A Tumor Microenvironment-Driven Network Regulated by STAT3 and p65 Negatively Controls the Enrichment of Cancer Stem Cells in Human HR+/HER2− Breast Cancer

SIMPLE SUMMARY: Hormone receptor-positive (HR+)/HER2− breast cancer is driven by extracellular cues within the tumor microenvironment (TME) including hormonal, inflammatory and growth-stimulating signals. Our past findings indicate that a “TME Stimulation” jointly addressing these three arms induces pro-metastatic traits in HR+/HER2− breast cancer cells, primarily with the enrichment of cancer stem cells (CSCs), driving metastasis in vivo. Here, we reveal intricate roles for STAT3 as a negative and positive regulator of TME Stimulation-induced pro-metastatic effects in HR+/HER2− cells. Of note, the two transcription factors STAT3 and p65 acted in cooperativity to limit CSC enrichment, and their down-regulation has led to enriched levels of chemotherapy-resistant CSCs. Moreover, STAT3 and p65 activation were inversely connected to a CSC signature and positively associated with improved survival in patient datasets. These findings suggest that we need to carefully consider the roles of STAT3 and p65 roles in regulating TME activities in malignant diseases, in efforts to identify novel targets for therapeutic intervention. ABSTRACT: Hormone receptor-positive and HER2-negative (HR+/HER2−; luminal A) tumors are prevalent in breast cancer. Our past studies demonstrated that “TME Stimulation” (estrogen + TNFα + EGF, representing three arms of the tumor microenvironment, TME) has enriched metastasis-forming cancer stem cells (CSCs) in HR+/HER2− human breast cancer cells. Here, following information obtained by RNAseq analyses of TME-stimulated CSCs and Non-CSCs, we found that TME Stimulation has induced the activation of S727-STAT3, Y705-STAT3, STAT1 and p65. Upon TME Stimulation, stattic (STAT3 inhibitor) usage demonstrated that Y705-STAT3 activation negatively controlled CSC enrichment and epithelial-to-mesenchymal transition (EMT) traits, while inducing CXCL8 (IL-8) and PD-L1 expression. However, STAT3 knock-down (siSTAT3) had no effect on these functions; in terms of CSC enrichment, p65 had down-regulatory roles that compensated for the loss of an entire STAT3 protein. Y705-STAT3 and p65 acted additively in reducing CSC enrichment, and Y705A-STAT3 variant + sip65 has enriched chemo-resistant CSCs. Clinical data analyses revealed an inverse correlation between Y705-STAT3 + p65 phosphorylation and CSC signature in luminal A patients, and connection to improved disease course. Overall, we find regulatory roles for Y705-STAT3 and p65 in TME-stimulated HR+/HER2− tumors, with the ability to limit CSC enrichment. These findings raise concerns about using inhibitors of STAT3 and p65 as therapeutic strategies in the clinic.