Post-Transcriptional and Epigenetic Regulation of Estrogen Signaling.

Post-translational and epigenetic regulation are important mechanisms controlling functions of genes and proteins. Although the "classic" estrogen receptors (ERs) have been acknowledged to function in mediating estrogen effects via transcriptional mechanisms, estrogenic agents modulate the turnover of several proteins via post-transcriptional and post-translational pathways including epigenetics. For instance, the metabolic and angiogenic action of G-protein coupled estrogen receptor (GPER) in vascular endothelial cells has been recently elucidated. By interacting with GPER, 17ß-estradiol and the GPER agonist G1 enhance endothelial stability of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) and capillary tube formation by increasing ubiquitin-specific peptidase 19 levels, thereby reducing PFKFB3 ubiquitination and proteasomal degradation. In addition to ligands, the functional expression and trafficking of ERs can be modulated by post-translational modification, including palmitoylation. MicroRNAs (miRNAs), the most abundant form of endogenous small RNAs in humans, regulate multiple target genes and are at the center of the multi-target regulatory network. This review also discusses the emerging evidence of how miRNAs affect glycolytic metabolism in cancer, as well as their regulation by estrogens. Restoring dysregulated miRNA expression represents a promising strategy to counteract the progression of cancer and other disease conditions. Accordingly, estrogen post-transcriptional regulatory and epigenetic mechanisms represent novel targets for pharmacological and nonpharmacological intervention for the treatment and prevention of hormone-sensitive noncommunicable diseases, including estrogen-sensitive cancers of the reproductive system in women. SIGNIFICANCE STATEMENT: The effects of estrogen are mediated by several mechanisms that are not limited to the transcriptional regulation of target genes. Slowing down the turnover of master regulators of metabolism by estrogens allows cells to rapidly adapt to environmental cues. Identification of estrogen-targeted microRNAs may lead to the development of novel RNA therapeutics that disrupt pathological angiogenesis in estrogen-dependent cancers.

Targeting of PFKFB3 with miR-206 but not mir-26b inhibits ovarian cancer cell proliferation and migration involving FAK downregulation.

Few studies explored the role of microRNAs (miRNAs) in the post-transcriptional regulation of glycolytic proteins and downstream effectors in ovarian cancer cells. We recently showed that the functional activation of the cytoskeletal regulator FAK in endothelial cells is fostered by the glycolytic enhancer 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). We tested the hypothesis that miR-206 and mir-26b, emerging onco-suppressors targeting PFKFB3 in estrogen-dependent tumors, would regulate proliferation and migration of serous epithelial ovarian cancer (EOC) cells via common glycolytic proteins, i.e., GLUT1 and PFKFB3, and downstream FAK. PFKFB3 was overexpressed in SKOV3, and its pharmacological inhibition with 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) significantly reduced cell proliferation and motility. Both miR-206 and miR-26b directly targeted PFKFB3 as evaluated by a luciferase reporter assay. However, endogenous levels of miR-26b were higher than those of miR-206, which was barely detectable in SKOV3 as well as OVCAR5 and CAOV3 cells. Accordingly, only the anti-miR-26b inhibitor concentration-dependently increased PFKFB3 levels. While miR-206 overexpression impaired proliferation and migration by downregulating PFKFB3 levels, the decreased PFKFB3 protein levels related to miR-26 overexpression had no functional consequences in all EOC cell lines. Finally, consistent with the migration outcome, exogenous miR-206 and miR-26b induced opposite effects on the levels of total FAK and of its phosphorylated form at Tyr576/577. 3PO did not prevent miR-26b-induced SKOV3 migration. Overall, these results support the inverse relation between endogenous miRNA levels and their tumor-suppressive effects and suggest that restoring miR-206 expression represents a potential dual anti-PFKFB3/FAK strategy to control ovarian cancer progression.

Leucocyte Abnormalities in Synovial Fluid of Degenerative and Inflammatory Arthropathies.

