Mitochondrial antiviral signaling protein enhances MASLD progression through the ERK/TNFα/NFκß pathway.

BACKGROUND AND AIMS: Mitochondrial antiviral signaling protein (MAVS) is a critical regulator that activates the host's innate immunity against RNA viruses, and its signaling pathway has been linked to the secretion of proinflammatory cytokines. However, the actions of MAVS on inflammatory pathways during the development of metabolic dysfunction-associated steatotic liver disease (MASLD) have been little studied. APPROACH AND RESULTS: Liver proteomic analysis of mice with genetically manipulated hepatic p63, a transcription factor that induces liver steatosis, revealed MAVS as a target downstream of p63. MAVS was thus further evaluated in liver samples from patients and in animal models with MASLD. Genetic inhibition of MAVS was performed in hepatocyte cell lines, primary hepatocytes, spheroids, and mice. MAVS expression is induced in the liver of both animal models and people with MASLD as compared with those without liver disease. Using genetic knockdown of MAVS in adult mice ameliorates diet-induced MASLD. In vitro, silencing MAVS blunts oleic and palmitic acid-induced lipid content, while its overexpression increases the lipid load in hepatocytes. Inhibiting hepatic MAVS reduces circulating levels of the proinflammatory cytokine TNFα and the hepatic expression of both TNFα and NFκß. Moreover, the inhibition of ERK abolished the activation of TNFα induced by MAVS. The posttranslational modification O -GlcNAcylation of MAVS is required to activate inflammation and to promote the high lipid content in hepatocytes. CONCLUSIONS: MAVS is involved in the development of steatosis, and its inhibition in previously damaged hepatocytes can ameliorate MASLD.

p63 controls metabolic activation of hepatic stellate cells and fibrosis via an HER2-ACC1 pathway.

The p63 protein has pleiotropic functions and, in the liver, participates in the progression of nonalcoholic fatty liver disease (NAFLD). However, its functions in hepatic stellate cells (HSCs) have not yet been explored. TAp63 is induced in HSCs from animal models and patients with liver fibrosis and its levels positively correlate with NAFLD activity score and fibrosis stage. In mice, genetic depletion of TAp63 in HSCs reduces the diet-induced liver fibrosis. In vitro silencing of p63 blunts TGF-ß1-induced HSCs activation by reducing mitochondrial respiration and glycolysis, as well as decreasing acetyl CoA carboxylase 1 (ACC1). Ectopic expression of TAp63 induces the activation of HSCs and increases the expression and activity of ACC1 by promoting the transcriptional activity of HER2. Genetic inhibition of both HER2 and ACC1 blunt TAp63-induced activation of HSCs. Thus, TAp63 induces HSC activation by stimulating the HER2-ACC1 axis and participates in the development of liver fibrosis.

Further Studies on the Highly Active Des-C-Ring and Aromatic-D-Ring Analogues of 1α,25-Dihydroxyvitamin D3 (Calcitriol): Refinement of the Side Chain.

Current efforts in the vitamin D field are directed toward the development of highly antiproliferative yet noncalcemic analogues of the natural hormone 1α,25-dihydroxyvitamin D3 (1,25D3). We have recently reported the design, synthesis, biological evaluation, and crystal structures of a series of novel analogues that both lack the steroidal C-ring and have an m-phenylene ring replacing the steroidal cyclopentane D-ring. We have now investigated the potentiating effects of incorporating selected modifications (hexafluorination and/or an internal triple bond) within the steroidal side chain in our series. An alternative synthetic strategy (Wittig-Horner approach instead of our previously used Pd-catalyzed tandem cyclization/cross-coupling) for the construction of the vitamin D triene system was found convenient for the target compounds 2, 3a, 3b, and 3c of this report. These modifications enhance vitamin D nuclear receptor (VDR) interactions and consequently VDR-associated biological properties compared to parental PG-136 compound while maintaining normal calcium levels.

Hepatic p63 regulates glucose metabolism by repressing SIRT1.

OBJECTIVE: p63 is a transcription factor within the p53 protein family that has key roles in development, differentiation and prevention of senescence, but its metabolic actions remain largely unknown. Herein, we investigated the physiological role of p63 in glucose metabolism. DESIGN: We used cell lines and mouse models to genetically manipulate p63 in hepatocytes. We also measured p63 in the liver of patients with obesity with or without type 2 diabetes (T2D). RESULTS: We show that hepatic p63 expression is reduced on fasting. Mice lacking the specific isoform TAp63 in the liver (p63LKO) display higher postprandial and pyruvate-induced glucose excursions. These mice have elevated SIRT1 levels, while SIRT1 knockdown in p63LKO mice normalises glycaemia. Overexpression of TAp63 in wild-type mice reduces postprandial, pyruvate-induced blood glucose and SIRT1 levels. Studies carried out in hepatocyte cell lines show that TAp63 regulates SIRT1 promoter by repressing its transcriptional activation. TAp63 also mediates the inhibitory effect of insulin on hepatic glucose production, as silencing TAp63 impairs insulin sensitivity. Finally, protein levels of TAp63 are reduced in obese persons with T2D and are negatively correlated with fasting glucose and homeostasis model assessment index. CONCLUSIONS: These results demonstrate that p63 physiologically regulates glucose homeostasis.

