Ivan Casaburi

For many types of cancer, pre-clinical studies have shown that dietary interventions and supplements can be effective in reducing the toxicity and increasing the efficacy of chemotherapeutics. In this context, the polyphenol curcumin is an attractive molecule. We have previously demonstrated that curcumin inhibits adrenocortical carcinoma (ACC) cell growth, has an impact on ACC cell metabolism, decreasing cholesterol availability and promoting glucose and glutamine metabolism. In this study, we evidenced that curcumin downregulates the transcription factors SF-1 and SREBPs and their targets, while inducing a ROS-dependent, HIF1α- and NRF2-mediated metabolic rewiring. NRF2 sustained an adaptive antioxidant mechanism dependent on glutamine, cysteine, and glycine uptake to support glutathione synthesis and avoid lipid peroxidation. Furthermore, the combination of curcumin with mitotane demonstrated synergistic effects in inhibiting ACC cell viability and reducing steroidogenic gene expression. These synergistic effects were observed with sub-therapeutic doses of mitotane, which are reached by patients who fail to attain the therapeutic plasma concentrations of the drug. Crucially, in vivo, curcumin administration to tumor-free mice significantly upregulated NRF2 expression and preserved liver tissue integrity. These results warrant further preclinical evaluation of the proposed combination therapy, particularly for those patients who fail to achieve or maintain mitotane plasma concentrations in the therapeutic range.

Glioblastoma (GB) is one of the most aggressive malignant brain tumors. Due to the high invasiveness of this cancer, surgical removal is often not possible, and relapses after surgery are very common, making current treatments ineffective. Developing new therapies or treatment combinations remains a major challenge in managing GB. Metformin (MET), an anti-diabetic medication, has recently gained attention for its potential anticancer effects. To better understand how MET inhibits GB growth at the molecular level, we studied its impact on survivin, a member of the inhibitor of apoptosis (IAP) family that is essential for GB cell survival, resistance to radio- and chemotherapy, and tumor recurrence. Using T98G and U87-MG cell lines, we performed cell viability, migration, and invasion assays, along with Western blot analysis, ChIP assays, and gene silencing experiments to examine key signaling pathways. We found that MET effectively inhibits the growth, viability, and invasiveness of GB cell lines through a molecular mechanism involving activation of the AMPK/FoxO3a/survivin pathway. In vivo studies support these findings, showing increased FoxO3a and decreased survivin in brain tissue sections from metformin-treated mice compared with untreated controls. These results suggest new possibilities for repurposing MET as an adjuvant treatment for GB.

Organoid culture is an emerging and promising 3D culture system by which three-dimensional cell aggregates have been produced from different organs and tissues. This new innovative culture technology preserves parental gene expression, as well as the biological features of parental cells in vitro and ensures maintenance of three-dimensional cell culture for prolonged periods, opening new encouraged scientific scenarios and making them a functioning and valid system for testing new drugs for tissue engineering studies and precision oncology medicine. Various research focused on organoids has been performed in perfusion bioreactors, an advanced device able to mimic the tumor environment, providing a physiological growth state and a long-term culture viability. Perfusion bioreactors have been used for the maintenance and growth of organoids as well as for tumor patient samples improving proliferation while supporting the development of extracellular matrix (ECM). The ability to mimic the tumor environment and to maintain patient-derived biopsies for a long time makes perfusion bioreactors an essential model for preclinical testing.

Androgen receptor (AR) expression in estrogen receptor-positive (ER+) breast cancer (BC) correlates with lower tumor grade and a better clinical outcome. Additionally, in normal mammary epithelium or ER+ BC preclinical models, androgens counteract basal/ER-dependent proliferation. Here, we report an additional mechanism, underlining the protective role exerted by AR. Specifically, the activation of intracellular AR upregulates the Bcl-2-family protein BAD, and TCGA database analyses show that in ER+ BC, BAD expression is associated with better disease-free survival. Ligand-activated AR influences its own and BAD cellular compartmentalization by enhancing levels in the nucleus, as well as in mitochondrial fractions. In both compartments, BAD exerts unconventional functions. In the nucleus, BAD and AR physically interact and, upon androgen stimulation, are recruited at the AP-1 and ARE sites within the cyclin D1 promoter region, contributing to explaining the anti-proliferative effect of androgens in BC cells. Androgens cause an enrichment in BAD and AR content in the mitochondria, correlated with a decrease in mitochondrial function. Thus, we have defined a novel mechanism by which androgens modulate BAD expression, its mitochondria localization, and nuclear content to force its ability to act as a cell cycle inhibitor, strengthening the protective role of androgen signaling in estrogen-responsive BCs.

Extensive research suggests that curcumin interferes with multiple cell signaling pathways involved in cancer development and progression. This study aimed to evaluate curcumin effects on adrenocortical carcinoma (ACC), a rare but very aggressive tumor. Curcumin reduced growth, migration and activated apoptosis in three different ACC cell lines, H295R, SW13, MUC-1. This event was related to a decrease in estrogen-related receptor-α (ERRα) expression and cholesterol synthesis. More importantly, curcumin changed ACC cell metabolism, increasing glycolytic gene expression. However, pyruvate from glycolysis was only minimally used for lactate production and the Krebs cycle (TCA). In fact, lactate dehydrogenase, extracellular acidification rate (ECAR), TCA genes and oxygen consumption rate (OCR) were reduced. We instead found an increase in Glutamic-Pyruvic Transaminase (GPT), glutamine antiport transporter SLC1A5 and glutaminase (GLS1), supporting a metabolic rewiring toward glutamine metabolism. Targeting this mechanism, curcumin effects were improved. In fact, in a low glutamine-containing medium, the growth inhibitory effects elicited by curcumin were observed at a concentration ineffective in default growth medium. Data from this study prove the efficacy of curcumin against ACC growth and progression and point to the concomitant use of inhibitors for glutamine metabolism to improve its effects.