Genome damage has been related to the induction of autoimmune processes, chronic inflammation, and apoptosis. Recent studies suggest that some rheumatological diseases are associated with overall genomic instability in the T cell compartment. However, no data regarding leucocyte abnormalities in synovial fluid (SF) and their relationship with inflammation are available. The aim of this study was to investigate cellular phenotypes in SF collected from patients with different inflammatory arthropathies, including rhematoid arthritis (RA), psoriatic arthritis (PsA), crystal-induced arthritis (CIA), and non-inflammatory arthropathies, such as osteoarthritis (OA). We found high percentage of micronuclei in SF from CIA compared to the other groups and a high frequency of pyknotic cell in RA and CIA patients. A correlation between pyknosis and immature polymorphonuclear cells with local inflammatory indices was observed. The study of the apoptosis process revealed an increased BAX expression in CIA and RA compared to OA and PsA, while Bcl-2 was higher in CIA. Caspase-3 activity was increased in SF from RA patients and correlates with inflammatory and anti-inflammatory cytokines. In conclusion, our results showed that inflammatory SF is associated with genomic instability and abnormal cell subsets.

Synovial Fluid from Patients with Osteoarthritis Shows Different Inflammatory Features Depending on the Presence of Calcium Pyrophosphate Crystals.

The role of calcium pyrophosphate (CPP) crystals in osteoarthritis (OA) is still a matter of debate. With this study we aimed to investigate the inflammatory features of synovial fluid (SF) collected from patients with OA with CPP crystals compared with those without crystals. We also explored the effect of OA SF on monocytes response. SFs were collected from adult patients with OA and subdivided according to the presence of crystals. Local cellular and humoral inflammatory mediators were analysed in the SF samples. The expression levels of IL-1ß, IL-18, CASP-1, NLRP3, and GAPDH were measured by RT-PCR in the cells obtained by pelleting the SF samples. For the in vitro study, a monocytic cell line was treated with selected SF samples. SF with CPP crystals showed a significant increase in inflammatory cellular indices and higher levels of IL-1ß, IL-8, and caspase-1 transcript with respect to SF without crystals. Higher concentrations of VEGF were also observed in the early stages of the whole OA patients. THP-1 cells stimulated with OA SF released a significant amount of IL-1 ß in culture supernatants. This study demonstrated that SF collected from patients with OA shows different inflammatory features depending on the presence of CPP crystals.

Non-genomic mechanisms in the estrogen regulation of glycolytic protein levels in endothelial cells.

Few studies have explored the mechanisms coupling estrogen signals to metabolic demand in endothelial cells. We recently showed that 17ß-estradiol (E2) triggers angiogenesis via the membrane G-protein coupled estrogen receptor (GPER) and the key glycolytic protein PFKFB3 as a downstream effector. We herein investigated whether estrogenic agents regulate the stability and/or degradation of glycolytic proteins in human umbilical vein endothelial cells (HUVECs). Similarly to E2, the GPER selective agonist G1 rapidly increased PFKFB3 protein amounts, without affecting mRNA levels. In the presence of cycloheximide, E2 and G1 treatment counteracted PFKFB3 degradation over time, whereas E2-induced PFKFB3 stabilization was abolished by the GPER antagonist G15. Inhibitors of selective SCF E3 ubiquitin ligase (SMER-3) and proteasome (MG132) rapidly increased PFKFB3 protein levels. Accordingly, ubiquitin-bound PFKFB3 was lower in E2- or G1-treated HUVECs. Both agents increased deubiquitinase USP19 levels through GPER signaling. Notably, USP 19 siRNA decreased PFKFB3 levels and abolished E2- and G1-mediated HUVEC tubularization. Finally, E2 and G1 treatments rapidly enhanced glucose transporter GLUT1 levels via GPER independent of transcriptional activation. These findings provide new evidence on mechanisms coupling estrogen signals with the glycolytic program in endothelium and unravel the role of USP19 as a target of the pro-angiogenic effect of estrogenic agents.

Estrogen Receptor Functions and Pathways at the Vascular Immune Interface.

