Notice: Wiley Online Library will be unavailable on Saturday 30th July 2016 from 08:00-11:00 BST / 03:00-06:00 EST / 15:00-18:00 SGT for essential maintenance. Apologies for the inconvenience.
Department of Science, Section Biomedical Sciences and Technology, University Roma Tre, Rome, Italy
Address correspondence to: Filippo Acconcia, Department of Science, Section Biomedical Sciences and Technology, University Roma Tre, Viale Guglielmo Marconi 446, I-00146 Rome, Italy. Tel: +39-0657336320. Fax: +39-0657336321. E-mail: email@example.com
If you can't find a tool you're looking for, please click the link at the top of the page to "Go to old article view". Alternatively, view our Knowledge Base articles for additional help. Your feedback is important to us, so please let us know if you have comments or ideas for improvement.
Breast cancer (BC) is a heterogeneous disease in which the sex steroid hormone 17β-estradiol (E2) plays a prominent role through the engagement of the estrogen receptor alpha (ERα) and through the E2:ERα complex consequent signaling. In particular, besides its role as transcriptional factor in the nucleus, ERα localizes in the cytoplasm and also at the plasma membrane of BC cells. Remarkably, it is now clear that the E2-dependent engagement of the plasma membrane-localized ERα in BC cells produces the activation of many signal transduction cascades (e.g., PI3K/AKT pathway) that ultimately result in the modulation of many genes involved in the regulation of cell cycle progression (e.g., cyclin D1) and in cell proliferation as well as in the remodeling of the actin cytoskeleton, which is the prerequisite for cell migration .
In recent years, mounting evidence indicated that the ubiquitin (Ub)-system could be a pharmacological option for cancer therapy . Thus, in principle, the Ub-system could be another signaling pathway regulated by E2 that could be pharmacologically exploited for BC treatment. However, the involvement of Ub-system enzymes (i.e., the Ub-activating enzyme E1, the Ub-conjugating E2, and the Ub-ligases E3) in E2:ERα signaling remains primordial in BC cells. Remarkably, the first cell-permeable inhibitor of the key initiating enzyme E1, 4[4-(5-nitro-furan-2-ylmethylene)−3,5-dioxo-pyrazolidin-1-yl]-benzoic acid ethyl ester (Pyr-41), on which the activation of the Ub-system relies on has been identified . Thus, the main aim of this work was to determine the involvement of the Ub-system in the ERα-mediated signals required for E2-induced proliferation and migration through the evaluation of the Pyr-41 effect in BC cells.
Cell culture and Reagents
All cell lines and reagents were previously described [4-6]. 4[4-(5-Nitro-furan-2-ylmethylene)-3,5-dioxo-pyrazolidin-1-yl]-benzoic acid ethyl ester (Pyr-41; ref. 3) and anti-tubulin (mouse; 1:10,000) and anti-vinculin (mouse; 1:10,000) antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies were used for Western blotting against ERα (HC-20 rabbit; 1:1,000), Ub (P4D1 mouse; 1:1,000), phospho-ERK1/2 (E-4 mouse; 1:1,000), ERK2 (C-14 rabbit; 1:1,000), p53 (DO-1 mouse; 1:1,000), cyclin D1 (H-295 rabbit; 1:200), and phospho-p38/MAPK (sc-17852-R rabbit; 1:1,000) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antiphospho-AKT Ser 473 (rabbit; 1:1,000), anti-AKT (rabbit; 1:1,000), and anti-p38 (rabbit; 1:1,000) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). p38/MAPK inhibitor, SB 203580 (SB), and the 26S proteasome inhibitor, Mg-132 (Mg), were purchased from Calbiochem (San Diego, CA, USA).
