Pierangela Totta

Survival versus apoptotic 17β‐estradiol effect: Role of ERα and ERβ activated non‐genomic signaling

The capability of 17β‐estradiol (E2) to induce the non‐genomic activities of its receptors (ERα and ERβ) and to evoke different signaling pathways committed to the regulation of cell proliferation has been analyzed in different cell cancer lines containing transfected (HeLa) or endogenous (HepG2, DLD1) ERα or ERβ. In these cell lines, E2 induced different effects on cell growth/apoptosis in dependence of ER isoforms present. The E2–ERα complex rapidly activated multiple signal transduction pathways (i.e., ERK/MAPK, PI3K/AKT) committed to both cell cycle progression and apoptotic cascade prevention. On the other hand, the E2–ERβ complex induced the rapid and persistent phosphorylation of p38/MAPK which, in turn, was involved in caspase‐3 activation and cleavage of poly(ADP‐ribose)polymerase, driving cells into the apoptotic cycle. In addition, the E2–ERβ complex did not activate any of the E2–ERα‐activated signal molecules involved in cell growth. Taken together, these results demonstrate the ability of ERβ isoform to activate specific signal transduction pathways starting from plasma membrane that may justify the effect of E2 in inducing cell proliferation or apoptosis in cancer cells. In particular this hormone promotes cell survival through ERα non‐genomic signaling and cell death through ERβ non‐genomic signaling.

Survival versus apoptotic 17beta-estradiol effect: role of ER alpha and ER beta activated non-genomic signaling.

The capability of 17β‐estradiol (E2) to induce the non‐genomic activities of its receptors (ERα and ERβ) and to evoke different signaling pathways committed to the regulation of cell proliferation has been analyzed in different cell cancer lines containing transfected (HeLa) or endogenous (HepG2, DLD1) ERα or ERβ. In these cell lines, E2 induced different effects on cell growth/apoptosis in dependence of ER isoforms present. The E2–ERα complex rapidly activated multiple signal transduction pathways (i.e., ERK/MAPK, PI3K/AKT) committed to both cell cycle progression and apoptotic cascade prevention. On the other hand, the E2–ERβ complex induced the rapid and persistent phosphorylation of p38/MAPK which, in turn, was involved in caspase‐3 activation and cleavage of poly(ADP‐ribose)polymerase, driving cells into the apoptotic cycle. In addition, the E2–ERβ complex did not activate any of the E2–ERα‐activated signal molecules involved in cell growth. Taken together, these results demonstrate the ability of ERβ isoform to activate specific signal transduction pathways starting from plasma membrane that may justify the effect of E2 in inducing cell proliferation or apoptosis in cancer cells. In particular this hormone promotes cell survival through ERα non‐genomic signaling and cell death through ERβ non‐genomic signaling.

Mechanisms of Naringenin‐induced Apoptotic Cascade in Cancer Cells: Involvement of Estrogen Receptor a and ß Signalling

The flavanone naringenin (Nar), especially abundant in the Mediterranean diet, is reported to have anti‐proliferative effects in many cancer cell lines. Antioxidant activities, kinase and glucose uptake inhibition have been proposed as molecular mechanisms for these effects. In addition, an anti‐estrogenic activity has been observed but, at the present, it is poorly understood whether this latter activity could play a role in the Nar anti‐tumoral effects. Here, we tested the ability of Nar to activate a specific, rapid signal transduction pathway committed to the generation of an apoptotic cascade in the presence of one of the two estrogen receptor (ER) isoforms (i.e., ERα or ERβ). Cancer cells containing transfected (human cervix epitheloid carcinoma HeLa cells) or endogenous ERα (human hepatoma HepG2 cells) or ERβ (human colon adenocarcinoma DLD‐1 cells) were used. Our results show that Nar exerts an anti‐proliferative effect only in the presence of ERα or ERβ. Moreover, Nar stimulation induces the activation of p38/MAPK leading to the pro‐apoptotic caspase‐3 activation and to the poly(ADP‐ribose) polymerase cleavage in all cancer cell lines considered. Notably, Nar shows an anti‐estrogenic effect only in ERα containing cells; whereas in ERβ containing cells, Nar mimics the 17β‐estradiol effects. These findings indicate new steps in the mechanism underlying ER‐dependent anti‐proliferative effects of Nar suggesting new potential chemopreventive actions of flavonoids on cancer growth. IUBMB Life, 56: 491‐499, 2004

