Tamoxifen resistance is a major clinical challenge in breast cancer treatment. Aromatase inhibitors are effective in women who progressed or recurred on tamoxifen, suggesting a role of local estrogen ...production by aromatase in driving tamoxifen-resistant phenotype. However, the link between aromatase activity and tamoxifen resistance has not yet been reported. We investigated whether long-term tamoxifen exposure may affect aromatase activity and/or expression, which may then sustain tamoxifen-resistant breast cancer cell growth. We employed MCF-7 breast cancer cells, tamoxifen-resistant MCF-7 cells (MCF-7 TR1 and TR2), SKBR-3 breast cancer cells, cancer-associated fibroblasts (CAFs1 and CAFs2). We used tritiated-water release assay, realtime-RT-PCR, and immunoblotting analysis for evaluating aromatase activity and expression; anchorage-independent assays for growth; reporter-gene, electrophoretic-mobility-shift, and chromatin-immunoprecipitation assays for promoter activity studies. We demonstrated an increased aromatase activity and expression, which supports proliferation in tamoxifen-resistant breast cancer cells. This is mediated by the G-protein-coupled receptor GPR30/GPER, since knocking-down GPER expression or treatment with a GPER antagonist reversed the enhanced aromatase levels induced by long-term tamoxifen exposure. The molecular mechanism was investigated in ER-negative, GPER/aromatase-positive SKBR3 cells, in which tamoxifen acts as a GPER agonist. Tamoxifen treatment increased aromatase promoter activity through an enhanced recruitment of c-fos/c-jun complex to AP-1 responsive elements located within the promoter region. As tamoxifen via GPER induced aromatase expression also in CAFs, this pathway may be involved in promoting aggressive behavior of breast tumors in response to tamoxifen treatment. Blocking estrogen production and/or GPER signaling activation may represent a valid option to overcome tamoxifen-resistance in breast cancers.
Breast cancer is a complex and heterogeneous disease, with distinct histologic features dictating the therapy. Although the clinical outcome of breast cancer patients has been considerably improved, ...the occurrence of resistance to common endocrine and chemotherapy treatments remains the major cause of relapse and mortality. Thus, efforts in identifying new molecules to be employed in breast cancer therapy are needed. As a "faster" alternative to reach this aim, we evaluated whether lamotrigine, a broadly used anticonvulsant, could be "repurposed" as an antitumoral drug in breast cancer. Our data show that lamotrigine inhibits the proliferation, the anchorage-dependent, and independent cell growth in breast cancer cells (BCC), including hormone-resistant cell models. These effects were associated with cell-cycle arrest and modulation of related proteins (cyclin D1, cyclin E, p27
, and p21
), all target genes of FoxO3a, an ubiquitous transcription factor negatively regulated by AKT. Lamotrigine also increases the expression of another FoxO3a target, PTEN, which, in turn, downregulates the PI3K/Akt signaling pathway, with consequent dephosphorylation, thus activation, of FoxO3a. Moreover, lamotrigine induces FoxO3a expression by increasing its transcription through FoxO3a recruitment on specific FHRE located on its own promoter, in an autoregulatory fashion. Finally, lamotrigine significantly reduced tumor growth
, increasing FoxO3a expression.
The anticonvulsant drug lamotrigine shows strong antiproliferative activity on breast cancer, both
and
Thus, drug repurposing could represent a valuable option for a molecularly targeted therapy in breast cancer patients.
.
Breast cancer stem cells (BCSCs) play crucial roles in tumor initiation, metastasis and therapeutic resistance. A strict dependency between BCSCs and stromal cell components of tumor microenvironment ...exists. Thus, novel therapeutic strategies aimed to target the crosstalk between activated microenvironment and BCSCs have the potential to improve clinical outcome. Here, we investigated how leptin, as a mediator of tumor-stromal interactions, may affect BCSC activity using patient-derived samples (n = 16) and breast cancer cell lines, and determined the potential benefit of targeting leptin signaling in these model systems. Conditioned media (CM) from cancer-associated fibroblasts and breast adipocytes significantly increased mammosphere formation in breast cancer cells and depletion of leptin from CM completely abrogated this effect. Mammosphere cultures exhibited increased leptin receptor (OBR) expression and leptin exposure enhanced mammosphere formation. Microarray analyses revealed a similar expression profile of genes involved in stem cell biology among mammospheres treated with CM and leptin. Interestingly, leptin increased mammosphere formation in metastatic breast cancers and expression of OBR as well as HSP90, a target of leptin signaling, were directly correlated with mammosphere formation in metastatic samples (r = 0.68/p = 0.05; r = 0.71/p = 0.036, respectively). Kaplan-Meier survival curves indicated that OBR and HSP90 expression were associated with reduced overall survival in breast cancer patients (HR = 1.9/p = 0.022; HR = 2.2/p = 0.00017, respectively). Furthermore, blocking leptin signaling by using a full leptin receptor antagonist significantly reduced mammosphere formation in breast cancer cell lines and patient-derived samples. Our results suggest that leptin/leptin receptor signaling may represent a potential therapeutic target that can block the stromal-tumor interactions driving BCSC-mediated disease progression.