Metabolic reprogramming is an emerging hallmark of cancer, involving the overexpression of metabolism-related proteins, such as glucose and monocarboxylate transporters and intracellular glycolytic enzymes. The biology of testicular germ cell tumors (TGCTs) is very complex, and although their metabolic profile has been scantily explored, some authors have recently reported that the metabolic rewiring of cancer cells resulted in an association with aggressive clinicopathological characteristics. In this study we have investigated, by immunohistochemical analysis, the expression of key proteins sustaining the hyperglycolytic phenotype in pure seminoma (SE, nr. 35), pure embryonal carcinoma (EC, nr. 17) tissues samples, and normal testes (nr. 5). GLUT1, CD44, PFK-1, MCT1, MCT4, LDH-A, and PDH resulted in more expression in EC cells compared to SE cells. TOM20 was more expressed in SE than in EC. GLUT1, MCT1, and MCT4 expression showed a statistically significant association with SE histology, while for EC, the association resulted in being significant only for GLUT1 and MCT4. Finally, we observed that EC resulted as negative for p53, suggesting that the GLUT1 and MTC overexpression observed in EC could be also attributed to p53 downregulation. In conclusion, our findings evidenced that EC exhibits a higher expression of markers of active aerobic glycolysis compared to SE, suggesting that the aggressive phenotype is associated with a higher glycolytic rate. These data corroborate the emerging evidence on the involvement of metabolic reprogramming in testicular malignancies as well, highlighting that the metabolic players should be explored in the future as promising therapeutic targets.

The aim of this study was to investigate the metabolic changes that occur in adrenocortical cancer (ACC) cells in response to the modulation of Estrogen Related Receptor (ERR)α expression and the impact on ACC progression. Proteomics analysis and metabolic profiling highlighted an important role for ERRα in the regulation of ACC metabolism. Stable ERRα overexpression in H295R cells promoted a better mitochondrial fitness and prompted toward a more aggressive phenotype characterized by higher Vimentin expression, enhanced cell migration and spheroids formation. By contrast, a decrease in ERRα protein levels, by molecular (short hairpin RNA) and pharmacological (inverse agonist XCT790) approaches modified the energetic status toward a low energy profile and reduced Vimentin expression and ability to form spheroids. XCT790 produced similar effects on two additional ACC cell lines, SW13 and mitotane-resistant MUC-1 cells. Our findings show that ERRα is able to modulate the metabolic profile of ACC cells, and its inhibition can strongly prevent the growth of mitotane-resistant ACC cells and the progression of ACC cell models to a highly migratory phenotype. Consequently, ERRα can be considered an important target for the design of new therapeutic strategies to fight ACC progression.

Cholesterol affects the proliferation of breast cancer (BC) and in particular of estrogen receptor-negative (ER−) BC. Cholesterol is converted to 27-hydroxycholesterol (27HC), which promotes the growth of ER+ BC. Potentially, 27HC can be involved in cholesterol-dependent ER− BC proliferation. Stable MDA-MB-231 silenced clones for CYP7B1 (27HC metabolizing enzyme) show an increased basal proliferation rate, which is not observed in the presence of lipoprotein-deprived serum. Furthermore, the treatment of SKBR3, MDA-MB-231 and MDA-MB-468 with 27HC increased cell proliferation that was prevented by G15, a selective G Protein-Coupled Estrogen Receptor (GPER) inhibitor, suggested this receptor to be a potential 27HC target. Binding experiments demonstrate that 27HC is a new ligand for GPER. We show that ERK1/2 and NFκB are part of the 27HC/GPER pathway. The stable silencing of GPER prevents NFκB activation and reduces basal and 27HC-dependent tumor growth. Additionally, conditioned medium from ER− BC cells treated with 27HC promotes tube formation, which does not occur with CM from GPER silenced cells. Collectively, these data demonstrate that cholesterol conversion into 27HC promotes ER− BC growth and progression, and the expression of GPER is required for its effects.

It is known that estrogen stimulates growth and inhibits apoptosis through estrogen receptor(ER)-mediated mechanisms in many cancer cell types. Interestingly, there is strong evidence that estrogens can also induce apoptosis, activating different ER isoforms in cancer cells. It has been observed that E2/ERα complex activates multiple pathways involved in both cell cycle progression and apoptotic cascade prevention, while E2/ERβ complex in many cases directs the cells to apoptosis. However, the exact mechanism of estrogen-induced tumor regression is not completely known. Nevertheless, ERs expression levels of specific splice variants and their cellular localization differentially affect outcome of estrogen-dependent tumors. The goal of this review is to provide a general overview of current knowledge on ERs-mediated apoptosis that occurs in main hormone dependent-cancers. Understanding the molecular mechanisms underlying the induction of ER-mediated cell death will be useful for the development of specific ligands capable of triggering apoptosis to counteract estrogen-dependent tumor growth.