Metabolic dysfunction-associated steatotic liver disease (MASLD) encompasses a spectrum of hepatic disorders, ranging from simple steatosis to steatohepatitis, with the most severe outcomes including cirrhosis, liver failure, and hepatocellular carcinoma. Notably, MASLD prevalence is lower in premenopausal women than in men, suggesting a potential protective role of estrogens in mitigating disease onset and progression. In this study, we utilized preclinical in vitro models—immortalized cell lines and hepatocyte-like cells derived from human embryonic stem cells—exposed to clinically relevant steatotic-inducing agents. These exposures led to lipid droplet (LD) accumulation, increased reactive oxygen species (ROS) levels, and mitochondrial dysfunction, along with decreased expression of markers associated with hepatocyte functionality and differentiation. Estrogen treatment in steatotic-induced liver cells resulted in reduced ROS levels and LD content while preserving mitochondrial integrity, mediated by the upregulation of mitochondrial thioredoxin 2 (TRX2), an antioxidant system regulated by the estrogen receptor. Furthermore, disruption of TRX2, either pharmacologically using auranofin or through genetic interference, was sufficient to counteract the protective effects of estrogens, highlighting a potential mechanism through which estrogens may prevent or slow MASLD progression.

Extracellular matrix (ECM) is a major component of the tumor environment, promoting the establishment of a pro-invasive behavior. Such environment is supported by both tumor- and stromal-derived metabolites, particularly lactate. In prostate cancer (PCa), cancer-associated fibroblasts (CAFs) are major contributors of secreted lactate, able to impact on metabolic and transcriptional regulation in cancer cells. Here, we describe a mechanism by which CAF-secreted lactate promotes in PCa cells the expression of genes coding for the collagen family. Lactate-exploiting PCa cells rely on increased α-ketoglutarate (α-KG) which activates the α-KG-dependent collagen prolyl-4-hydroxylase (P4HA1) to support collagen hydroxylation. De novo synthetized collagen plays a signaling role by activating discoidin domain receptor 1 (DDR1), supporting stem-like and invasive features of PCa cells. Inhibition of lactate-induced collagen hydroxylation and DDR1 activation reduces the metastatic colonization of PCa cells. Overall, these results provide a new understanding of the link between collagen remodeling/signaling and the nutrient environment exploited by PCa.

Metabolic disfunction-associated steatotic liver disease (MASLD) encompasses a plethora of hepatic disorders ranging from steatosis to steatohepatitis with the worst clinical outcome represented by cirrhosis, liver failure, and hepatocellular carcinoma. According to the lower MASLD prevalence reported in pre-menopausal women compared to men, we identified a potential protective role of estrogens in counteracting the oxidative stress during disease induction and progression. We have used preclinical relevant in vitro models [i.e., immortalized cells and hepatocyte-like cells (HLC) derived from human embryonic stem cells (hESC)], exposed to sodium lactate, sodium pyruvate, and octanoic acid (LPO) to induce hepatic steatosis. This established practice of MASLD induction resulted in lipid droplet (LD) accumulation and increased mitochondrial and cytosolic reactive oxygen species (ROS) levels, paralleled by the reduction of several markers of hepatocyte function and differentiation. Here we found that estrogen replacement reduced ROS levels and LD content through the upregulation of mitochondrial thioredoxin 2 (TRX2), an antioxidant system that is under the control of the estrogen receptor alpha (hereafter referred as ER). Last, disrupting the TRX2 system using auranofin was sufficient to revert the scavenging effects exerted by estrogens, thus identifying a potential mechanism that could prevent or delay the progression of the disease.

Targeting aromatase deprives ER + breast cancers of estrogens and is an effective therapeutic approach for these tumors. However, drug resistance is an unmet clinical need. Lipidomic analysis of long-term estrogen-deprived (LTED) ER + breast cancer cells, a model of aromatase inhibitor resistance, revealed enhanced intracellular lipid storage. Functional metabolic analysis showed that lipid droplets together with peroxisomes, which we showed to be enriched and active in the LTED cells, controlled redox homeostasis and conferred metabolic adaptability to the resistant tumors. This reprogramming was controlled by acetyl-CoA-carboxylase-1 (ACC1), whose targeting selectively impaired LTED survival. However, the addition of branched- and very long–chain fatty acids reverted ACC1 inhibition, a process that was mediated by peroxisome function and redox homeostasis. The therapeutic relevance of these findings was validated in aromatase inhibitor–treated patient-derived samples. Last, targeting ACC1 reduced tumor growth of resistant patient-derived xenografts, thus identifying a targetable hub to combat the acquisition of estrogen independence in ER + breast cancers.