Resistance to endocrine therapy is still a major clinical challenge in the management of estrogen receptor α-positive (ERα+) breast cancer (BC). Here, the role of the Forkhead box class O (FoxO)3a ...transcription factor in tumor progression has been evaluated in tamoxifen-resistant BC cells (TamR), expressing lower levels of FoxO3a compared to sensitive ones. FoxO3a re-expression reduces TamR motility (wound-healing and transmigration assays) and invasiveness (matrigel transwell invasion assays) through the mRNA (qRT-PCR) and protein (Western blot) induction of the integrin α5 subunit of the α5β1 fibronectin receptor, a well-known membrane heterodimer controlling cell adhesion and signaling. The induction occurs through FoxO3a binding to a specific Forkhead responsive core sequence located on the integrin α5 promoter (cloning, luciferase, and ChIP assays). Moreover, FoxO3a failed to inhibit migration and invasion in integrin α5 silenced (siRNA) cells, demonstrating integrin α5 involvement in both processes. Finally, using large-scale gene expression data sets, a strong positive correlation between FoxO3a and integrin α5 in ERα+, but not in ER-negative (ERα-), BC patients emerged. Altogether, our data show how the oncosuppressor FoxO3a, by increasing the expression of its novel transcriptional target integrin α5, reverts the phenotype of endocrine-resistant BC toward a lower aggressiveness.
Leptin, a product of adipocytes, is involved in the regulation of body weight and results strongly correlated to body fat content. An excess of fat mass represents a breast cancer risk factor ...particularly in postmenopausal women, where estrogen production by adipose tissue through its own aromatase activity stimulates tumor progression. Leptin stimulates estrogen production through the increase of aromatase expression and activity in human luteinized granulosa cells and adipose stromal cells. In the present study, we have examined the possible link that exists between leptin and breast cancer, focusing our attention on the direct effect of leptin on aromatase activity, which may enhance estrogen production and induce tumor cell growth stimulation. We have shown that leptin enhances aromatase mRNA expression, aromatase content, and its enzymatic activity in MCF-7. Aromatase expression appears to be regulated by tissue-specific promoter. It has been demonstrated that promoters II and 1.3 are the major promoters that drive aromatase expression in MCF-7. Transient transfection experiments using vector containing human aromatase promoters II and 1.3 sequence fused with luciferase reporter gene demonstrated that leptin is able to activate this promoter. In the presence of either mitogen-activated protein kinase inhibitor PD 98059 or ERK2 dominant negative as well as in the presence of STAT3 dominant negative, the stimulatory effects of leptin on aromatase promoter, enzymatic activity, and aromatase protein content were inhibited. Functional studies of mutagenesis and electrophoretic mobility shift assay revealed that the AP-1 motif is important in determining the up-regulatory effects induced by leptin on aromatase expression in MCF-7.
Natural products and herbal therapies represent a thriving field of research, but methods for the production of plant-derived compounds with a significative biological activity by synthetic methods ...are required. Conventional commercial production by chemical synthesis or solvent extraction is not yet sustainable and economical because toxic solvents are used, the process involves many steps, and there is generally a low amount of the product produced, which is often mixed with other or similar by-products. For this reason, alternative, sustainable, greener, and more efficient processes are required. Membrane processes are recognized worldwide as green technologies since they promote waste minimization, material diversity, efficient separation, energy saving, process intensification, and integration. This article describes the production, characterization, and utilization of bioactive compounds derived from renewable waste material (olive leaves) as drug candidates in breast cancer (BC) treatment. In particular, an integrated membrane process composed by a membrane bioreactor (MBR) and a membrane emulsification (ME) system was developed to produce a purified non-commercially available phytotherapic compound: the oleuropein aglycone (OLA). This method achieves a 93% conversion of the substrate (oleuropein) and enables the extraction of the compound of interest with 90% efficiency in sustainable conditions. The bioderived compound exercised pro-apoptotic and antiproliferative activities against MDA-MB-231 and Tamoxifen-resistant MCF-7 (MCF-7/TR) cells, suggesting it as a potential agent for the treatment of breast cancer including hormonal resistance therapies.
