Current treatment options for triple-negative breast cancer (TNBC) are limited to toxic drug combinations of low efficacy. We recently identified an aryl-substituted fatty acid analogue, termed CTU, that effectively killed TNBC cells in vitro and in mouse xenograft models in vivo without producing toxicity. However, there was a residual cell population that survived treatment. The present study evaluated the mechanisms that underlie survival and renewal in CTU-treated MDA-MB-231 TNBC cells. RNA-seq profiling identified several pro-inflammatory signaling pathways that were activated in treated cells. Increased expression of cyclooxygenase-2 and the cytokines IL-6, IL-8 and GM-CSF was confirmed by real-time RT-PCR, ELISA and Western blot analysis. Increased self-renewal was confirmed using the non-adherent, in vitro colony-forming mammosphere assay. Neutralizing antibodies to IL-6, IL-8 and GM-CSF, as well as cyclooxygenase-2 inhibition suppressed the self-renewal of MDA-MB-231 cells post-CTU treatment. IPA network analysis identified major NF-κB and XBP1 gene networks that were activated by CTU; chemical inhibitors of these pathways and esiRNA knock-down decreased the production of pro-inflammatory mediators. NF-κB and XBP1 signaling was in turn activated by the endoplasmic reticulum (ER)-stress sensor inositol-requiring enzyme 1 (IRE1), which mediates the unfolded protein response. Co-treatment with an inhibitor of IRE1 kinase and RNase activities, decreased phospho-NF-κB and XBP1s expression and the production of pro-inflammatory mediators. Further, IRE1 inhibition also enhanced apoptotic cell death and prevented the activation of self-renewal by CTU. Taken together, the present findings indicate that the IRE1 ER-stress pathway is activated by the anti-cancer lipid analogue CTU, which then activates secondary self-renewal in TNBC cells.

BACKGROUND: Mammosphere formation assay has become a versatile tool to quantify the activity of putative breast cancer stem cells in non-adherent in vitro cultures. However, optimizing the suspension culture system is crucial to establish mammosphere cultures from primary breast tumors.
METHODS: This study aimed at determining the self-renewal and sphere-forming potential of breast cancer stem-like cells derived from human primary invasive ductal carcinoma and normal breast tissue samples, and MCF-7 breast cancer cell line using an optimal suspension culture system. Mammosphere-forming efficiency of the mammospheres generated from the tissue samples and cell line were compared. We evaluated the expression of CD44+/CD24-/low and CD49f+/EpCAM-/low phenotypes in the stem-like cells by flow cytometry. CK-18, CK-19, α-SMA, and EpCAM marker expression was assessed using immunohistochemical staining.
RESULTS: Breast epithelial cells isolated from the three samples formed two-dimensional spheroids in suspension cultures. Interestingly, mammospheres formed from patient-derived primary breast tumors were enriched in breast cancer stem-like cells with the phenotype CD44+/CD24-/low and exhibited a relatively more number of large spheres when compared to the normal breast stem cells. MCF-7-derived SCs were more aggressive and resulted in the formation of a significantly higher number of spheroids. The expression of CK-18/CK-19 and α-SMA/EpCAM proteins was confirmed in breast cancer tissues.
CONCLUSIONS: Thus, the use of primary tumor specimens and breast cancer cell lines as suitable models for elucidating the breast cancer stem cell activity was validated using mammosphere culture system.

[This corrects the article DOI: 10.3389/fgene.2019.00500.].

Breast cancer tumors display different cellular phenotypes. A growing body of evidence points toward a population of cancer stem cells (CSCs) that is important for metastasis and treatment resistance, although the characteristics of these cells are incomplete. We used mammosphere formation assay and label-retention assay as functional cellular approaches to enrich for cells with different degree of CSC properties in the breast cancer cell line MDA-MB-231 and performed single-cell RNA sequencing. We clustered the cells based on their gene expression profiles and identified three subpopulations, including a CSC-like population. The cell clustering into these subpopulations overlapped with the cellular enrichment approach applied. To molecularly define these groups, we identified genes differentially expressed between the three subpopulations which could be matched to enriched gene sets. We also investigated the transition process from CSC-like cells into more differentiated cell states. In the CSC population we found 14 significantly upregulated genes. Some of these potential breast CSC markers are associated to reported stem cell properties and clinical survival data, but further experimental validation is needed to confirm their cellular functions. Detailed characterization of CSCs improve our understanding of mechanisms for tumor progression and contribute to the identification of new treatment targets.