Metabolic Reprogramming by Folate Restriction Leads to a Less Aggressive Cancer Phenotype

Folate coenzymes are involved in biochemical reactions of one-carbon transfer, and deficiency of this vitamin impairs cellular proliferation, migration and survival in many cell types. Here the effect of folate restriction on mammary cancer was evaluated using three distinct breast cancer subtypes differing in their aggressiveness and metastatic potential: non-invasive basal-like (E-Wnt), invasive but minimally metastatic claudin-low (M-Wnt), and highly metastatic claudin-low (metM-Wntliver) cell lines, each derived from the same pool of MMTV-Wnt-1 transgenic mouse mammary tumors. NMR-based metabolomics was used to quantitate 41 major metabolites in cells grown in folate-free medium versus standard medium. Each cell line demonstrated metabolic reprogramming when grown in folate-free medium. In E-Wnt, M-Wnt and metM-Wntliver cells 12, 29, and 25 metabolites, respectively, were significantly different (p<0.05 and at least 1.5-fold change). The levels of eight metabolites (aspartate, ATP, creatine, creatine phosphate, formate, serine, taurine and β-alanine) were changed in each folate-restricted cell line. Increased glucose, decreased lactate, and inhibition of glycolysis, cellular proliferation, migration and invasion occurred in M-Wnt and metM-Wntliver cells (but not E-Wnt cells) grown in folate-free versus standard medium. These effects were accompanied by altered levels of several folate-metabolizing enzymes, indicating that the observed metabolic reprogramming may result from both decreased folate availability and altered folate metabolism. These findings reveal that folate restriction results in metabolic and bioenergetic changes and a less aggressive cancer cell phenotype

Effects of folic acid withdrawal on transcriptomic profiles in murine triple-negative breast cancer cell lines

We have previously shown that withdrawal of folic acid led to metabolic reprogramming and a less aggressive phenotype in a mouse cell model of triple-negative breast cancer (TNBC). Herein, we evaluate the effects of folic acid withdrawal on transcriptomic profiles in these cells. Murine cell lines were originally derived from a pool of spontaneous mammary tumors grown in MMTV-Wnt1 transgenic mice. Based on their differential molecular characteristics and metastatic potential, these cell lines were previously characterized as non-metastatic epithelial (E-Wnt), non-metastatic mesenchymal (M-Wnt) and metastatic mesenchymal (metM-Wntliver) cells. Using custom two-color 180K Agilent microarrays, we have determined gene expression profiles for three biological replicates of each subtype kept on standard medium (2.2 μM folic acid) or folic acid-free medium for 72 h. The analyses revealed that more genes were differentially expressed upon folic acid withdrawal in M-Wnt cells (1884 genes; Benjamini-Hochberg-adjusted P-value liver cells (108 and 222 genes, respectively). Pathway analysis has identified that type I interferon signaling was strongly affected by folic acid withdrawal, with interferon-responsive genes consistently being upregulated upon folic acid withdrawal in M-Wnt cells. Of note, repressed interferon signaling has been established as one of the characteristics of aggressive human TNBC, and hence reactivation of this pathway may be a promising therapeutic approach. Overall, while our study indicates that the response to folic acid withdrawal varies by molecular subtype and cellular phenotype, it also underscores the necessity to further investigate one-carbon metabolism as a potential therapeutic means in the treatment of advanced TNBC.</p

Deleterious mutations in ALDH1L2 suggest a novel cause for neuro-ichthyotic syndrome

International audienceNeuro-ichthyotic syndromes are a group of rare genetic diseases mainly associated with perturbations in lipid metabolism, intracellular vesicle trafficking, or glycoprotein synthesis. Here, we report a patient with a neuro-ichthyotic syndrome associated with deleterious mutations in the ALDH1L2 (aldehyde dehydrogenase 1 family member L2) gene encoding for mitochondrial 10-formyltetrahydrofolate dehydrogenase. Using fibroblast culture established from the ALDH1L2-deficient patient, we demonstrated that the enzyme loss impaired mitochondrial function affecting both mitochondrial morphology and the pool of metabolites relevant to β-oxidation of fatty acids. Cells lacking the enzyme had distorted mitochondria, accumulated acylcarnitine derivatives and Krebs cycle intermediates, and had lower ATP and increased ADP/AMP indicative of a low energy index. Re-expression of functional ALDH1L2 enzyme in deficient cells restored the mitochondrial morphology and the metabolic profile of fibroblasts from healthy individuals. Our study underscores the role of ALDH1L2 in the maintenance of mitochondrial integrity and energy balance of the cell, and suggests the loss of the enzyme as the cause of neuro-cutaneous disease. npj Genomic Medicine (2019) 4:17 ; https://doi

