AP-1 regulates heterogeneous cellular dormancy in TNBC

BackgroundDormant or slow cycling cells (SSCs) pre-exist in tumor and responsible for chemo-resistant and tumor recurrence. Due to their low differentiation and dormancy characteristics, SCCs are resistant to standard chemotherapy and targeted therapy. Label-retaining is a common method used to identify and isolate live SCCs. However, it remains unclear whether different label-retaining methods yield distinct SCC subpopulations. In this study, we investigated that various label-retaining methods result in overlapping yet heterogeneous subpopulations of SCCs. Additionally, we explored the molecular mechanisms regulating dormancy in triple-negative breast cancer (TNBC). MethodsWe employed multiple label-retaining methods to simultaneously label MDA-MB-231 cells, thereby generating distinct subpopulations of SCCs. We subsequently analyzed these subpopulations for heterogeneity in cell cycle distribution, drug resistance, invasive capacity, and other characteristics using real-time PCR, flow cytometry, and Transwell assays. RNA-seq analysis was performed to characterize the gene expression profiles of the SCCs. Furthermore, we used real-time PCR, Western blotting, immunofluorescence, and luciferase assays to investigate the role of characteristic AP-1 expression in dormancy regulation. Finally, the therapeutic effects of targeting AP-1 in the treatment of TNBC were assessed using a cell-derived xenograft model. ResultsWe labeled and separated three overlapping but non-identical SCCs subpopulations. We found that all three SCCs subgroups are cell cycle arrested. Additionally, Violet enriched SCCs showed stronger drug resistance and more G1 phase arrest, while Claret enriched SCCs demonstrated enhanced migratory and invasive abilities, along with more G2/M phase arrest. Furthermore, we observed upregulation of AP-1 expression in SCCs, and the JunB subunit of AP-1 promoted the expression of CDKN1A and GADD45A, thereby maintaining cell cycle arrest. CC-930 can inhibit AP-1 transcriptional activity by suppressing JNK activity, ultimately improving the therapeutic efficacy and prognosis of TNBC when used in combination with chemotherapy drugs. ConclusionsWe obtained three subpopulations of SCCs with heterogeneous drug resistance. Our findings suggest that AP-1 plays a regulatory role in dormancy regulation in TNBC, and elucidated the molecular function of JunB subunit. Targeting AP-1 with CC-930 has the potential to improve the treatment and prognosis of TNBC. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/566980v2_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@129ce76org.highwire.dtl.DTLVardef@1b1cfa1org.highwire.dtl.DTLVardef@b68315org.highwire.dtl.DTLVardef@57faf1_HPS_FORMAT_FIGEXP M_FIG C_FIG TNBC harbors both fast-cycling cells (FCCs) and functionally overlapping slow-cycling cell (SCC) subpopulations that manifest differential drug sensitivities and motility (A) but are commonly regulated by the JunB-containing AP1 complex (B). (A). Slow-cycling (quiescent) TNBC cells in culture (a) identified by different label-retaining approaches phenotypically overlap (b), display differential drug sensitivities (c, d) and motility (e) but share common gene expression profiles (f). (B). Schematic depicting regulation of proliferation in FCCs by the c-Jun/c-Fos AP1 complex (left) and regulation of cellular dormancy in SCCs by c-Jun/JunB AP1 complex.