Background Breast cancer patients diagnosed at the age younger than 45 years old show poor prognosis. High exposure of young women to plasticizers is one of major risk factors for the early-onset of breast cancer (EOBC). Breast tumors from younger patients showed higher FOXP3⁺ tumor-infiltrating lymphocytes (TILs), suggesting a role of dysregulation of immune surveillance in the tumorigenesis of EOBC. The impact of plasticizer exposure on the immune dysregulation for the early onset of breast cancers and the underlying mechanisms were investigated to develop appropriate therapeutic strategies for this disease.

Methods Clinical specimens were obtained from Chung Shan Medical University and DEHP levels in hair were measured in GC/Mass analysis and the correlation between PD-L1, ERβ, and TILs expression in human breast cancer tissues were detected in IHC analysis. The induction of PD-L1 by DEHP, one of the phthalate plasticizers, in breast epithelial and cancer cells was tested by western blot and flow cytometry. The role of plasticizer in regulating the expressions of various immune checkpoints and the cytotoxic activity of T cells was studied by using both in vitro cell line models and in vivo mice models. The involved transcription factor and signaling pathways were identified by using various molecular biological analyses.

Results The hair level of DEHP was higher in EOBC patients, and was positively associated with PD-L1 and ERβ expressions but negatively associated with TILs. In mouse models, the treatment with DEHP accelerated tumorigenesis and tumor growth and repressed T cell-mediated cytotoxicity due to the upregulations of immune checkpoint PD-L1. Anti-PD-L1 antibody reversed the exhaustion of CD8⁺ T cells by DEHP-treated breast cancer cells. Our data, for the first time, further demonstrated that DEHP transcriptionally induced PD-L1 expression through ERβ activation even in triple-negative breast cancer (TNBC) cells. Moreover, DEHP also increased CCND1-CDK4 expressions, and CDK4/6 inhibitor Palbociclib prevented DEHP-mediated CD8⁺ T cell exhaustion. Finally, combination therapy with anti-PD-1 antibody and Palbociclib showed a synergistic anti-cancer activity in DEHP-associated allograft 4T1-TNBC mice model in vivo.

Conclusions DEHP suppressed the cytotoxicity of CD8⁺ T cells and facilitates the tumor onset and growth of breast cancer through the ERβ-dependent upregulation of PD-L1 expression and CDK4/6 and CCND1 signaling axis in cancer cells. Co-treatment with anti-PD-1 antibody and CDK4/6 inhibitor showed promising therapeutic activity in plasticizer-associated breast cancers.

Ethics Approval Above study has been approved with the full-board review by Institutional Review Board of the Chung Shan Medical University Hospital on Jun 23, 2022. CSMUH No: CS1–22107

The animal use protocol listed below has been reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). CMUIACUC-2022–051-2