Triple-negative breast cancer (TNBC) has limited therapeutic options, is highly metastatic and characterized by early recurrence. Lipid metabolism is generally deregulated in TNBC and might reveal vulnerabilities to be targeted or used as biomarkers with clinical value. Ferroptosis is a type of cell death caused by iron-dependent lipid peroxidation which is facilitated by the presence of polyunsaturated fatty acids (PUFA). Here we identify fatty acid desaturases 1 and 2 (FADS1/2), which are responsible for PUFA biosynthesis, lipid susceptible to peroxidation, to be highly expressed in a subset of TNBC with a poorer prognosis. Lipidomic analysis, coupled with functional metabolic assays, showed that FADS1/2 high-expressing TNBC are susceptible to ferroptosis-inducing agents and that targeting FADS1/2 renders those tumors ferroptosis-resistant. These findings were validated in vitro and in vivo in mouse and human-derived clinically relevant models and in a retrospective cohort of TNBC patients. One sentence summary The availability of intracellular PUFA depends on FADS1/2 desaturases, expressed at higher levels in aggressive triple-negative breast cancers highly susceptible to ferroptosis.

Supplementary Figure S6. 2-DG increases MCF7-LTED tumor spheroid invasion in Matrigel.

Supplementary Figure S5. MCF7-LTED cells are not undergoing EMT and do not activate MMPs or enhance collagen degradation.

Supplementary Table S1. Combination Index (CI) analysis of MCF7-2A cells treated with letrozole and 2-deoxyglucose (2-DG).

Background and Aims: Cancer cells reprogram their metabolic pathways to support bioenergetic and biosynthetic needs and to maintain their redox balance. In several human tumors, the Keap1-Nrf2 system controls proliferation and metabolic reprogramming by regulating the pentose phosphate pathway (PPP). However, whether this metabolic reprogramming also occurs in normal proliferating cells is unclear. Approach and Results: To define the metabolic phenotype in normal proliferating hepatocytes, we induced cell proliferation in the liver by 3 distinct stimuli: liver regeneration by partial hepatectomy and hepatic hyperplasia induced by 2 direct mitogens: lead nitrate (LN) or triiodothyronine. Following LN treatment, well-established features of cancer metabolic reprogramming, including enhanced glycolysis, oxidative PPP, nucleic acid synthesis, NAD + /NADH synthesis, and altered amino acid content, as well as downregulated oxidative phosphorylation, occurred in normal proliferating hepatocytes displaying Nrf2 activation. Genetic deletion of Nrf2 blunted LN-induced PPP activation and suppressed hepatocyte proliferation. Moreover, Nrf2 activation and following metabolic reprogramming did not occur when hepatocyte proliferation was induced by partial hepatectomy or triiodothyronine. Conclusions: Many metabolic changes in cancer cells are shared by proliferating normal hepatocytes in response to a hostile environment. Nrf2 activation is essential for bridging metabolic changes with crucial components of cancer metabolic reprogramming, including the activation of oxidative PPP. Our study demonstrates that matured hepatocytes exposed to LN undergo cancer-like metabolic reprogramming and offers a rapid and useful in vivo model to study the molecular alterations underpinning the differences/similarities of metabolic changes in normal and neoplastic hepatocytes.

Supplementary Figure Legends

Supplementary Figure S4. Fulvestrant (ICI 182,780) treatment impairs glucose uptake in MCF7-2A, wt-MCF7 and MCF7-LTED and increases CO2 production in wt-MCF7 and MCF7-LTED.

Supplementary Figure S7. miR155 is ER-dependent and control HK2 expression.

Resistance to endocrine treatments and CDK4/6 inhibitors is considered a near-inevitability in most patients with estrogen receptor positive breast cancers (ER + BC). By genomic and metabolomics analyses of patients’ tumours, metastasis-derived patient-derived xenografts (PDX) and isogenic cell lines we demonstrate that a fraction of metastatic ER + BC is highly reliant on oxidative phosphorylation (OXPHOS). Treatment by the OXPHOS inhibitor IACS-010759 strongly inhibits tumour growth in multiple endocrine and palbociclib resistant PDX. Mutations in the PIK3CA / AKT1 genes are significantly associated with response to IACS-010759. At the metabolic level, in vivo response to IACS-010759 is associated with decreased levels of metabolites of the glutathione, glycogen and pentose phosphate pathways in treated tumours. In vitro, endocrine and palbociclib resistant cells show increased OXPHOS dependency and increased ROS levels upon IACS-010759 treatment. Finally, in ER + BC patients, high expression of OXPHOS associated genes predict poor prognosis. In conclusion, these results identify OXPHOS as a promising target for treatment resistant ER + BC patients.

Supplementary Figure S3. 2-DG and metformin treatment do not cause apoptosis to the ER+ MCF7-LTED cells but impair survival fraction of the ER- ZR75-LTED.

Supplementary Data from Lactate Rewires Lipid Metabolism and Sustains a Metabolic–Epigenetic Axis in Prostate Cancer

Supplementary Figure S10. Tamoxifen-treated patients expressing high levels of HK2, GLUT1, MCT4 and miR155 show worst prognosis.

EDITORIAL article Front. Endocrinol., 04 September 2023Sec. Cancer Endocrinology Volume 14 - 2023 | https://doi.org/10.3389/fendo.2023.1270199

Supplementary Figure S1. 3-brome-pyruvate (3-BP) in combination with letrozole synergistically inhibits cancer cell growth.