Cellular and Biochemical Assays
Cells were grown in 1% charcoal-stripped fetal calf serum medium for 24 h and then stimulated with E2 at the indicated time points; where indicated, inhibitors (Pyr-41, Ly, and SB) were added 30 min before E2 administration. Unless otherwise indicated, cell were treated with E2 (10−8 M), epidermal growth factor (EGF; 1 µg/mL), Pyr-41 (1 µM), Ly (1 µM), or SB (1 µM). Protein extraction and biochemical assays were performed as previously described [5, 6]. Cell counts were performed by plating 40,000 cells in six-well plates. Twenty-four hours after plating, cells were serum starved, and then E2 and inhibitors were added to the starvation medium. After 48 h, cell were harvested with trypsin and counted in a Berthold hemocytometer. Each condition was set in triplicate, at least five counts per well was done, and the experiment was repeated twice. XTT assay kit was purchased from Roche (Indianapolis, IN, USA) and used according to manufacturer's instructions. Colony assay formation was performed by plating 500 cells per well in 60-mm dishes in triplicate for each condition. Twenty-four hours after plating, cells were washed in PBS, and then E2 and inhibitors were added to cells in fresh starvation medium. Medium supplemented with E2 and inhibitors was changed every 3 days. After 15 days, cells were stained with crystal violet. Following extensive washes, stained cells were acquired by computer scanning, and retained crystal violet was quantitated by solubilizing cell with 1% SDS and by reading the corresponding absorbance at spectrophotometer at 575 nm. Western blotting analyses were performed as reported in refs. 5 and 6. Proteins were transferred onto precasted nitrocellulose or PVDF membranes using the transblot turbo transfer system (Biorad Laboratories, Hercules, CA, USA) for 10 min at room temperature. Band acquisition was performed by using the C-Digit® Blot Scanner (Li-Cor Lincon, NE, USA).
Wound Healing Assays
Cell migration potential was assessed using an established wound healing assay as previously described . Briefly, MCF-7 cells were plated in 60-mm dishes in 10% fetal calf serum–DMEM media. When cells were monolayer, they were rinsed twice in PBS and then starved for 24 h. The confluent monolayer of cells was then wounded by scraping a narrow 10-µL Pipetman tip across the plate in three parallel lines. Cells were rinsed twice in PBS and then grown in 1% DCC media or media supplemented with E2 in the presence or absence of the inhibitors Pyr-41, Ly, or SB. After an additional 48 h, each plate was examined by phase contrast microscopy for the amount of wound closure by measuring the physical separation remaining between the original wound widths. Each plate was examined by phase contrast microscopy for wound closure. Five separate measurements were made per plate, and each experiment was performed in duplicate.
Confocal Microscopy Analysis
MCF-7 cells were plated on coverslips serum starved for 24 h and then treated with E2 (5 min) or inhibitors. Fixed cells were stained with phalloidin (1:400; Invitrogen, Carlsbad, CA, USA) as reported in ref. . In detail, cells were grown on 30-mm glass gelatin-coated coverslips in six-well plates (5 × 105 cells per well). Cells were then fixed with paraformaldehyde (4%) for 10 min and permeabilized with Triton-X 100 (0.1%) for 5 min. After the permeabilization process, cells were incubated with bovine serum albumin (2%) for 1 h and then stained with phalloidin at room temperature. Following extensive washes, coverslips were mounted, and confocal analysis was performed using LCS (Leica Microsystems, Heidelberg, Germany).
A statistical analysis was performed using the ANOVA test with the InStat version 3 software system (GraphPad Software, San Diego, CA, USA). Densitometric analyses were performed using the freeware software Image J by quantifying the band intensity of the protein of interest with respect to the relative loading control band (i.e., vinculin or tubulin) intensity. Data are means of three independent experiments ± SD.
Role of E1 in the Inhibition of the Ubiquitination Cascade
To begin to unravel the role of E1 in BC cells, we have initially evaluated the ability of the E1 inhibitor 4[4-(5-nitro-furan-2-ylmethylene)-3,5-dioxo-pyrazolidin-1-yl]-benzoic acid ethyl ester (Pyr-41) to affect the cellular content of both p53 and cyclin D1, which rapidly undergo Ub-dependent degradation . As previously reported , treatment of MCF-7 cells with different doses of Pyr-41 induced the cellular accumulation of both p53 and cyclin D1, whereas no changes in the total content of ubiquitinated species were observed at any of the doses of Pyr-41 tested (Fig. 1A). The Pyr-41 impact on basal MCF-7 cell proliferation was assessed by treating cells with different doses of Pyr-41. Figures 1B and 1C show a dose-dependent reduction in the cell number and cell viability after Pyr-41 treatment, with a maximum effect occurring when MCF-7 cells were exposed to 50 µM of the drug. To link the inhibition of E1 activity with ERα signaling, we next evaluated ERα degradation in MCF-7 cells treated with different doses of Pyr-41. Surprisingly, the treatment of MCF-7 cells with Pyr-41 determined a dose-dependent reduction in the basal ERα intracellular content (Fig. 1D). MCF-7 cells were then treated with Pyr-41 (1 µM) in the presence or absence of a 26S proteasome inhibitor (Mg-132), which is known to increase total cellular ubiquitination . Consistent with its proposed function , Pyr-41 reduced the accumulation of cellular Ub conjugates in response to Mg-132 over a period of 2 h (Figs. 1E and 1E′).