Nutritional Flavonoids Modulate Estrogen Receptor α Signaling

Estrogen receptor α (ERα) mediates 17β‐estradiol (E2) actions through the transcription of E2‐sensitive target genes. In addition, rapid non‐genomic signaling (e.g., MAPK/ERK) occurs. It is now well accepted that these rapid membrane‐initiated responses account for E2‐related cancer. Beside many beneficial effects on human health, nutritional flavonoids exert protective and anticarcinogenic effects on E2‐related cancer. The mechanism underlying these effects seems to be related to flavonoids antioxidant properties and/or to their ability to alter signal transduction protein kinases. In addition, an antiestrogenic activity has been proposed but not yet defined. However, the identification and characterization of the responsible mechanisms for flavonoid antitumoral effects is poorly understood. Here, we investigated the possibility that the antimitogenic effects of flavonoids are transduced by modulating ERα‐mediated rapid signaling. The ability of two flavonoids, the flavanone naringenin and the flavanol quercetin, with respect of E2, to induce ERα activities has been studied in the human cervix epitheloid carcinoma cell line (HeLa) devoid of any estrogen receptors and rendered E2‐sensitive by transient transfection with a human ERα expression vector. Our results indicate that flavonoids act as E2 mimetic on ERα transcriptional activity, whereas they impair the activation of rapid signaling pathways committed to E2‐induced proliferation. The resulting decoupling of ERα signal transduction could be proposed as a new mechanism in the protective effects of flavonoids against E2‐related cancer. IUBMB Life, 56: 145‐151, 2004

Neuroglobin, a pro-survival player in estrogen receptor α-positive cancer cells

Recently, we reported that human neuroglobin (NGB) is a new player in the signal transduction pathways that lead to 17β-estradiol (E2)-induced neuron survival. Indeed, E2 induces in neuron mitochondria the enhancement of NGB level, which in turn impairs the activation of a pro-apoptotic cascade. Nowadays, the existence of a similar pathway activated by E2 in non-neuronal cells is completely unknown. Here, the role of E2-induced NGB upregulation in tumor cells is reported. E2 induced the upregulation of NGB in a dose- and time-dependent manner in MCF-7, HepG2, SK-N-BE, and HeLa cells transfected with estrogen receptor α (ERα), whereas E2 was unable to modulate the NGB expression in the ERα-devoid HeLa cells. Both transcriptional and extranuclear ERα signals were required for the E2-dependent upregulation of NGB in MCF-7 and HepG2 cell lines. E2 stimulation modified NGB intracellular localization, inducing a significant reduction of NGB in the nucleus with a parallel increase of NGB in the mitochondria in both HepG2 and MCF-7 cells. Remarkably, E2 pretreatment did not counteract the H2O2-induced caspase-3 and poly (ADP-ribose) polymerase 1 (PARP-1) cleavage, as well as Bcl-2 overexpression in MCF-7 and HepG2 cells in which NGB was stably silenced by using shRNA lentiviral particles, highlighting the pivotal role of NGB in E2-induced antiapoptotic pathways in cancer cells. Present results indicate that the E2-induced NGB upregulation in cancer cells could represent a defense mechanism of E2-related cancers rendering them insensitive to oxidative stress. As a whole, these data open new avenues to develop therapeutic strategies against E2-related cancers.