Estrogen receptor (ER) activity mediates multiple physiological processes in the cardiovascular system. ERα and ERß are ligand-activated transcription factors of the nuclear hormone receptor superfamily, while the G protein-coupled estrogen receptor (GPER) mediates estrogenic signals by modulating non-nuclear second messengers, including activation of the MAP kinase signaling cascade. Membrane localizations of ERs are generally associated with rapid, non-genomic effects while nuclear localizations are associated with nuclear activities/transcriptional modulation of target genes. Gender dependence of endothelial biology, either through the action of sex hormones or sex chromosome-related factors, is becoming increasingly evident. Accordingly, cardiometabolic risk increases as women transition to menopause. Estrogen pathways control angiogenesis progression through complex mechanisms. The classic ERs have been acknowledged to function in mediating estrogen effects on glucose metabolism, but 17ß-estradiol also rapidly promotes endothelial glycolysis by increasing glucose transporter 1 (GLUT1) and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) levels through GPER-dependent mechanisms. Estrogens alter monocyte and macrophage phenotype(s), and induce effects on other estrogen-responsive cell lineages (e.g., secretion of cytokines/chemokines/growth factors) that impact macrophage function. The pharmacological modulation of ERs for therapeutic purposes, however, is particularly challenging due to the lack of ER subtype selectivity of currently used agents. Identifying the determinants of biological responses to estrogenic agents at the vascular immune interface and developing targeted pharmacological interventions may result in novel improved therapeutic solutions.

Bisdemethoxycurcumin and Its Cyclized Pyrazole Analogue Differentially Disrupt Lipopolysaccharide Signalling in Human Monocyte-Derived Macrophages.

Several studies suggest that curcumin and related compounds possess antioxidant and anti-inflammatory properties including modulation of lipopolysaccharide- (LPS-) mediated signalling in macrophage cell models. We here investigated the effects of curcumin and the two structurally unrelated analogues GG6 and GG9 in primary human blood-derived macrophages as well as the signalling pathways involved. Macrophages differentiated from peripheral blood monocytes for 7 days were activated with LPS or selective Toll-like receptor agonists for 24 h. The effects of test compounds on cytokine production and immunophenotypes evaluated as CD80+/CCR2+ and CD206+/CD163+ subsets were examined by ELISA and flow cytometry. Signalling pathways were probed by Western blot. Curcumin (2.5-10 µM) failed to suppress LPS-induced inflammatory responses. While GG6 reduced LPS-induced IκB-α degradation and showed a trend towards reduced interleukin-1ß release, GG9 prevented the increase in proinflammatory CD80+ macrophage subset, downregulation of the anti-inflammatory CD206+/CD163+ subset, increase in p38 phosphorylation, and increase in cell-bound and secreted interleukin-1ß stimulated by LPS, at least in part through signalling pathways not involving Toll-like receptor 4 and nuclear factor-κB. Thus, the curcumin analogue GG9 attenuated the LPS-induced inflammatory response in human blood-derived macrophages and may therefore represent an attractive chemical template for macrophage pharmacological targeting.

Estrogen, Angiogenesis, Immunity and Cell Metabolism: Solving the Puzzle.

Estrogen plays an important role in the regulation of cardiovascular physiology and the immune system by inducing direct effects on multiple cell types including immune and vascular cells. Sex steroid hormones are implicated in cardiovascular protection, including endothelial healing in case of arterial injury and collateral vessel formation in ischemic tissue. Estrogen can exert potent modulation effects at all levels of the innate and adaptive immune systems. Their action is mediated by interaction with classical estrogen receptors (ERs), ERα and ERß, as well as the more recently identified G-protein coupled receptor 30/G-protein estrogen receptor 1 (GPER1), via both genomic and non-genomic mechanisms. Emerging data from the literature suggest that estrogen deficiency in menopause is associated with an increased potential for an unresolved inflammatory status. In this review, we provide an overview through the puzzle pieces of how 17ß-estradiol can influence the cardiovascular and immune systems.

The Glycolytic Enzyme PFKFB3 Is Involved in Estrogen-Mediated Angiogenesis via GPER1.

