Dear Editor,

We report here that 17-β-estradiol (E2), through ERα (estrogen receptor α), inhibits IFN-γ-induced surface PD-L1 (programmed death-ligand 1, CD274) level in advanced ER⁺/HER2⁻ breast cancer (BC) but not in triple negative BC (TNBC) or endometrial cancer (EC). This study implies that mTOR and MAPK pathways can reduce the surface PD-L1 level and diminish immune evasion via ERα. It also has important implications on endocrine resistance and the limitations of mTOR or MAPK-targeted therapies for treating advanced ER⁺/HER2^– BC.

Tumour cell surface PD-L1 binds to the PD-1 (programmed death 1, CD279) receptor on the surface of activated tumour-infiltrating lymphocytes (TILs), leading to tumour immune evasion. However, high-level surface PD-L1 is observed in ER⁻ BC and EC tissues and the TILs^{1, 2} but rarely in ER⁺ BC tumors.² Additionally, it remains unclear why ER⁺ patients are less responsive than those with ER⁻ cancers to anti-PD-1/PD-L1 therapy.

E2 functions primarily through the two receptors of ERα and ERβ in both cancers. Approximately 60%–65% of BC cases are ER⁺/HER2^– luminal A subtype,³ and 80% of EC are of type 1 ER⁺ cancers.⁴ E2 reduces the IRF-1 protein and mRNA levels in murine splenocytes⁵ and MCF-7 cell line,⁶ respectively, implying that E2 may reduce the surface PD-L1 in both diseases. However, how E2 regulates tumour cell surface PD-L1 expression in ER⁺ BC and type1 EC is elusive.

By qRT-PCR, BC cell lines MCF-7 (ERα⁺/PD-L1^trace) and MDA-MB-231 (ERα⁻/PD-L1^high), and EC cell lines Ishikawa (ERα⁺/PD-L1^trace, type 1) and TEN (ERα⁻/PD-L1^trace, type 2) were selected; flow cytometry was employed for this study. We examined the PD-L1 transcripts in a cohort of 25 EC specimens and detected a significantly higher PD-L1 transcriptional level in the type 2 EC group than in non-EC, but not for type 1 EC (Tables S1–S4 and Figures S1 and S2).

Since IFN-γ is a major driver of surface PD-L1 expression through the IFN-γ-JAK-STAT-IRF1 pathway in multiple cancers,⁷ we exposed these cells to different concentrations of IFN-γ for 24 h and demonstrated that IFN-γ strongly increased the surface PD-L1 level in MCF-7, MDA-MB-231 and TEN. This did not happen to Ishikawa (Figure 1A–D), even after 3 days of exposure to 50 ng/ml IFN-γ (Figure 1G,H). Comparatively, MCF-7 showed a strong response, with the surface PD-L1 level peaking at 48 h and declining non-significantly at 72 h (Figure 1E,F). The same trend occurred after the cells were exposed to 10 ng/ml IFN-γ in a phenol-red free medium containing 5% CSFBS (charcoal-stripped fetal bovine serum) for 3 days (Figure 1I,J).

We exposed the four cell lines to 10 ng/ml IFN-γ, 10 nM E2, and a combination of both in a phenol-red free medium containing 5% CSFBS during an eight-day culture. We showed that E2 alone did not alter the PD-L1 level on the surface of all four cell lines but greatly downregulated the IFN-γ-induced PD-L1 at the surface and transcriptional levels in MCF-7 co-exposed to E2 and IFN-γ, compared with IFN-γ exposure alone (Figure 2A–F), suggesting that it is not endogenous but the IFN-γ-induced surface PD-L1 that was downregulated by E2 in the advanced ER⁺/HER2⁻ BC in vitro.

As E2 affected ERα⁺/ERβ⁺ MCF-7 instead of the ERα⁻/ERβ⁺ MDA-MB-231, we used two ERα antagonists tamoxifen (Figure 3A) and ICI 182,780 (Fulvestrant) (Figure 3B), and the ERβ antagonist PHTPP (Figure 3C) to block both receptors, respectively, in MCF-7. We demonstrated that ERα contributed to the E2's downregulation at the surface and transcriptional (Figure 3D) levels. Since the PD-L1 level in the ERα⁻/ERβ⁺ A549 lung cancer cell line is strongly upregulated by IFN-γ via the JAK/STAT/IRF-1 pathway,⁸ our outcome was further validated in this line that ERβ was not involved (Figure 3E).

Our finding accords with a newly published work by Hühn et al.,⁹ who showed that total E2 deprivation or fulvestrant treatment increased the surface PD-L1 expression in MCF-7 through ERα. Their report designated changes in MFI, however, we observed changes in the proportion (%) of the PD-L1 expressing cells. This suggests that E2 may protect ER⁺/HER2^– BC by interacting with ERα and reducing the quantity of IFN-γ-induced surface PD-L1 expressing cells. Thus, such a protective role by E2 may slow the progression of ER⁺ BC's deterioration. This may explain why ER⁺/HER2^– BC expresses less PD-L1, less malignance than the basal TNBC subtype, and less sensitivity to anti-PD-1/PD-L1 therapy than ER⁻ BC.

We confirmed that the JAK/STAT/IRF1 pathway regulates the IFN-γ-induced surface PD-L1 level (Figure 4,1 and 2A). Co-exposure to BEZ235 or U0126 combined with E2 and IFN-γ significantly restored the proportion of the E2-downregulated IFN-γ-induced surface PD-L1 expressing cells in MCF-7 (Figure 4,2C), but LY294002 or other kinase inhibitors (Table S5) were not capable (Figure 4,2B,C). Additionally, BEZ235 performed at a much greater recovery rate than U0126, significantly higher than those exposed to IFN-γ in MCF-7 (Figure 4,2D). This outcome implies that both the mTOR and the MAPK pathways cooperate in inhibiting immune evasion through ERα by reducing the IFN-γ-induced surface PD-L1 expression in ER⁺ BC.

We found that tamoxifen or ICI 182,780 potentiates IFN-γ to upregulate surface PD-L1 expression, even when E2 is present in MCF-7 (Figure 3A,B). The higher the antiestrogen dose, the greater the PD-L1 surface expression (Figure 3A,B) as ICI 182, 780 inhibited ER in a dose-dependent manner.¹⁰ This indicates that prolonged administration of antiestrogens may induce tumour cell surface PD-L1 expression by disrupting a dynamic protective effect built by the E2-ERα-IFN-γ-mTOR-MAPK axis in the tumour milieu of the advanced ER⁺/HER2^– BC patients, triggering tumour immune evasion. Together, these findings may provide causal reasons for antiestrogen resistance and the limited efficacy of mTOR or MAPK-targeted therapies for treating advanced ER⁺/HER2^– BC patients. Additionally, the increased tumour cell surface PD-L1 induced by the inhibitors of ERα, mTOR or MAPK may potentially augment the sensitivity of ER⁺ BC cells to immune checkpoint inhibitors.