ABSTRACT: Triple-negative breast cancer (TNBC) initially exhibits a robust response to chemotherapy; however paradoxically, the treatment enriches a population of drug-tolerant persister (DTP) cells, which are known to drive therapy resistance. Therefore, it is crucial to, foremost, identify and subsequently target the signaling pathways implicated in therapy-induced drug resistance and the emergence of DTP cells. In this study, by combining the use of human TNBC cell lines, 3D patient-derived organoid models, and in vivo xenograft models, we identified activation of the Wnt/β-catenin signaling pathway as a common mechanism underlying early DTP cell(s) enrichment in response to different chemotherapeutic agents. Live-cell imaging using Wnt-reporter TNBC cell lines validated diverse patterns of Wnt-transcriptional enrichment, encompassing both intrinsic selection and acquired de novo activation in response to chemotherapy treatment. Purification of differential Wnt-transcriptional populations revealed that post-treatment WntLow cells succumb to apoptosis, while, in contrast, chemotherapy-treated Wnt-active (WntHigh) cells adopt stem cell-like traits, exhibit reduced proliferation, and are, in turn, refractory to treatment. The transition to a post-treatment WntHigh state is orchestrated by increased expression of Wnt ligands, R-spondins, and factors facilitating Wnt ligand secretion, such as PORCN. Consequently, genetic or concomitant - but not sequential - pharmacological inhibition of Wnt ligand secretion alongside chemotherapy prevents treatment-induced WntHigh enrichment and sensitizes TNBC tumors to chemotherapy. Taken together, these observations provide a strong rationale for testing dual treatment comprising chemotherapy and Wnt ligand secretion inhibitors in clinical trials, extending to all TNBC patients.