Overall, these data confirm that Pyr-41 efficiently inhibits the ubiquitination cascade (i.e., E1 activity) in BC cells  and further indicate that high doses of this E1 inhibitor are toxic for MCF-7 cells. For these reasons, next experiments were performed using low doses of Pyr-41 (1 µM).
Role of E1 in ERα Degradation
Because Pyr-41 induced a dose-dependent reduction in ERα cellular levels, we decided to better investigate the role of E1 in E2-dependent ERα degradation. As shown in Figure 2A, time course analysis revealed that treatment of MCF-7 cells with E2 induces a time-dependent reduction in ERα cellular content within the first 2 h of hormone treatment (Fig. 2A), as previously reported . A further increase in the E2-induced ERα breakdown was observed when MCF-7 cells were treated with Pyr-41 (Fig. 2A). The analysis of ERα degradation after 24 h of E2 treatment revealed that administration of Pyr-41 fastens the E2-evoked reduction in ERα intracellular content (Fig. 2B). Notably, MCF-7 cells treated with Pyr-41 (1 µM) alone did not display any significant changes in ERα cellular levels. These data indicate that inhibition of the ubiquitination cascade affects E2-dependent ERα degradation.
Role of E1 in E2-Induced Breast Cancer Cell Proliferation
Because E2 controls cell proliferation through the activation of the ERα-mediated extranuclear signaling , we next tested the effect of nontoxic doses of the E1 inhibitor Pyr-41 (1 µM) on this E2-induced pathway in MCF-7 cells. As shown in Figures 3A and 3A′, Pyr-41 prevented the E2-induced rapid increase in AKT and p38/MAPK phosphorylation, whereas it was ineffective on the E2-evoked ERK1/2 phosphorylation. The EGF-dependent ERK1/2, AKT, and p38/MAPK activation was also intact (Figs. 3B and 3B′), thus excluding a Pyr-41-dependent general impairment of the kinase cascades. Notably, no significant changes in the basal signaling kinase phosphorylation and total cellular levels were detected under Pyr-41 administration (Figs. 3B and 3B′). These data demonstrate that E1 activity is specifically involved in E2-induced ERα-mediated p38/MAPK and PI3K/AKT extranuclear activation and further suggest that E1 activity could play a role in E2-induced cell proliferation.
Colony formation assay demonstrated that Pyr-41 blocked the effect of E2 in increasing the number of the MCF-7 cell colonies (Figs. 4A and 4A′). Moreover, the E2 ability to induce a significant increase in the MCF-7 cell number was prevented when cells were treated with the hormone and Pyr-41 or the PI3K inhibitor Ly 294002 (Ly) but not in the presence of the p38/MAPK inhibitor SB 203580 (SB; Fig. 4B). Notably, similar results were obtained in another ERα-containing BC cell line (i.e., T47D-1) treated with Pyr-41, whereas both E2 and Pyr-41 did not change the number of ERα-negative BC cells (i.e., MDA-MB-231; Supporting Information Fig. S1). Accordingly, the E2-induced increase in cyclin D1 levels, a cell cycle-regulated gene, was prevented by E1 and PI3K inhibitor (i.e., Pyr-41 and Ly, respectively) but not by the p38/MAPK inhibitor (SB) in MCF-7 cells (Figs. 4C and 4D). Therefore, E1 is involved in the E2-dependent regulation of BC cell proliferation through the modulation of the ERα-mediated activation of the PI3K/AKT pathway.
Role of E1 in E2-Induced Breast Cancer Cell Migration
Besides cell proliferation, another distinctive trait of cancer is cell migration . Remarkably, E2 is a promigratory factor for BC cells , and E2-induced cell migration requires the activation of the ERα extranuclear signaling kinase cascades [1, 7, 13-20]. Thus, the role of E1 in E2-induced cell migration was evaluated. As expected , E2 induced rapid actin cytoskeleton remodeling both in MCF-7 and T47D-1 cells in a manner similar to EGF (Fig. 5A and Supporting Information Fig. S2). On the contrary, the hormone failed to trigger actin changes in MDA-MB-231 cells (Supporting Information Fig. S2). Moreover, E2 increased the motility of MCF-7 cells (Fig. 5B), as previously reported . Interestingly, these effects were prevented by the E1 inhibitor (Pyr-41), the PI3K inhibitor (Ly), or the p38/MAPK inhibitor (SB) only in MCF-7 cells (Figs. 5A and 5B and Supporting Information Fig. S2). Thus, E1 is involved in the E2-dependent regulation of BC cell migration through the modulation of the E2:ERα complex-dependent activation of both the PI3K/AKT and the p38/MAPK pathways.
