RSL3 and erastin do not exhibit synergistic effects with elacestrant or fulvestrant treatment in MCF7-LTED WT cells

ER+/HER2 − breast cancer is commonly treated with endocrine therapy combined with CDK4/6 inhibitors (CDK4/6i), including palbociclib, ribociclib, and abemaciclib. Although these combinations have improved clinical outcome, resistance remains frequent, highlighting the need to identify vulnerabilities that enhance CDK4/6i response. To uncover metabolic dependencies associated with CDK4/6i sensitivity, we performed an unbiased loss-of-function screen targeting ∼2,800 metabolic genes. This identified lipase H (LIPH), a phosphatidic acid-selective phospholipase A1 that generates lysophosphatidic acid (LPA), as a candidate vulnerability in palbociclib-treated ER+/HER2- breast cancer cells. LIPH depletion reduced LPA levels and sensitized multiple ER+ models to palbociclib, ribociclib, and abemaciclib. Mechanistically, LIPH-derived LPA activated LPAR2-dependent AMPK/p38 signaling, sustaining CD36 expression. CD36 regulated cyclin D1 levels and cell-cycle progression, while LIPH or CD36 depletion reduced cyclin D1, induced G0/G1 accumulation, and enhanced CDK4/6i sensitivity. Conversely, exogenous LPA restored CD36 and cyclin D1 expression in LIPH-silenced cells. Genetic or pharmacological CD36 targeting potentiated CDK4/6i efficacy in endocrine-sensitive and therapy-resistant ER+ models, as well as in patient-derived organoids. Clinically, high LIPH/CD36 scores were associated with poor response to CDK4/6i-based therapy, while elevated LIPH, CD36, or combined LIPH/LPAR2/CD36 expression correlated with worse outcome in ER+/HER2 − breast cancer cohorts. Together, our findings identify the LIPH–LPA–LPAR2–CD36–cyclin D1 axis as a lipid-signaling vulnerability that modulates CDK4/6i response and propose LIPH and CD36 as candidate therapeutic targets and biomarkers in ER+/HER2- breast cancer.

Integrative functional analysis complementary to that described in the main Figure 3, showing that ACSL4 targeting prevents ferroptosis execution in MCF7 Y537C and T47D Y537C cells

Prostate cancer (PCa) progression is strongly influenced by the metabolites available in the tumor microenvironment (TME), including lactic acid (LA), which is actively imported by PCa cells to boost mitochondrial metabolism and drive de novo collagen synthesis, sustaining increased malignancy. LA exploitation promotes the unbalance of tricarboxylic acid (TCA) cycle intermediates, particularly succinate and fumarate, well-known epigenetic modifiers for histone (de)methylation. Here, we show that the LA-induced increase in succinate levels affects the activating H3K4me3 methylation mark in PCa cells, promoting a pro-invasive phenotype. Notably, pharmacological targeting of H3K4me3 using OICR-9429 reduces LA-enhanced PCa cell invasiveness. Moreover, LA-induced H3K4me3 enrichment regulates the expression of procollagen-Lysine,2-Oxoglutarate 5-Dioxygenase 1 (PLOD1), a key enzyme involved in collagen maturation. Genetic impairment of PLOD1 reduces the LA-driven invasive potential of PCa cells, thereby highlighting PLOD1 as a crucial epigenetically regulated mediator of tumor invasion. Overall, our findings uncover a novel LA-fuelled metabolic-epigenetic axis that promotes the H3K4me3-mediated PLOD1 upregulation, consequently fostering PCa aggressiveness and unveiling a potential therapeutic vulnerability.

Endocrine therapy (ET) is the standard of care for estrogen receptor (ER)-positive breast cancer. Point mutations in the ligand-binding domain of the gene encoding the estrogen receptor (ESR1) are rare in naïve ER+ breast cancer while becoming common in the ET-resistant setting. In this study, we found that ESR1 mutations expose breast cancers to critical vulnerabilities related to lipid metabolism. Particularly, ESR1 mutations that induce constitutive ER activation drove aberrant lipid biogenesis and lipid upload in parallel with increased expression of acyl-CoA synthetase long-chain family member 4 (ACSL4), which plays a crucial role in fatty acid activation and has been shown to correlate with increased ferroptosis susceptibility. Although ER+ breast cancer cells displayed ferroptosis resistance, the presence of ESR1 mutations rendered tumor cells sensitive to ferroptosis induction. Importantly, ferroptosis inducers potentiated the effects of the selective ER degraders fulvestrant and elacestrant, which are the standard of care for breast cancers carrying ESR1 mutations. These findings, validated both in preclinical models and in patient-derived material, identify a combinatory therapeutic approach in the setting of ET resistance and establish ACSL4 as an important biomarker to recognize ER+ breast cancers susceptible to ferroptosis induction. SIGNIFICANCE:ESR1 mutations in breast cancer induce metabolic changes that trigger ferroptosis sensitivity, enabling ferroptosis inducers to enhance selective ER degraders' efficacy and positioning ACSL4 as a biomarker for guiding therapy in endocrine-resistant disease.

