ATP citrate lyase knockdown impacts cancer stem cells in vitro

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.

Targeting Lactate Dehydrogenase-A Inhibits Tumorigenesis and Tumor Progression in Mouse Models of Lung Cancer and Impacts Tumor-Initiating Cells

“…To extend our results, we also used another previously published stem cell system i.e. MCF10A cells expressing shPTEN or over-expressing PIK3CA (Hanai et al, 2013) to demonstrate that an LDH-A inhibitor reduced the stem cell population as assessed by the tumorsphere assay (Figure 6C). …”

“…Immortalized human breast epithelial cells (HMLE), were gifts from Dr. Weinberg (Whitehead Institute). All cell lines were grown as described earlier (Hanai et al, 2013). …”

Chemical inhibition of acetyl-CoA carboxylase suppresses self-renewal growth of cancer stem cells

“…These results suggest that ACLY deficiency forces cancer cells to upregulate the expression of downstream genes in fatty acid and cholesterol synthesis pathways to compensate for the loss of ACLY. Of note, this type of compensation that occurs during the therapeutic targeting of lipogenesis-related enzymes such as ACLY might be unacceptable for cancer treatment [43, 46]. We acknowledge that our study failed to evaluate whether the chemical perturbation of ACACA promoted major changes in the expression profile of other metabolic genes.…”

Mitochondrial substrates in cancer: Drivers or passengers?

“…In addition to its role in regulating catabolic processes, cytosolic citrate stimulates anabolic reactions such as lipid synthesis by enhancing acetyl-CoA carboxylase activity and by serving as a source for acetyl-CoA itself via cleavage by ATP-citrate lyase (ACLY), ultimately inducing fatty acid synthesis. ACLY was further shown to be important for metabolic modulation mediated by oncogenic PI3K/AKT signaling , and its knockdown caused tumor suppression and cell differentiation (Hanai et al, 2013;Hatzivassiliou et al, 2005). The level of citrate in the cytosol is determined by the mitochondrial export rate and can be modulated by differential expression of the CIC gene.…”