is the correlation coefficient. cancer cells. Molecularly, SIRT1 deacetylates and stabilizes PRRX1, an EMT inducer, whose destabilization promotes KLF4 transcription. KLF4 upregulates transcription and thus induces CSCs. KLF4 inhibitor Kenpaullone overcomes Paclitaxel (PTX) resistance imposed by deficiency and reduces lung metastasis in mouse models. Our data identify a SIRT1-PRRX1-KLF4-ALDH1 circuitry as a central regulator of CSCs and highlight its therapeutic potential in targeting the progression and metastasis of breast cancer. Results A SIRT1-centered circuitry underlies age-related CSC induction To understand potential links between aging and breast cancer stemness, we employed the GenAge Human Genes list A 967079 to screen for genes that are correlated with core stemness factor OCT4, SOX2, NANOG, and KLF4 in a cohort of breast cancer cell lines collected from the TCGA database [11, 22, 23]. ALDH1A1/3 and CD44 were included as CSC markers in the analysis. As shown, 75 out of 300 genes are negatively correlated with the core stemness program, and 8 of the 75 are reversely correlated with or (Table S1). Interestingly, the top enrichment list includes triangle of functionally interacting network (Fig. ?(Fig.1a).1a). Further, nine genes in the circuitry and breast cancer CSC markers were analyzed and the correlation was assessed in breast cancer cell lines [11]. A strong reverse correlation between and was obtained (Fig. ?(Fig.1c1c and Table S2, was associated with high (Fig. 1a, b and Table S1, deficiency activates TGF- signaling, enhances EMT and promotes lung metastasis [24], and induces mesenchymal-like CSCs marked with CD44+CD24- in breast cancers (Figure S1), providing a proof of concept of aging-promoted CSC induction. To confirm the findings, we did pathway enrichment analysis via STRING v10 [25]. The KEGG analysis showed an enrichment of pathways that safeguard genome integrity, wherein KLF4 and ALDH1A1/3 are the most correlated, followed by NANOG, then SOX2, and CD44 and OCT3/4 are the least and even lack of correlation (Fig. ?(Fig.1b1b and A 967079 Table S3). Interestingly, EMT-type CSCs (CD44+) are associated with low and share similar set of genes with OCT3/4, suggesting differential roles of Sirtuins on breast cancer stemness: SIRT1 is related to MET-type CSCs (ALDH1+), whereas SIRT7 is correlated with EMT-type (CD44+). Together, the data points to a SIRT1-KLF4-ALDH1 circuitry, which couples aging and CSCs. Open in a separate window Fig. 1 A SIRT1-KLF4-ALDH1A1-ALDH1A3 circuitry dictates age-related breast CSCs. a Functionally interacting network modules constructed from genes belonging to Age Human Genes list and stemness-associated genes were analyzed by Prism A 967079 5.0 tool (based on Pearsons correlation coefficient) and visualized in Cytoscape. A graph that nodes have a power law distribution for their number of links, showing correlation between four factors, CSC markers and age-related genes in breast cancer cells (see Table S1). Bigger and darker colored nodes represent proteins with more links. Noted SIRT1 as the node at the tail end of the distribution on the graph. b Pathway Goat polyclonal to IgG (H+L) enrichment analysis of four factors, CSC A 967079 markers and their associated aging-related proteins by STRING database. Noted a significant enrichment of pathways that safeguard genome integrity, to which KLF4 and ALDH1A1/3 are the most correlated, followed by NANOG, then SOX2, and CD44 and OCT3/4 are the least. CD44?+?CSCs are associated with low SIRT7 and share similar set of genes with OCT3/4. c Pearson correlation between and mRNA levels in 52 breast cancer cell lines. is the correlation coefficient Genetic ablation of induces ALDH1+ CSCs via upregulating and level predicts poor survival, chemotherapy-resistance and metastasis of breast cancer (Figure S2B). We are particularly interested in SIRT1, whose precise function in CSCs remains less well documented. To determine its A 967079 molecular function in CSCs, was knocked out in triple-negative basal-like breast cancer BT-549 cells via a CRISPR/Cas9 procedure. As predicted, loss of increased the mammosphere-forming capacity by more than 3 folds (Fig. 2aCc). Similarly, knocking down in a murine triple-negative basal-like breast cancer cell line 4T1 significantly promoted mammosphere-formation (Figure S3ACC). We next did RNAseq and gene set enrichment analysis (GSEA) in depletion were positively associated with.