The endogenous estrogen 17ß-estradiol (E2) is a key factor in promoting endothelial healing and angiogenesis. Recently, proangiogenic signals including vascular endothelial growth factor and others have been shown to converge in endothelial cell metabolism. Because inhibition of the glycolytic enzyme activator phosphofructokinase-2/fructose-2,6-bisphosphatase 3 (PFKFB3) reduces pathologic angiogenesis and estrogen receptor (ER) signaling stimulates glucose uptake and glycolysis by inducing PFKFB3 in breast cancer, we hypothesized that E2 triggers angiogenesis in endothelial cells via rapid ER signaling that requires PFKFB3 as a downstream effector. We report that treatment with the selective G protein-coupled estrogen receptor (GPER1) agonist G-1 (10-10 to 10-7 M) mimicked the chemotactic and proangiogenic effect of E2 as measured in a number of short-term angiogenesis assays in human umbilical vein endothelial cells (HUVECs); in addition, E2 treatment upregulated PFKFB3 expression in a time- and concentration-dependent manner. Such an effect peaked at 3 hours and was also induced by G-1 and abolished by pretreatment with the GPER1 antagonist G-15 or GPER1 siRNA, consistent with engagement of membrane ER. Experiments with the PFKFB3 inhibitor 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one showed that PFKFB3 activity was required for estrogen-mediated HUVEC migration via GPER1. In conclusion, E2-induced angiogenesis was mediated at least in part by the membrane GPER1 and required upregulation of the glycolytic activator PFKFB3 in HUVECs. These findings unravel a previously unrecognized mechanism of estrogen-dependent endocrine-metabolic crosstalk in HUVECs and may have implications in angiogenesis occurring in ischemic or hypoxic tissues.

The antiangiogenic effect of digitoxin is dependent on a ROS-elicited RhoA/ROCK pathway activation.

We previously showed that digitoxin inhibits angiogenesis and cancer cell proliferation and migration and these effects were associated to protein tyrosine kinase 2 (FAK) inhibition. Considering the interactions between FAK and Rho GTPases regulating cell cytoskeleton and movement, we investigated the involvement of RhoA and Rac1 in the antiangiogenic effect of digitoxin. Phalloidin staining of human umbilical vein endothelial cells (HUVECs) showed the formation of stress fibers in cells treated with 10 nM digitoxin. By Rhotekin- and Pak1- pull down assays, detecting the GTP-bound form of GTPases, we observed that digitoxin (10-25 nM) induced sustained (0.5-6 h) RhoA activation with no effect on Rac1. Furthermore, inhibition of HUVEC migration and capillary-like tube formation by digitoxin was counteracted by hindering RhoA-ROCK axis with RhoA silencing or Y-27632 treatment. Digitoxin did not decrease p190RhoGAP phosphorylation at Tyr1105 (a site targeted by FAK), suggesting that RhoA activation was independent from FAK inhibition. Because increasing evidence points to a redox regulation of RhoA, we measured intracellular ROS and found that digitoxin treatment enhanced ROS levels in a concentration-dependent manner (1-25 nM). Notably, the flavoprotein inhibitor DPI or the pan-NADPH oxidase (NOX) inhibitor VAS-2870 antagonized both ROS increase and RhoA activation by digitoxin. Our results provide evidence that inhibition of HUVEC migration and tube formation by digitoxin is dependent on ROS production by endothelial NOX, which leads to the activation of RhoA/ROCK pathway. Digitoxin effects on proteins regulating cytoskeletal organization and cell motility could have a wider impact on cancer progression, beyond the antiangiogenic activity.

MiR-206 inhibits estrogen signaling and ovarian cancer cell migration without affecting GPER.

AIMS: Estrogen-regulated pathways are involved in the etiology and progression of epithelial ovarian cancer (EOC), but the relative contribution of estrogen receptor isoforms is unclear. Only a subset of patients responds to antiestrogens including tamoxifen. Based on our previous evidence that miR-206 behaves as an oncosuppressor in EOC, we hypothesized that miR-206 would interfere with G protein-coupled estrogen receptor (GPER)-mediated signaling and cell motility. MAIN METHODS: PFKFB3 and FAK proteins from OC cells challenged with selective estrogen receptor agonist and antagonist were measured by Western blotting. Cell proliferation and motility were analyzed by MTT and Boyden chamber, respectively. Estrogen-dependent cells were transfected with miR-206 mimic or control using Lipofectamine. KEY FINDINGS: The migration of SKOV3 and OVCAR5 cells significantly increased following treatment with 17ß-estradiol (E2) and the selective GPER agonist G1. However, tamoxifen failed to inhibit E2 effect and even promoted SKOV3 cell migration. Estrogen receptor ligands did not affect SKOV3 proliferation. The GPER antagonist G15 significantly prevented E2-mediated upregulation of PFKFB3 expression, while G1 concentration-dependently upregulated PFKFB3 levels. Consistent with the functional link between PFKFB3 and FAK activation, E2 and G1 increased FAK phosphorylation at Tyr397. Transfection with miR-206 abolished estrogen-induced EOC migration and down-regulated PFKFB3 protein levels. Notably, miR-206 transfection reduced ERα protein abundance, whereas GPER amount was unchanged. SIGNIFICANCE: By blocking estrogen signaling and G1-induced EOC cell invasiveness with no direct interference with GPER levels, miR-206 mimics have the potential to act as pathway-selective antagonists and deserve further testing as RNA therapeutics in estrogen-dependent EOC.