In this work, we determined the role of the Ub-system on E2:ERα signaling to proliferation and migration of BC cells by using the new E1 inhibitor Pyr-41. In human cells, there is a single essential E1 enzyme that controls the entire ubiquitination cascade [3, 21] that is targeted by Pyr-41. Although we did not directly test the effect of Pyr-41 on the endogenous E1 enzymatic function in BC cells, our results confirm all the functional effects of the E1 inhibitor initially characterized in different cell lines  and strongly sustain that low doses of Pyr-41 (1 µM) inhibit the E1 activity in MCF-7 cells. Notably, Pyr-41 treatment can result in a dose-dependent accumulation of sumoylated species in cells . For these reasons, we used 1 µM of Pyr-41, which do not affect the total level of protein sumoylation . Moreover, Pyr-41 treatment of MCF-7 cells reverts the 26S proteasome inhibitor (Mg-132)-dependent accumulation of total cellular ubiquitinated species and induces the accumulation of two known ubiquitinated proteins [i.e., p53 and cyclin D1; ref. ]. Altogether, these results sustain that Pyr-41 treatment efficiently prevents E1 activity in MCF-7 cells.
Unexpectedly, we found that the inhibition of E1 activity in MCF-7 cells increases the ability of E2 to induce ERα degradation. Although this could appear as a paradoxical effect of a drug that inhibits the ubiquitination cascades but increases the degradation of a 26S proteasome-eliminated protein, these results indicate that other degradation pathways exist in the process of E2-induced receptor breakdown. In this respect, we have recently reported that E2 addresses ERα to the lysosomes for proteolytic degradation . Thus, it is possible that under the condition in which the ubiquitination cascade is inhibited, the ERα is routed to the lysosome for degradation. Whatever the case, the obtained evidence directly demonstrates that the Pyr-41-dependent inhibition of E1 affects E2-dependent ERα degradation.
In addition, we report for the first time that E1 activity is required for E2-induced proliferation and migration of ERα-positive BC cells. Indeed, the inhibition of E1 prevents MCF-7 cell proliferation by impairing the E2-induced upregulation of the cell cycle-regulating gene cyclin D1 and thus cell cycle progression [22, 23]. As a consequence, cells do not respond to the proliferative E2-dependent stimuli. On the other hand, E1 inhibitor Pyr-41 prevents E2-induced cytoskeleton remodeling and cell motility. According to the notion that E2:ERα extranuclear signaling regulates BC cell proliferation and migration [1, 7, 13-20], our data also indicate that E2-induced ERα-mediated extranuclear signaling is specifically sensitive to E1 inhibition, which does not affect the general ability of the cells to activate signaling pathways in response to extracellular stimuli (e.g., EGF). Indeed, although E1 activity is dispensable for E2-dependent ERK/MAPK activation, it is necessary for E2-induced activation of both the PI3K/AKT and p38/MAPK pathways. In many cell lines, the ERK/MAPK and PI3K/AKT pathways are activated by E2 through a mechanism that requires the E2-dependent association of the ERα with transmembrane growth factor receptors (e.g., IGF1-R) as well as with several downstream signaling proteins . On the contrary, E2-dependent p38/MAPK activation appears not to be dependent on ERα localization at the plasma membrane . We observed that in the presence of Pyr-41, E2 fails to induce AKT and p38/MAPK phosphorylation but still determines ERK1/2 activation, thus the mechanism by which the E2:ERα complex transduces the intracellular signal to the activation of ERK, AKT, and p38 could be separable. A differential recruitment of ERα extranuclear Ub-dependent binding partners could allow signaling specificity, as in the case of G-protein coupled receptors . However, although the involvement of E1 in the activation of the E2-dependent PI3K/AKT pathway is critical for the regulation of both cell proliferation and cell migration, the E1 activity is required only for the p38/MAPK pathway activation by E2, which then controls cell migration.
In conclusion, we report that E1 is involved in E2:ERα signaling to BC proliferation and migration by modulating the E2-evoked activation of the PI3K/AKT and the p38/MAPK pathways. All neoplastic diseases have distinctive traits (i.e., hallmarks), including sustained proliferative signaling and activation of cell migration and invasion . Remarkably, targeting the Ub-system has been recently recognized as a pharmacological option for cancer treatment . The data reported here strongly suggest that E1 activity is involved in two E2-dependent BC hallmarks . Thus, the current evidence indicates a novel molecular circuitry that can be explored to define new opportunities for BC treatment.
This study was supported by grants from Associazione Italiana Ricerca sul Cancro (AIRC; MFAG12756) and Ateneo Roma Tre (to F. Acconcia).