Role of Scaffold Protein Proline-, Glutamic Acid-, and Leucine-Rich Protein 1 (PELP1) in the Modulation of Adrenocortical Cancer Cell Growth

PELP1 acts as an estrogen receptor (ER) coactivator that exerts an essential role in the ER's functions. ER coregulators have a critical role in the progression and response to hormonal treatment of estrogen-dependent tumors. We previously demonstrated that, in adrenocortical carcinoma (ACC), ER\u3b1 is upregulated and that estradiol activates the IGF-II/IGF1R signaling pathways defining the role of this functional cross-talk in H295R ACC cell proliferation. The aim of this study was to determine if PELP1 is expressed in ACC and may play a role in promoting the interaction between ER\u3b1 and IGF1R allowing the activation of pathways important for ACC cell growth. The expression of PELP1 was detected by Western blot analysis in ACC tissues and in H295R cells. H295R cell proliferation decrease was assessed by A3-(4,5-Dimethylthiaoly)-2,5-diphenyltetrazolium bromide (MTT) assay and [3H] thymidine incorporation. PELP1 is expressed in ACC tissues and in H295R cells. Moreover, treatment of H295R with E2 or IGF-II induced a multiprotein complex formation consisting of PELP1, IGF1R, ER\u3b1, and Src that is involved in ERK1/2 rapid activation. PELP1/ER/IGF1R/c-Src complex identification as part of E2- and IGF-II-dependent signaling in ACC suggests PELP1 is a novel and more efficient potential target to reduce ACC growth

GPER agonist G-1 decreases adrenocortical carcinoma (ACC) cell growth in vitro and in vivo

We have previously demonstrated that estrogen receptor (ER) alpha (ESR1) increases proliferation of adrenocortical carcinoma (ACC) through both an estrogen-dependent and -independent (induced by IGF-II/IGF1R pathways) manner. Then, the use of tamoxifen, a selective estrogen receptor modulator (SERM), appears effective in reducing ACC growth in vitro and in vivo. However, tamoxifen not only exerts antiestrogenic activity, but also acts as full agonist on the G protein-coupled estrogen receptor (GPER). Aim of this study was to investigate the effect of a non-steroidal GPER agonist G-1 in modulating ACC cell growth. We found that G-1 is able to exert a growth inhibitory effect on H295R cells both in vitro and, as xenograft model, in vivo. Treatment of H295R cells with G-1 induced cell cycle arrest, DNA damage and cell death by the activation of the intrinsic apoptotic mechanism. These events required sustained extracellular regulated kinase (ERK) 1/2 activation. Silencing of GPER by a specific shRNA partially reversed G-1-mediated cell growth inhibition without affecting ERK activation. These data suggest the existence of G-1 activated but GPER-independent effects that remain to be clarified. In conclusion, this study provides a rational to further study G-1 mechanism of action in order to include this drug as a treatment option to the limited therapy of ACC

Leptin and leptin receptor in pig spermatozoa: evidence of their involvement in sperm capacitation and survival

Several studies have recently investigated the role of leptin, the adipocyte-secreted hormone, in the growth and reproduction of rodents, humans, and domestic animals. The present study was designed to explore the expression of leptin and its receptor in pig spermatozoa. Successful Western blot evidenced a 16 kDa band for leptin and six isoforms, ranging from 120 to 40 kDa, for the leptin receptor. Both leptin and leptin receptor were interestingly located at sperm acrosomal level, suggesting their involvement in the oocyte fertilization events. In fact, both capacitation indexes and acrosin activity were enhanced by leptin, and these effects were reduced by the anti-leptin receptor antibody. Afterwards, we investigated the main transduction pathways regulated by the hormone. Our results showed that, in pig sperm, leptin can trigger the signal transducer and activator of transcription 3, a classical component of cytokine signal transduction pathways, whose expression has not been previously reported in male gamete; in addition it was found constitutively activated. Besides, leptin was able to induce the activation of phosphatidylinositol phosphate kinase 3 and MAP kinase pathways as well as of BCL2, a known antiapoptotic protein. These data address to a role of leptin and its receptor on pig sperm survival. The presence of leptin and its receptor in pig sperm suggests that they, through an autocrine short loop, may induce signal transduction and molecular changes associated with sperm capacitation and survival

Antioxidant, enzyme-inhibitory and antitumor activity of the wild dietary plant Muscari comosum (L.) Mill.

Conventional medicines used to treat obesity and cancer frequently exhibit high side effects, so that researchers are focusing on new therapies and drugs based on natural products. Total extracts from bulbs of Muscari comosum were tested for i) free radical scavenging activity, ii) in vitro enzymatic inhibition of pancreatic α-amylase and lipase, and iii) inhibition of the growth of breast adenocarcinoma cells. Three treatments were considered: bulbs boiled in water for 15 min (traditional cooking method; BB); bulbs steam-cooked for 15 min (alternative cooking method; SB); raw bulbs (RB). The polyphenol content and antioxidant capacity of bulb extracts were related to the inhibition of pancreatic lipase and α-amylase, whose activities have been found to have a half maximal inhibitory concentration (IC50) of 0.28, 2.14 and 3.22 mg/mL for lipase, and 0.16, 0.73 and 0.69 mg/mL for α-amylase in RB, SB and BB, respectively. The analysis on breast adenocarcinoma MCF-7 cells revealed that RB extracts, and in a lesser extent BB, exerted a dose-dependent inhibition on cell proliferation. Considering that the potential of natural products for the treatment of obesity are under exploration, M. comosum could be an excellent plant for the development of future anti-obesity drugs, also able to prevent cancer

