, niclosamide has been reported to regulate multiple signaling pathways involved in cancer, so it is possible that niclosamide may also affect the other proteins involved in ICB resistance or immune evasion. Secondly, the precise molecular mechanisms underlying the interruption of PD-L1 glycosylation by niclosamide remain unclear. Further studies are necessary to identify the specific downstream targets of niclosamide in this context and elucidate the precise mechanisms by which it regulates PD-L1 glycosylation. Additional studies are needed to fully understand the potential off-target effects of niclosamide and its impact on other cellular processes.

In summary, we demonstrate a novel regulation mechanism of PD-L1 by HuR, which might be a promising new therapeutic target. The FDA-approved drug niclosamide, acting on both HuR translocation and PD-L1 glycosylation, may be a promising repurposed drug to overcome immune resistance to ICB and promote survival in vivo. Our study offers a strong proof-of-principle of repurposing niclosamide as the first HuR inhibitor to test in clinic, to modulate HuR-PD-L1 signaling to improve the response of ICB immunotherapy, especially for the immune “cold” breast cancer.