Lysosomal function is involved in 17β-estradiol-induced estrogen receptor α degradation and cell proliferation

The homeostatic control of the cellular proteome steady-state is dependent either on the 26S proteasome activity or on the lysosome function. The sex hormone 17β-estradiol (E2) controls a plethora of biological functions by binding to the estrogen receptor α (ERα), which is both a nuclear ligand-activated transcription factor and also an extrinsic plasma membrane receptor. Regulation of E2-induced physiological functions (e.g., cell proliferation) requires the synergistic activation of both transcription of estrogen responsive element (ERE)-containing genes and rapid extra-nuclear phosphorylation of many different signalling kinases (e.g., ERK/MAPK; PI3K/AKT). Although E2 controls ERα intracellular content and activity via the 26S proteasome-mediated degradation, biochemical and microscopy-based evidence suggests a possible cross-talk among lysosomes and ERα activities. Here, we studied the putative localization of endogenous ERα to lysosomes and the role played by lysosomal function in ERα signalling. By using confocal microscopy and biochemical assays, we report that ERα localizes to lysosomes and to endosomes in an E2-dependent manner. Moreover, the inhibition of lysosomal function obtained by chloroquine demonstrates that, in addition to 26S proteasome-mediated receptor elimination, lysosome-based degradation also contributes to the E2-dependent ERα breakdown. Remarkably, the lysosome function is further involved in those ERα activities required for E2-dependent cell proliferation while it is dispensable for ERα-mediated ERE-containing gene transcription. Our discoveries reveal a novel lysosome-dependent degradation pathway for ERα and show a novel biological mechanism by which E2 regulates ERα cellular content and, as a consequence, cellular functions.

Abnormal Kinetochore-Generated Pulling Forces from Expressing a N-Terminally Modified Hec1

Background Highly Expressed in Cancer protein 1 (Hec1) is a constituent of the Ndc80 complex, a kinetochore component that has been shown to have a fundamental role in stable kinetochore-microtubule attachment, chromosome alignment and spindle checkpoint activation at mitosis. HEC1 RNA is found up-regulated in several cancer cells, suggesting a role for HEC1 deregulation in cancer. In light of this, we have investigated the consequences of experimentally-driven Hec1 expression on mitosis and chromosome segregation in an inducible expression system from human cells. Methodology/Principal Findings Overexpression of Hec1 could never be obtained in HeLa clones inducibly expressing C-terminally tagged Hec1 or untagged Hec1, suggesting that Hec1 cellular levels are tightly controlled. On the contrary, a chimeric protein with an EGFP tag fused to the Hec1 N-terminus accumulated in cells and disrupted mitotic division. EGFP- Hec1 cells underwent altered chromosome segregation within multipolar spindles that originated from centriole splitting. We found that EGFP-Hec1 assembled a mutant Ndc80 complex that was unable to rescue the mitotic phenotypes of Hec1 depletion. Kinetochores harboring EGFP-Hec1 formed persisting lateral microtubule-kinetochore interactions that recruited the plus-end depolymerase MCAK and the microtubule stabilizing protein HURP on K-fibers. In these conditions the plus-end kinesin CENP-E was preferentially retained at kinetochores. RNAi-mediated CENP-E depletion further demonstrated that CENP-E function was required for multipolar spindle formation in EGFP-Hec1 expressing cells. Conclusions/Significance Our study suggests that modifications on Hec1 N-terminal tail can alter kinetochore-microtubule attachment stability and influence Ndc80 complex function independently from the intracellular levels of the protein. N-terminally modified Hec1 promotes spindle pole fragmentation by CENP-E-mediated plus-end directed kinetochore pulling forces that disrupt the fine balance of kinetochore- and centrosome-associated forces regulating spindle bipolarity. Overall, our findings support a model in which centrosome integrity is influenced by the pathways regulating kinetochore-microtubule attachment stability.

Survival versus apoptotic 17?-estradiol effect: Role of ER? and ER? activated non-genomic signaling

The capability of 17β‐estradiol (E2) to induce the non‐genomic activities of its receptors (ERα and ERβ) and to evoke different signaling pathways committed to the regulation of cell proliferation has been analyzed in different cell cancer lines containing transfected (HeLa) or endogenous (HepG2, DLD1) ERα or ERβ. In these cell lines, E2 induced different effects on cell growth/apoptosis in dependence of ER isoforms present. The E2–ERα complex rapidly activated multiple signal transduction pathways (i.e., ERK/MAPK, PI3K/AKT) committed to both cell cycle progression and apoptotic cascade prevention. On the other hand, the E2–ERβ complex induced the rapid and persistent phosphorylation of p38/MAPK which, in turn, was involved in caspase‐3 activation and cleavage of poly(ADP‐ribose)polymerase, driving cells into the apoptotic cycle. In addition, the E2–ERβ complex did not activate any of the E2–ERα‐activated signal molecules involved in cell growth. Taken together, these results demonstrate the ability of ERβ isoform to activate specific signal transduction pathways starting from plasma membrane that may justify the effect of E2 in inducing cell proliferation or apoptosis in cancer cells. In particular this hormone promotes cell survival through ERα non‐genomic signaling and cell death through ERβ non‐genomic signaling.

