Association of NR2F1, a tumor dormancy marker, with cancer cell proliferation and lymph node metastasis, and expression in cancer-associated fibroblasts in breast cancer.

Abstract

e12550 Background: It is now well accepted that some cancer cells remain in quiescent state and survive through adjuvant systemic treatments, also known as tumor dormancy, which preserve the potential for late recurrence. NR2F1 is a known biomarker for dormant cancer cells, which suppress cell proliferation in experimental models. Here, we hypothesized that patient breast cancer (BC) with high NR2F1 gene expression is associated with less cell proliferation and worse survival. Methods: More than 7000 patients from 3 large-scale primary and 5 metastatic BC patient cohorts and 2 single-cell sequencing cohorts were analyzed. Results: NR2F1 expression was upregulated in dormant cell lines and in bone metastatic clone cells compared with mother 4T1 cells. High NR2F1 BC enriched multiple metastasis-related pathways such as TGFβ, EMT, angiogenesis, as well as cancer stem cell-related signaling (NOTCH, HEDGEHOG, and Wntβ-catenin). As expected, all 5 cell proliferation-related gene sets including E2F Targets and G2M Checkpoints were enriched in low NR2F1 BC, and so was MKI67 expression. Unexpectedly, there were no consistent association between NR2F1 expression and survival among the 3 cohorts. Three out of four cohorts demonstrated significantly high NR2F1 expression in primary BC with lymph node metastasis. There was no difference in NR2F1 expression in primary BC by the distant metastasis in the four cohorts, by the metastatic sites, or between the primary and metastatic BC neither. Given the discrepancy between the biology and clinical outcome of high NR2F1 BC, we performed the functional analysis exclusively . Surprisingly, there were no consistent enrichment of metastasis-related gene sets nor downregulation of cell proliferation-related gene sets to high NR2F1 expression cancer cells, unlike the analyses of bulk tumors. Intratumoral heterogeneity, HRD, proliferation score, and mutation load were all less in high NR2F1 BC, along with IFN-γ response and TIL fractions. Infiltration of immune cells such as Th1 and Th2 T-cells, M1 and M2 macrophages were all less, while stromal cells such as adipocytes, fibroblasts, stromal cells and endothelial cells were more abundant in high NR2F1 BC. Surprisingly, NR2F1 was barely expressed in cancer cells and was most highly expressed in cancer-associated fibroblasts consistently regardless of subtypes in two independent single-cell sequence cohorts. The expression of NR2F1 was higher in inflammatory than myofibroblastic CAFs, and that of CAFs, but not cancer cells, was associated with lymph node metastasis or advanced stage. Conclusions: The tumor dormancy marker NR2F1 expression in primary BC is associated with mechanism of metastasis and suppression of cancer growth, but is most expressed in cancer-associated fibroblasts in the tumor microenvironment.