Tamoxifen-resistant breast cancer cells (TamR-BCCs) are characterized by an enhanced metabolic phenotype compared to tamoxifen-sensitive cells. FoxO3a is an important modulator of cell metabolism, ...and its deregulation has been involved in the acquisition of tamoxifen resistance. Therefore, tetracycline-inducible FoxO3a was overexpressed in TamR-BCCs (TamR/TetOn-AAA), which, together with their control cell line (TamR/TetOn-V), were subjected to seahorse metabolic assays and proteomic analysis. FoxO3a was able to counteract the increased oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) observed in TamR by reducing their energetic activity and glycolytic rate. FoxO3a caused glucose accumulation, very likely by reducing LDH activity and mitigated TamR biosynthetic needs by reducing G6PDH activity and hindering NADPH production via the pentose phosphate pathway (PPP). Proteomic analysis revealed a FoxO3a-dependent marked decrease in the expression of LDH as well as of several enzymes involved in carbohydrate metabolism (e.g., Aldolase A, LDHA and phosphofructokinase) and the analysis of cBioPortal datasets of BC patients evidenced a significant inverse correlation of these proteins and FoxO3a. Interestingly, FoxO3a also increased mitochondrial biogenesis despite reducing mitochondrial functionality by triggering ROS production. Based on these findings, FoxO3a inducing/activating drugs could represent promising tools to be exploited in the management of patients who are refractory to antiestrogen therapy.
This paper explores the inequality related to the lifetime redistributive impact of public sector intervention across living generations. While fiscal policies are typically assessed with respect to ...the inter-personal dimension of inequality, they may well have heterogeneous impacts across different cohorts, thus raising the issue of intergenerational fairness. We take the case of Italy over the period 1990-2008 to show how such possibly unfair effects can be measured by adapting the methods developed in the context of the generational accounting literature. Importantly, we aim to work on a comprehensive approach, so that reforms involving several taxation and spending programmes, possibly implemented over several years, can be assessed. We find that a significant improvement in public finance sustainability came at the price of an unequal distribution of sacrifices across living generations, with younger cohorts contributing far more over their residual life-cycle.
Resistance to endocrine therapy is still a major clinical challenge in the management of estrogen receptor positive (ER+) breast cancer (BC) patients, whose poor outcome demands additional studies. ...Forkhead box class O (FoxO)3a transcription factor, a bona fide oncosuppressor, has been involved in BC metastasis as well as in antiestrogen resistance. Here we demonstrate that the α5 subunit of the integrin α5β1, a well‐known membrane heterodimer controlling cell surface adhesion and signaling, is a novel FoxO3a transcriptional target. FoxO3a re‐expression reduces motility (wound healing and transmigration assays) of different Tamoxifen resistant BC cell lines, through the induction of α5 mRNA (qRT‐PCR) and protein (Western blot) levels. FoxO3a transcriptionally regulates α5 expression by binding to specific Forkhead responsive elements located on the α5 promoter (Luciferase and ChIP assays). Accordingly, using a large‐scale BC gene expression datasets from The Cancer Genome Atlas (TCGA) database, a strong positive correlation between FoxO3a and α5 in ER+ BC patients emerged. Altogether, our data unveil an additional mechanism through which FoxO3a activation/induction, by increasing α5 expression, restores a less aggressive phenotype in BC refractory to endocrine therapy.
ABSTRACT
Obesity is a risk factor for breast cancer, largely due to altered expression of various adipocytokines. As it concerns adiponectin, there are not univocal results regarding its role in ...breast cancer occurrence and progression. Here, we demonstrate that in animals injected with human estrogen receptor (ER)‐α‐negative MDA‐MB‐231 cells pre treated with adiponectin (1 and 5 μg/ml), a significant reduction (60 and 40%, respectively) in tumor volume is observed, whereas an increased tumor growth (54 and 109%, respectively) is evidenced in the animals receiving human ER‐α‐positive MCF‐7 cells. Moreover, cyclin D1 (CD1) mRNA and protein levels are decreased in MDA‐MB‐231 cells, whereas they are up‐regulated in ER‐α‐positive cells by adiponectin. These findings fit with the opposite effects of adiponectin on CD1 promoter: 0.44‐ and 0.34‐fold decrease in MDA‐MB‐231 cells and 0.63‐ and 0.95‐fold increase in MCF‐7 cells, treated with 1 and 5 μg/ml, respectively. Functional studies indicate that these effects are mediated by the specific protein 1 motif located in the CD1 promoter. In the absence of ER‐α, the adiponectin‐mediated down‐regulation of CD1 involves the recruitment of corepressors. In the presence of ER‐α, the adiponectin‐induced expression of CD1 requires the involvement of an activator complex. In conclusion, we propose that a possible mechanism through which adiponectin differently affects breast cancer growth is the opposite modulation of CD1 levels accordingly to ER‐α expression.—Mauro, L., Pellegrino, M., Giordano, F., Ricchio, E., Rizza, P., De Amicis, F., Catalano, S., Bonofiglio, D., Panno, M. L., Andò, S. Estrogen receptor‐α drives adiponectin effects on cyclin D1 expression in breast cancer cells. FASEB J. 29, 2150‐2160 (2015). www.fasebj.org
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