Adrenocortical Carcinoma (ACC) Cells Rewire Their Metabolism to Overcome Curcumin Antitumoral Effects Opening a Window of Opportunity to Improve Treatment

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

Estrogen Related Receptor Alpha (ERRα) a Bridge between Metabolism and Adrenocortical Cancer Progression

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 and Its Metabolites in Tumor Growth: Therapeutic Potential of Statins in Cancer Treatment

Cholesterol is essential for cell function and viability. It is a component of the plasma membrane and lipid rafts and is a precursor for bile acids, steroid hormones, and Vitamin D. As a ligand for estrogen-related receptor alpha (ESRRA), cholesterol becomes a signaling molecule. Furthermore, cholesterol-derived oxysterols activate liver X receptors (LXRs) or estrogen receptors (ERs). Several studies performed in cancer cells reveal that cholesterol synthesis is enhanced compared to normal cells. Additionally, high serum cholesterol levels are associated with increased risk for many cancers, but thus far, clinical trials with 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) have had mixed results. Statins inhibit cholesterol synthesis within cells through the inhibition of HMG-CoA reductase, the rate-limiting enzyme in the mevalonate and cholesterol synthetic pathway. Many downstream products of mevalonate have a role in cell proliferation, since they are required for maintenance of membrane integrity; signaling, as some proteins to be active must undergo prenylation; protein synthesis, as isopentenyladenine is an essential substrate for the modification of certain tRNAs; and cell-cycle progression. In this review starting from recent acquired findings on the role that cholesterol and its metabolites fulfill in the contest of cancer cells, we discuss the results of studies focused to investigate the use of statins in order to prevent cancer growth and metastasis

Cholesterol as an Endogenous ERRα Agonist: A New Perspective to Cancer Treatment

The estrogen-related receptors (ERRs) are important members of nuclear receptors which contain three isoforms (α, β, and γ). ERRα is the best-characterized isoform expressed mainly in high-energy demanding tissues where it preferentially works in association with the peroxisome proliferator-activated receptor-γ co-activator 1α (PGC-1α) and PGC-1β. ERRα together with its cofactors modulates cellular metabolism, supports the growth of rapidly dividing cells, directs metabolic programs required for cell differentiation and maintains cellular energy homeostasis in differentiated cells. In cancer cells, the functional association between ERRα and PGC-1s is further influenced by oncogenic signals and induces metabolic programs favoring cell growth and proliferation as well as tumor progression. Recently, cholesterol has been identified as a natural ERRα ligand using a combined biochemical strategy. This new finding highlighted some important physiological aspects related to the use of cholesterol-lowering drugs such as statins and bisphosphonates. Even more meaningful is the link between increased cholesterol levels and certain cancer phenotypes characterized by an overexpressed ERRα such as mammary, prostatic, and colorectal cancers, where the metabolic adaptation affects many cancer processes. Moreover, high-energy demanding cancer-related processes are strictly related to the cross-talk between tumor cells and some key players of tumor microenvironment, such as tumor-associated macrophage that fuels cancer progression. Some evidence suggests that high cholesterol content and ERRα activity favor the inflammatory environment by the production of different cytokines. In this review, starting from the most recent observations on the physiological role of the new signaling activated by the natural ligand of ERRα, we propose a new hypothesis on the suitability to control cholesterol levels as a chance in modulating ERRα activity in those tumors in which its expression and activity are increased

Statins reduce intratumor cholesterol affecting adrenocortical cancer growth

Mitotane causes hypercholesterolemia in ACC patients. We suppose that cholesterol increases within the tumor and can be used to activate proliferative pathways. In this study, we used statins to decrease intratumor cholesterol and investigated the effects on ACC growth related to ER\u3b1 action at the nuclear and mitochondrial levels. We first used microarray to investigate mitotane effect on genes involved in cholesterol homeostasis and evaluated their relationship with patients' survival in ACC TCGA. We then blocked cholesterol synthesis with simvastatin and determined the effects on H295R cell proliferation, estradiol production and ER\u3b1 activity in vitro and in xenograft tumors. We found that mitotane increases intratumor cholesterol content and expression of genes involved in cholesterol homeostasis, among them INSIG, whose expression affects patients' survival. Treatment of H295R cells with simvastatin to block cholesterol synthesis decreased cellular cholesterol content and this affected cell viability. Simvastatin reduced estradiol production and decreased nuclear and mitochondrial ER\u3b1 function. A mitochondrial target of ER\u3b1, the respiratory complex IV (COX IV) was reduced after simvastatin treatment, which profoundly affected mitochondrial respiration activating apoptosis. In vivo experiments confirmed the ability of simvastatin to reduce tumor volume and weight of grafted H295R cells, intratumor cholesterol content, Ki-67 and ER\u3b1, COX IV expression and activity and increase TUNEL positive cells. Collectively these data demonstrate that a reduction in intratumor cholesterol content prevents estradiol production, inhibits mitochondrial respiratory chain inducing apoptosis in ACC cells. Inhibition of mitochondrial respiration by simvastatin represents a novel strategy to counteract ACC growth