Loss of Hematopoietic Cell-Derived Oncostatin M Worsens Diet-Induced Dysmetabolism in Mice.

Innate immune cells infiltrate growing adipose tissue and propagate inflammatory clues to metabolically distant tissues, thereby promoting glucose intolerance and insulin resistance. Cytokines of the IL-6 family and gp130 ligands are among such signals. The role played by oncostatin M (OSM) in the metabolic consequences of overfeeding is debated, at least in part, because prior studies did not distinguish OSM sources and dynamics. Here, we explored the role of OSM in metabolic responses and used bone marrow transplantation to test the hypothesis that hematopoietic cells are major contributors to the metabolic effects of OSM. We show that OSM is required to adapt during the development of obesity because OSM concentrations are dynamically modulated during high-fat diet (HFD) and Osm-/- mice displayed early-onset glucose intolerance, impaired muscle glucose uptake, and worsened liver inflammation and damage. We found that OSM is mostly produced by blood cells and deletion of OSM in hematopoietic cells phenocopied glucose intolerance of whole-body Osm-/- mice fed a HFD and recapitulated liver damage with increased aminotransferase levels. We thus uncovered that modulation of OSM is involved in the metabolic response to overfeeding and that hematopoietic cell-derived OSM can regulate metabolism, likely via multiple effects in different tissues.

Gender differences and pharmacological regulation of angiogenesis induced by synovial fluids in inflammatory arthritis.

Several mediators including cytokines, growth factors and metalloproteinases (MMP) modulate pathological angiogenesis associated with inflammatory arthritis. The biological factors underlying sex disparities in the incidence and severity of rheumatic musculoskeletal diseases are only partially understood. We hypothesized that synovial fluids (SFs) from rheumatoid arthritis (RA) and psoriatic arthritis (PsA) patients would impact on endothelial biology in a sexually dimorphic fashion. Immune cell counts and levels of pro-angiogenic cytokines found in SFs from RA and PsA patients (n = 17) were higher than in osteoarthritis patients (n = 6). Synovial VEGF concentration was significantly higher in male than in female RA patients. Zymography revealed that SFs comprised solely MMP-9 and MMP-2, with significantly higher MMP-9 levels in male than female RA patients. Using in vitro approaches that mimic the major steps of the angiogenic process, SFs from RA and PsA patients induced endothelial migration and formation of capillary-like structures compared to control. Notably, endothelial cells from female donors displayed enhanced angiogenic response to SFs with respect to males. Treatment with the established anti-angiogenic agent digitoxin prevented activation of focal adhesion kinase and SF-induced in vitro angiogenesis. Thus, despite higher synovial VEGF and MMP-9 levels in male patients, the responsiveness of vascular endothelium to SF priming was higher in females, suggesting that gender differences in angiogenic responses were mainly related to the endothelial genotype. These findings may have implications for pathogenesis and targeted therapies of inflammatory arthritis.

Clinical efficacy and safety of angiogenesis inhibitors: sex differences and current challenges.

Vasoactive molecules, such as vascular endothelial growth factor (VEGF) and endothelins, share cytokine-like activities and regulate endothelial cell (EC) growth, migration, and inflammation. Some endothelial mediators and their receptors are targets for currently approved angiogenesis inhibitors, drugs that are either monoclonal antibodies raised towards VEGF, or inhibitors of vascular receptor protein kinases and signalling pathways. Pharmacological interference with the protective functions of ECs results in a similar spectrum of adverse effects. Clinically, the most common side effects of VEGF signalling pathway inhibition include an increase in arterial pressure, left ventricular dysfunction facilitating the development of heart failure, thromboembolic events including pulmonary embolism and stroke, and myocardial infarction. Sex steroids, such as androgens, progestins, and oestrogens and their receptors (ERα, ERß, GPER; PR-A, PR-B; AR) have been identified as important modifiers of angiogenesis, and sex differences have been reported for anti-angiogenic drugs. This review article discusses the current challenges clinicians are facing with regard to angiogenesis inhibitor therapy, including the need to consider sex differences affecting clinical efficacy and safety. We also propose areas for future research taking into account the role of sex hormone receptors and sex chromosomes. Development of new sex-specific drugs with improved target- and cell-type selectivity likely will open the way to personalized medicine in men and women requiring anti-angiogenic therapy to reduce adverse effects and to improve therapeutic efficacy.