Xenoestrogens Alter Estrogen Receptor (ER) α Intracellular Levels

17β-estradiol (E2)-dependent estrogen receptor (ER) α intracellular concentration is a well recognized critical step in the pleiotropic effects elicited by E2 in several target tissues. Beside E2, a class of synthetic and plant-derived chemicals collectively named endocrine disruptors (EDs) or xenoestrogens bind to and modify both nuclear and extra-nuclear ERα activities. However, at the present no information is available on the ability of EDs to hamper ERα intracellular concentration. Here, the effects of bisphenol A (BPA) and naringenin (Nar), prototypes of synthetic and plant-derived ERα ligands, have been evaluated on ERα levels in MCF-7 cells. Both EDs mimic E2 in triggering ERα Ser118 phosphorylation and gene transcription. However, only E2 or BPA induce an increase of cell proliferation; whereas 24 hrs after Nar stimulation a dose-dependent decrease in cell number is reported. E2 or BPA treatment reduces ERα protein and mRNA levels after 24 hrs. Contrarily, Nar stimulation does not alter ERα content but reduces ERα mRNA levels like other ligands. Co-stimulation experiments indicate that 48 hrs of Nar treatment prevents the E2-induced ERα degradation and hijacks the physiological ability of E2:ERα complex to regulate gene transcription. Mechanistically, Nar induces ERα protein accumulation by preventing proteasomal receptor degradation via persistent activation of p38/MAPK pathway. As a whole these data demonstrate that ERα intracellular concentration is an important target through which EDs hamper the hormonal milieu of E2 target cells driving cells to different outcomes or mimicking E2 even in the absence of the hormone.

Modulation of 17β-Estradiol Signaling on Cellular Proliferation by Caveolin-2.

The sex hormone 17β‐estradiol (E2) exerts pleiotropic effects by binding to the ligand‐activated transcription factor estrogen receptor α (ERα). The E2:ERα complex regulates several physiological processes, including cell survival and proliferation, through transcriptional effects (i.e., estrogen responsive element [ERE]‐based gene transcription) and non‐transcriptional membrane‐initiated effects (i.e., the activation of extra‐nuclear signaling cascades), which derive from the activation of the pool of ERα that is localized to plasma membrane caveolae. Caveolae are ω‐shaped membrane sub‐domains that are composed of scaffold proteins named caveolins (i.e., caveolin‐1, caveolin‐2, and caveolin‐3). Although caveolin‐3 is exclusively expressed in muscles, caveolin‐1 and caveolin‐2 are co‐expressed in all human tissues. From a functional point of view, caveolin‐2 can operate both dependently on and independently of caveolin‐1, which is the main coat component of caveolae. Interestingly, while a functional interplay between caveolin‐1 and ERα has been reported in the control of E2‐induced physiological effects, the role of caveolin‐2 in E2:ERα signaling within the cell remains poorly understood. This study shows that siRNA‐mediated caveolin‐2 depletion in breast ductal carcinoma cells (MCF‐7) reduces E2‐induced ERα phosphorylation at serine residue 118 (S118), controls intracellular receptor levels, precludes ERα‐mediated extra‐nuclear activation of signaling pathways, reduces ERα transcriptional activity, and prevents cellular proliferation. Meanwhile, the impact of caveolin‐1 depletion on ERα signaling in MCF‐7 cells is shown to be similar to that elicited by siRNA‐mediated caveolin‐2 depletion. Altogether, these data demonstrate that caveolin‐2 expression is necessary for the control of E2‐dependent cellular proliferation. J. Cell. Physiol. 231: 1219–1225, 2016.