Abstract
ATP citrate lyase (ACL) knockdown (KD) causes tumor suppression and induces differentiation. We have previously reported that ACL KD reverses epithelial–mesenchymal transition (EMT) in lung cancer cells. Because EMT is often associated with processes that induce stemness, we hypothesized that ACL KD impacts cancer stem cells. By assessing tumorsphere formation and expression of stem cell markers, we showed this to be the case in A549 cells, which harbor a Ras mutation, and in two other non-small-cell lung cancer cell lines, H1975 and H1650, driven by activating EGFR mutations. Inducible ACL KD had the same effect as stable ACL KD. Similar effects were noted in another well-characterized Ras-induced mammary model system (HMLER). Moreover, treatment with hydroxycitrate phenocopied the effects of ACL KD, suggesting that the enzymatic activity of ACL was critical. Indeed, acetate treatment reversed the ACL KD phenotype. Having previously established that ACL KD impacts signaling through the phosphatidylinositol 3-kinase (PI3K) pathway, not the Ras-mitogen-activated protein kinase (MAPK) pathway, and that EMT can be reversed by PI3K inhibitors, we were surprised to find that stemness in these systems was maintained through Ras-MAPK signaling, and not via PI3K signaling. Snail is a downstream transcription factor impacted by Ras-MAPK signaling and known to promote EMT and stemness. We found that snail expression was reduced by ACL KD. In tumorigenic HMLER cells, ACL overexpression increased snail expression and stemness, both of which were reduced by ACL KD. Furthermore, ACL could not initiate either tumorigenesis or stemness by itself. ACL and snail proteins interacted and ACL expression regulated the transcriptional activity of snail. Finally, ACL KD counteracted stem cell characteristics induced in diverse cell systems driven by activation of pathways outside of Ras-MAPK signaling. Our findings unveil a novel aspect of ACL function, namely its impact on cancer stemness in a broad range of genetically diverse cell types.
Highlights
cancer stem cells (CSCs) utilize many of the same signaling pathways that are found in normal stem cells, including Hedgehog, Notch and Wnt signaling.[6]
We previously reported that ATP citrate lyase (ACL) KD reverses epithelial–mesenchymal transition (EMT) in NSCLC,[13] we hypothesized that ACL KD might impact CSCs
We used the aldefluor[17,18,19] assay, and assessed CD133,20,21 the verapamil sensitive side population[21,22,23,24] and CD44 expression (Supplementary Figure 1) to detect lung CSCs, using control and ACL KD cells generated for the human NSCLC cell lines A549, H1975, and H1650.13 A549 cells bear a K-ras mutation, whereas the last two cell types bear EGFR mutations
Summary
ATP citrate lyase (ACL) knockdown (KD) causes tumor suppression and induces differentiation. Snail is a downstream transcription factor impacted by Ras-MAPK signaling and known to promote EMT and stemness. A definitive role for ACL in tumorigenesis has emerged from the Thompson group,[14,15] which reported that decreasing the expression of ACL by shRNA or its activity by a small-molecule inhibitor suppressed tumor growth (leading to a cytostatic effect) and promoted differentiation in numerous glycolytic tumors.[14,15] We have previously extended these findings and reported that ACL knockdown (KD) in non-small-cell lung cancer (NSCLCs) cells reverses EMT in vitro and in vivo, and suppresses tumor growth, through intercepting PI3K/AKT signaling, but without effect on Ras-MAPK signaling.[13] Here, we show that ACL KD suppresses cancer stemness in numerous cell types, and does this at least partially through affecting snail expression and function
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