Sex Differences in the Pro-Angiogenic Response of Human Endothelial Cells: Focus on PFKFB3 and FAK Activation.

Female hormones and sex-specific factors are established determinants of endothelial function, yet their relative contribution to human endothelium phenotypes has not been defined. Using human umbilical vein endothelial cells (HUVECs) genotyped by donor's sex, we investigated the influence of sex and estrogenic agents on the main steps of the angiogenic process and on key proteins governing HUVEC metabolism and migratory properties. HUVECs from female donors (fHUVECs) showed increased viability (p < 0.01) and growth rate (p < 0.01) compared with those from males (mHUVECs). Despite higher levels of G-protein coupled estrogen receptor (GPER) in fHUVECs (p < 0.001), treatment with 17ß-estradiol (E2) and the selective GPER agonist G1 (both 1-100 nM) did not affect HUVEC viability. Migration and tubularization in vitro under physiological conditions were higher in fHUVECs than in mHUVECs (p < 0.05). E2 treatment (1-100 nM) upregulated the glycolytic activator PFKFB3 with higher potency in fHUVECs than in mHUVECs, despite comparable baseline levels. Moreover, Y576/577 phosphorylation of focal adhesion kinase (FAK) was markedly enhanced in fHUVECs (p < 0.001), despite comparable Src activation levels. While the PI3K inhibitor LY294002 (25 µM) inhibited HUVEC migration (p < 0.05), Akt phosphorylation levels in fHUVECs and mHUVECs were comparable. Finally, digitoxin treatment, which inhibits Y576/577 FAK phosphorylation, abolished sexual dimorphism in HUVEC migration. These findings unravel complementary modulation of HUVEC functional phenotypes and signaling molecules involved in angiogenesis by hormone microenvironment and sex-specific factors, and highlight the need for sex-oriented pharmacological targeting of endothelial function.

Effects of digitoxin on cell migration in ovarian cancer inflammatory microenvironment.

Clinical and experimental evidence supports a role for cardiac glycosides (CGs) as potential novel anticancer drugs. However, there are no studies reporting the effect of CGs on the inflammatory tumor microenvironment (TME), which plays a central role in tumor progression and invasiveness. We investigated whether digitoxin affects a) specific pathways involved in motility and/or activation of different cell types shaping TME, and b) cancer cell growth and invasiveness in response to TME-associated factors. To test our hypothesis, conditioned media (CM) from polarized macrophages, and apoptotic or non-apoptotic ovarian cancer cells (SKOV3) were tested as chemoattractants for endothelial cells, monocytes and cancer cells. We demonstrated that CM from M1 (LPS/IFNγ) and M2 (IL-4/IL-13) polarized macrophages, which mimic inflammatory TME, increased both HUVEC migration and tubularization. Treatment of HUVECs with digitoxin at concentrations within its plasma therapeutic range counteracted these effects. Digitoxin affected the expression of neither M1 (CD80/CD68) nor M2 (CD206/CD163) activation markers, nor the amount of cell-bound IL-1ß and CCL22. Accordingly, HUVEC migration in response to CM from digitoxin-treated activated macrophages was unchanged. These data point to a direct effect of digitoxin on HUVEC signaling rather than on the modulation of the cytokine profile released from activated macrophages. At variance with what observed for HUVECs, digitoxin did not prevent monocyte migration induced by SKOV3 CM. In addition, digitoxin significantly impaired SKOV3 growth and migration in response to M1 or M2 macrophage CM. Finally, we showed that digitoxin inhibited FAK phosphorylation in SKOV3 but not PYK2 phosphorylation in monocytes, thus providing a molecular mechanism accounting for the observed differential anti-migratory effect. Overall, digitoxin counteracted salient features of the inflammatory ovarian cancer microenvironment, laying the ground for potential digitoxin repositioning as an anticancer drug.