ChIP-seq data relative to ESR1-mutant and its neomorphic transcriptional activity in regulating ACSL4, a feature linked to transcriptional reprogramming associated with ferroptosis

Prostate carcinoma (PCa) progression is strongly influenced by the surrounding tumor microenvironment, where cancer-associated fibroblasts (CAFs) represent the most abundant and functionally relevant stromal population. Despite their importance, the lack of stable cell lines representing CAF phenotypes limits the study of stromal-tumor interactions. To address this limitation, we provide an optimized protocol for isolating CAFs from fresh human PCa biopsies based on a mechanical procedure exploiting the specific CAF ability to migrate out from the tumor explants. This approach preserves tissue architecture and maintains CAF viability and phenotype. The resulting ex vivo CAF cultures provide a suitable model to investigate CAF biology within the tumor microenvironment. Key features • The protocol provides an optimized workflow for isolating CAFs from prostate tumor explants by exploiting their selective outgrowth. • It is an optimized, enzymatic-free procedure that minimizes fibroblast cell stress while preserving cell phenotypic features.

Ferroptosis is a form of regulated cell death dependent on iron‐driven phospholipid peroxidation and is controlled by both cell autonomous and non‐cell autonomous mechanisms. In prostate cancer (PCa), tumor cells engage in a metabolic crosstalk with cancer‐associated fibroblasts (CAFs), resulting in increased utilization of CAF‐secreted lactic acid, that ultimately supports cancer aggressiveness. In this context, the effect of the prostate tumor microenvironment in modulating ferroptosis sensitivity has not yet been extensively investigated. Here, we demonstrate that CAF‐secreted lactic acid protects PCa cells from ferroptosis induction and supports the upregulation of the antioxidant enzyme glutathione peroxidase 4 (GPX4). Interestingly, targeting carbonic anhydrase IX/XII (CA IX/XII), the main regulators of microenvironmental acidosis, in tumor and stromal compartments hinders lactic acid shuttle within the tumor–stroma interplay and thus, prevents ferroptosis resistance induced by lactic acid. Analyses of tissue samples from PCa patients also revealed that GPX4, CA IX, and CA XII expression levels increase during PCa progression. Overall, these findings support a role for stromal lactic acid in mediating ferroptosis resistance in PCa, identifying CA IX/XII as potential therapeutic targets regulating ferroptosis sensitivity.

Developing three-dimensional (3D) tumor models that accurately mimic the tumor microenvironment (TME) and its heterogeneity remains a significant challenge in preclinical research. Advancing these models holds the potential to improve the study of cancer pathologies in vitro, while reducing dependence on animal models. To tackle this challenge, in this work, we report on the development of an in vitro 3D stromalized prostate cancer model using gelatin porous microparticles as microscaffolds for cell attachment and growth. Gelatin porous microparticles were prepared by a double emulsion method and cross-linked with a biocompatible cross-linking agent, that is, glyceraldehyde, to prevent dissolution under physiological conditions. Then, we developed a stromalized 3D gelatin-based microscaffold biomimicking the interplay between human prostate cancer (PCa) and stromal cells by coculturing 22Rv1 cells and fibroblasts with gelatin porous microparticles. Overall, our results demonstrate the feasibility of gelatin microscaffolds in reproducing a 3D stromalized model of PCa progression (e.g., metabolic reprogramming), resulting from the tumor-stroma interaction. Thus, these systems represent a valuable platform and an effective tool for the study of cancer progression, such as TME biomimetics, while simultaneously offering a valid alternative to minimize the reliance on animal studies in preclinical research.

BACKGROUND & AIMS:GD2, a member of the ganglioside (GS) family (sialic acid-containing glycosphingolipids), is a potential biomarker of cancer stem cells (CSC) in several tumours. However, the possible role of GD2 and its biosynthetic enzyme, GD3 synthase (GD3S), in intrahepatic cholangiocarcinoma (iCCA) has not been explored. METHODS:The stem-like subset of two iCCA cell lines was enriched by sphere culture (SPH) and compared to monolayer parental cells (MON). GS profiles were evaluated by chromatography, after feeding with radioactive sphingosine. Membrane GD2 expression was evaluated by FACS, and the expression of enzymes of GS biosynthesis was analysed by RT-qPCR. The modulation of stem features by GS was investigated in vitro and in vivo using GD3S-overexpressing cells and corroborated by global transcriptomic analysis. RESULTS:GS composition was markedly different comparing SPH and MON. Among complex GS, iCCA-SPH showed increased GD2 levels, in agreement with the high expression levels of GD3 and GM2/GD2 synthases. iCCA cells overexpressing GD3S had higher sphere-forming ability, invasive properties and drug resistance than parental cells. NOD/SCID mice implanted with CCLP1 cells overexpressing GD3S developed larger tumours than control cells. By global transcriptomic analysis, ontology investigation identified 74 processes shared by the iCCA-SPH and GD3S-transfected cells, with enrichment for development and morphogenesis processes, MAPK signalling and locomotion. In a cohort of patients with iCCA, GD3S expression was correlated with lymph node invasion, indicating a possible relevance of GD3S in the clinical setting. CONCLUSIONS:The profile of GS derivatives regulates the stem-like properties of iCCA cells.