CHIP E3 ligase mediates proteasomal degradation of the proliferation regulatory protein ALDH1L1 during the transition of NIH3T3 fibroblasts from G₀/G₁ to S-phase

<div><p>ALDH1L1 is a folate-metabolizing enzyme abundant in liver and several other tissues. In human cancers and cell lines derived from malignant tumors, the <i>ALDH1L1</i> gene is commonly silenced through the promoter methylation. It was suggested that ALDH1L1 limits proliferation capacity of the cell and thus functions as putative tumor suppressor. In contrast to cancer cells, mouse cell lines NIH3T3 and AML12 do express the ALDH1L1 protein. In the present study, we show that the levels of ALDH1L1 in these cell lines fluctuate throughout the cell cycle. During S-phase, ALDH1L1 is markedly down regulated at the protein level. As the cell cultures become confluent and cells experience increased contact inhibition, ALDH1L1 accumulates in the cells. In agreement with this finding, NIH3T3 cells arrested in G₁/S-phase by a thymidine block completely lose the ALDH1L1 protein. Treatment with the proteasome inhibitor MG-132 prevents such loss in proliferating NIH3T3 cells, suggesting the proteasomal degradation of the ALDH1L1 protein. The co-localization of ALDH1L1 with proteasomes, demonstrated by confocal microscopy, supports this mechanism. We further show that ALDH1L1 interacts with the chaperone-dependent E3 ligase CHIP, which plays a key role in the ALDH1L1 ubiquitination and degradation. In NIH3T3 cells, silencing of CHIP by siRNA halts, while transient expression of CHIP promotes, the ALDH1L1 loss. The downregulation of ALDH1L1 is associated with the accumulation of the ALDH1L1 substrate 10-formyltetrahydrofolate, which is required for <i>de novo</i> purine biosynthesis, a key pathway activated in S-phase. Overall, our data indicate that CHIP-mediated proteasomal degradation of ALDH1L1 facilitates cellular proliferation.</p></div

ALDH1L1 is ubiquitinated in NIH3T3 cells.

<p><b>A</b>, ALDH1L1 pulled-down from NIH3T3 cell lysates using ALDH1L1-specific antibody and protein A beads; elution with glycine buffer (<i>lane 1</i>), followed by elution with SDS-PAGE loading buffer (<i>lane 2</i>). Proteins were resolved on a 7.5% SDS-PAGE gel followed by Western blot assay with ubiquitin-specific antibody (<i>left panel</i>) or ALDH1L1-specific antibody (<i>right panel</i>). Lane <i>St</i> is purified recombinant ALDH1L1. <b>B</b>, ALDH1L1 was immunoprecipitated from NIH3T3 cell lysates using an ALDH1L1-specific antibody and Protein A Magnetic beads; samples were resolved on a 7.5% SDS-PAGE followed by Western blot assay with anti-ubiquitin monoclonal antibody. Cells were harvested at different time points after splitting (as indicated); lysates were treated with deubiquitinase inhibitor (4.0 μM recombinant human ubiquitin aldehyde C-terminal derivative) prior to immunoprecipitation. After immunoprecipitation, eluates were treated with deubiquitinase (200 nM of recombinant human USP2 catalytic domain); <i>control</i>, untreated lysates. <b>C</b>, ALDH1L1 was immunoprecipitated from NIH3T3 cells as in <b>B</b> and treated with USP7. Cells were treated with 10 μM MG-132 for 4 h before the pull-down. After treatment with USP7, we have repeated the pull-down with ALDH1L1-specific antibody and detected ubiquitinated species as in <b>B</b>.</p

ALDH1L1 interacts with E3 ligase CHIP and CHIP-assisting chaperone HSP90 <i>in vivo</i>.

<p><b>A</b>, schematic depicting the pull-down assay on a folate affinity column. <b>B</b>, endogenous ALDH1L1 interacts strongly with immobilized folinic acid (after loading NIH3T3 cell lysate on the affinity column, the following eluted fractions were collected and analyzed by SDS-PAGE/Western blot assay with ALDH1L1-specific antibody: <i>lane 1</i>, washing buffer; <i>lane 2</i>, 0.5 M KCl; <i>lane 3</i>, 1.0 M KCl; <i>lane 4</i>, 2.0 KCl; <i>lane 5</i>, 5 mM folic acid in 2.0 KCl; <i>lane 6</i>, 20 mM folic acid in 2.0 M KCl; <i>lane 7</i>, purified ALDH1L1 standard; <i>St</i>, molecular masses standards (indicated by numbers in kDa on the <i>left</i>). <b>C</b>, samples as in <b>B</b> probed with CHIP-specific antibody. <b>D</b>, samples as in <i>B</i> probed with HSP90-specific antibody. <b>E</b>, Immunoprecipitation of NIH3T3 cell lysate with ALDH1L1-specific (<i>left panels</i>) or HSP90-specific (<i>right panels</i>) antibody followed by SDS-PAGE/Western blot assay with ALDH1L1-specific, HSP90-specific and CHIP-specific antibodies. In each experiment (ALDH1L1 or HSP90 pull-down) the same blot was stripped twice and reprobed. Numbers indicate molecular masses (kDa) for standards.</p

<i>In vitro</i> ubiquitination of purified ALDH1L1.

