Transcriptomic profiling of curcumin-treated human breast stem cells identifies a role for stearoyl-coa desaturase in breast cancer prevention

Abstract

Curcumin is a potential agent for both the prevention and treatment of cancers. Curcumin treatment alone, or in combination with piperine, limits breast stem cell self-renewal while remaining non-toxic to normal differentiated cells. We paired fluorescence activated cell sorting with RNA sequencing to characterize the genome-wide changes induced specifically in normal breast stem cells following treatment with these compounds. We generated genome-wide maps of the transcriptional changes that occur in epithelial-like (ALDH+) and mesenchymal-like (ALDH−/CD44+/CD24−) normal breast stem/progenitor cells following treatment with curcumin and piperine. We show that curcumin targets both stem cell populations by down-regulating expression of breast stem cell genes including ALDH1A3, CD49f, PROM1, and TP63. We also identified novel genes and pathways targeted by curcumin, including downregulation of SCD. Transient siRNA knockdown of SCD in MCF10A cells significantly inhibited mammosphere formation and the mean proportion of CD44+/CD24− cells, suggesting that SCD is a regulator of breast stemness and a target of curcumin in breast stem cells. These findings extend previous reports of curcumin targeting stem cells, here in two phenotypically distinct stem/progenitor populations isolated from normal human breast tissue. We identified novel mechanisms by which curcumin and piperine target breast stem cell self-renewal, such as by targeting lipid metabolism, providing a mechanistic link between curcumin treatment and stem cell self renewal. These results elucidate the mechanisms by which curcumin may act as a cancer preventive compound and provide novel targets for cancer prevention and treatment.

Heterogeneity of normal human breast stem and progenitor cells as revealed by transcriptional profiling

“…Despite the lack of overlap between these populations, these cell maintain a cellular plasticity allowing them to inter-convert in a process regulated by the tumor microenvironment (Liu et al, 2013). Similarly, we previously showed that ALDH + or CD44 + normal mammary cells cultured as mammospheres also have distinct epithelial or mesenchymal phenotypes relative to each other (Colacino et al, 2016). In the current study, we expanded upon these observations by using CD24 + cells as a differentiated cell anchor to compare the gene expression signatures of ALDH + and CD44 + cells.…”

“…Normal mammary cells were stained for CD44, and CD24 expression and ALDH activity accessed by the Aldefluor assay as described previously (Colacino et al, 2016) and analyzed or sorted on a Beckman Coulter MoFlo Astrios at the University of Michigan Flow Cytomtery core facility. Briefly, single mammary cells were first incubated with a lineage depletion cocktail that consisted of biotinylated antibodies targeted against CD45, HLA-DR, CD14, CD31, CD41, CD19, CD235a, CD56, CD3, CD16, and CD140b (all from eBioscience, except for CD140b (Biolegend) and CD41 (Acris)).…”

“…We have previously shown that ALDH + cells cultured as mammospheres for 24 hours had a global gene expression pattern reflecting an epithelial phenotype relative to CD44 + cells, which have a generally mesenchymal phenotype (Colacino et al, 2016). To further explore the relative phenotypes of the ALDH + , CD44 + , and CD24 + cells, we compared the expression of genes associated with an epithelial or mesenchymal phenotype across these populations ( Figure 3).…”

Cadmium exposure inhibits branching morphogenesis and causes alterations consistent with HIF-1α inhibition in human primary breast organoids

“…Non-pathogenic breast tissue was isolated from women undergoing voluntary reduction mammoplasty at the University of Michigan. Breast tissue was mechanically and enzymatically digested as previously described (Colacino et al 2016;Dontu et al 2003) to yield a viable single cell suspension of human mammary cells. This study was reviewed and approved by the University of Michigan Institutional Review Board (HUM00042409).…”

“…The primary mammosphere formation assay is a surrogate readout of breast stem cell activity, while the secondary mammosphere formation assay is a readout of breast stem cell self-renewal capacity (Dontu et al 2003). Single primary human breast cells were plated in mammosphere formation conditions, in the presence of 0.25µM or 2.5µM cadmium chloride or vehicle control following our previously established protocol (Colacino et al 2016). Briefly, cells were plated at a concentration of 100,000/mL in ultralow attachment plates (Corning) conditions in MammoCult (StemCell) media.…”