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.

Background:Podocytes and podocyte progenitors are interdependent components of the kidney's glomerular structure, with podocytes forming the glomerular filtration barrier and progenitors being key players in podocyte regeneration during pathophysiological processes. Both cell types are subjected to constant mechanical forces, whose alterations can initiate podocytopathy and worsen glomerular injury. Despite this, the specific mechanosensors and mechanotransduction pathways involved in their response to mechanical cues remain only partially explored. Methods:We used transcriptomics, immunofluorescence, and silencing experiments on human primary podocyte progenitor cell cultures to demonstrate the expression and function of Piezo1 channels. We generated inducible podocyte- and podocyte progenitor-specific Piezo1 knockout mice to evaluate the effects of Piezo1 loss in the context of Adriamycin nephropathy and over 10 months of aging. Results:Silencing of Piezo1 in progenitors triggered F-actin remodelling, induced cell shape modification and nuclear envelope defects with accumulation of DNA damage that led to mitotic catastrophe in differentiated podocytes. Podocyte-specific knockout of Piezo1 induced higher susceptibility to podocyte injury in Adriamycin nephropathy and led to accumulation of DNA damage and mild albuminuria starting from adult age. Podocyte progenitor-specific knockout of Piezo1 in mouse resulted in severe albuminuria during Adriamycin nephropathy, leading to the generation of defective podocytes. Conclusions:These results demonstrated that Piezo1, thanks to its role in F-actin cytoskeleton maintenance, is essential for the survival of podocytes exposed to mechanical stress conditions and for their correct regeneration.

Melanoma is more aggressive in male patients than female ones and this is associated with sexual dimorphism in immune responses. Taking into consideration the impact tumour metabolic alterations in affecting the immune landscape, we aimed to investigate the effect of the sex-dependent metabolic profile of melanoma in re-shaping immune composition. Melanoma is characterised by Warburg metabolism, and secreted lactate has emerged as a key driver in the establishment of an immunosuppressive environment. Here, we identified lactate dehydrogenase A (LDH-A) as a crucial player in modulating sex-related differences in melanoma immune responses, both in vitro and in patient-derived specimens. LDH-A is associated with higher lactate secretion in male melanoma cells, which leads to a significant enrichment in pro-tumoural regulatory T cells (Treg) with a concurrent decrease in the number and activity of anti-tumour CD8+ T cells. Remarkably, pharmacological and genetic impairment of LDH-A in male melanoma cells normalises Treg and CD8+ infiltration. In keeping with this, in vivo pharmacological targeting of LDH-A in melanoma-bearing male mice impairs tumour growth and lung colonisation, with a concomitant modulation of Treg and CD8+ T cells infiltration. Taken together, our findings highlight the sex-related differences promoted by LDH-A in immune reshaping in melanoma, and suggest that therapeutic targeting of LDH-A could be leveraged as an effective strategy to abolish the sex-gap in melanoma progression.

The enrichment of specific metabolites within the tumor microenvironment is emerging as a driver of tumor progression. Specifically, in prostate cancer (PCa), increased abundance of lactate is associated with primary-to-metastasis tumor spreading by supporting cancer cell invasiveness. Here, we highlight that the endocannabinoid receptor GPR55 is able to sense lactate and consequently trigger PCa cell amoeboid-like invasiveness, through the activation of the pro-migratory RhoA/MLC2 signaling pathway. These findings uncover a new role for GPR55 in sustaining lactate-driven PCa cell motility.

Cancer cachexia is a multifactorial syndrome characterized by a progressive loss of body weight occurring in about 80% of cancer patients, frequently representing the leading cause of death. Dietary intervention is emerging as a promising therapeutic strategy to counteract cancer-induced wasting. Serine is the second most-consumed amino acid (AA) by cancer cells and has emerged to be strictly necessary to preserve skeletal muscle structure and functionality. Here, we demonstrate that decreased serine availability during tumor progression promotes myotubes diameter reduction in vitro and induces muscle wasting in in vivo mice models. By investigating the metabolic crosstalk between colorectal cancer cells and muscle cells, we found that incubating myotubes with conditioned media from tumor cells relying on exogenous serine consumption triggers pronounced myotubes diameter reduction. Accordingly, culturing myotubes in a serine-free medium induces fibers width reduction and suppresses the activation of the AKT-mTORC1 pathway with consequent impairment in protein synthesis, increased protein degradation, and enhanced expression of the muscle atrophy-related genes Atrogin1 and MuRF1. In addition, serine-starved conditions affect myoblast differentiation and mitochondrial oxidative metabolism, finally inducing oxidative stress in myotubes. Consistently, serine dietary deprivation strongly strengthens cancer-associated weight loss and muscle atrophy in mice models. These findings uncover serine consumption by tumor cells as a previously undisclosed driver in cancer cachexia, opening new routes for possible therapeutic approaches.

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.