<p><b>A</b>, purified ALDH1L1 (1.1 μg) was ubiquitinated using the <i>in vitro</i> ubiquitination kit that included CHIP E3 ligase (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199699#sec002" target="_blank">Materials and methods</a>). Reaction products were resolved by 7.5% SDS-PAGE followed by Western blot assay with ALDH1L1-specific antibody (<i>left panel</i>) or Ub-specific antibody (<i>right panel</i>). <i>Arrows</i> indicate positions of 95 kDa and 130 kDa pre-stained molecular mass protein standards. <b>B</b>, lanes 1–4, increased amount of ALDH1L1 (0.6, 1.2, 2.4 and 4.8 μg) were subjected to <i>in vitro</i> ubiquitination with NIH3T3 cell lysate; ubiquitination with CHIP was the positive control. <b>C</b>, negative control for panel <b>B</b>, lanes 1–4, untreated purified ALDH1L1 (0.6, 1.2, 2.4 and 4.8 μg) prior to ubiquitination. Each experiment was repeated at least three times. Molecular mass standards (<i>St</i>) are the same for all panels.</p

Levels of ALDH1L1 protein in NIH3T3 cells arrested at difference phases.

<p><b>A</b>, NIH3T3 cells arrested in G₀/G₁ (serum starvation), S-phase (double thymidine block) or G₂/M (double thymidine block and nocodazole treatment) phase. Asynchronous cells shown as a control. Numbers on the panels indicate distribution of cells between cell cycle phases. Fitted peaks are: <i>Blue</i>, calculated G₀/G₁ phase; <i>yellow</i>, S phase; <i>green</i>, G₂/M phase. Cell cycle data were analyzed using FlowJo software. <b>B</b>, Western blot assay of ALDH1L1 in NIH3T3 cells arrested in indicated phase (20 μg of total cell lysate was loaded in each lane). Actin is shown as loading control. Arrows indicate molecular weight standards (St). Numbers show ALDH1L1 band intensity (arbitrary densitometry units) normalized to actin. Experiments were performed three times.</p

Omitting components of ubiquitination machinery prevents <i>in vitro</i> ALDH1L1 ubiquitination.

<p>Purified ALDH1L1 (1.1 μg) was incubated for 1 h with the <i>in vitro</i> ubiquitination kit that included all components or with omission of ATP, E1, E2 or E3 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199699#sec002" target="_blank">Materials and methods</a>). Reaction products were resolved by 7.5% SDS-PAGE followed by Western blot assay with ALDH1L1-specific antibody and Ubiquitin (Ub)-specific antibody. <i>St</i>, pre-stained molecular mass protein standards (numbers on the right indicates standard molecular masses, kDa). Experiment was performed three times with the same outcome.</p

Inhibition of collagen XI alpha 1-induced fatty acid oxidation triggers apoptotic cell death in cisplatin-resistant ovarian cancer.

Collagen type XI alpha 1 (COL11A1) is a novel biomarker associated with cisplatin resistance in ovarian cancer. However, the mechanisms underlying how COL11A1 confers cisplatin resistance in ovarian cancer are poorly understood. We identified that fatty acid β-oxidation (FAO) is upregulated by COL11A1 in ovarian cancer cells and that COL11A1-driven cisplatin resistance can be abrogated by inhibition of FAO. Furthermore, our results demonstrate that COL11A1 also enhances the expression of proteins involved in fatty acid synthesis. Interestingly, COL11A1-induced upregulation of fatty acid synthesis and FAO is modulated by the same signaling molecules. We identified that binding of COL11A1 to its receptors, α1β1 integrin and discoidin domain receptor 2 (DDR2), activates Src-Akt-AMPK signaling to increase the expression of both fatty acid synthesis and oxidation enzymes, although DDR2 seems to be the predominant receptor. Inhibition of fatty acid synthesis downregulates FAO despite the presence of COL11A1, suggesting that fatty acid synthesis might be a driver of FAO in ovarian cancer cells. Taken together, our results suggest that COL11A1 upregulates fatty acid metabolism in ovarian cancer cells in a DDR2-Src-Akt-AMPK dependent manner. Therefore, we propose that blocking FAO might serve as a promising therapeutic target to treat ovarian cancer, particularly cisplatin-resistant recurrent ovarian cancers which typically express high levels of COL11A1