Design, Synthesis, Biological Activity, and Structural Analysis of Novel Des-C-Ring and Aromatic-D-Ring Analogues of 1α,25-Dihydroxyvitamin D3.

The toxic calcemic effects of the natural hormone 1α,25-dihydroxyvitamin D3 (1,25D3, 1,25-dihydroxycholecalciferol) in the treatment of hyperproliferative diseases demand the development of highly active and noncalcemic vitamin D analogues. We report the development of two highly active and noncalcemic analogues of 1,25D3 that lack the C-ring and possess an m-phenylene ring that replaces the natural D-ring. The new analogues (3a, 3b) are characterized by an additional six-carbon hydroxylated side chain attached either to the aromatic nucleus or to the triene system. Both compounds were synthesized by the Pd-catalyzed tandem cyclization/cross coupling approach starting from alkyne 6 and diphenol 8. Key steps include a stereoselective Cu-assisted addition of a Grignard reagent to an aromatic alkyne and a Takai olefination of an aromatic aldehyde. The new compounds are noncalcemic and show transcriptional and antiproliferative activities similar to 1,25D3. Structural analysis revealed that they induce a large conformational rearrangement of the vitamin D receptor around helix 6.

In Vivo Effects of Conditioned Medium from Human Uterine Cervical Stem Cells in an Ovarian Cancer Xenograft Mouse Model.

BACKGROUND/AIM: Ovarian cancer is the most lethal of all gynecological cancers, despite advances in surgical techniques and medical treatments. During the last years, therapies based on mesenchymal stem cells and particularly their secretome (conditioned medium, CM) have emerged as promising treatments for various types of tumors. MATERIALS AND METHODS: In the present study, we evaluated the in vivo antitumor effect of human uterine cervical stem cell conditioned medium (hUCESC-CM) after intraperitoneal administration in an ovarian cancer mouse model. RESULTS: We found that intraperitoneal injection of hUCESC-CM in immunodeficient mice, injected fifty days previously with the human ovarian adenocarcinoma SKOV-3 cell line, significantly reduced abdominal tumor growth, and significantly increased overall survival, compared to control mice. CONCLUSION: hUCESC-CM could be an alternative approach to intraperitoneal treatment of ovarian cancer, either administered alone and/or with conventional chemotherapy.

Conditioned Medium from Human Uterine Cervical Stem Cells Regulates Oxidative Stress and Angiogenesis of Retinal Pigment Epithelial Cells.

INTRODUCTION: Retinal homeostasis is essential to avoid retinal pigment epithelium (RPE) damage resulting in photoreceptor death and blindness. Mesenchymal stem cells-based cell therapy could contribute to the maintenance of the retinal homeostasis. We have explored the effect of human uterine cervical stem cells (hUCESCs)-conditioned medium (hUCESC-CM) on RPE cells under oxidative stress condition. METHODS: ARPE-19 cells were treated with hydrogen peroxide (H2O2) in the presence or absence of hUCESC-CM. qRT-PCR and Western blot were used to evaluate the expression of oxidative stress-related (HO-1, GCLC, and HSPB1) and vasculogenesis-related (VEGFA, PDGFA, and PDGFB) factors. Also, we assessed in vitro effects of hUCESC-CM on endothelial-cell (HUVEC) tube formation. RESULTS: mRNA expression of HO-1, GCLC, HSPB1, VEGFA, PDGFA, and PDGFB were significantly increased in ARPE-19 cells treated with H2O2 + hUCESC-CM compared to cells treated with H2O2 only. Regarding the tube formation assay, HUVEC treated with supernatant from ARPE-19 cells treated with H2O2 + hUCESC-CM showed a significant increase in average vessel length, number of capillary-like junctions, and average of vessels area compared with HUVEC treated with supernatant from ARPE-19 cells treated with H2O2 only. CONCLUSION: Our results show potential therapeutic effects of hUCESC-CM on RPE, such as protection from damage by oxidative stress, stimulation of detoxifying genes, and a better vascularization.

Anti-Inflammatory Effect of Tacrolimus/Hydroxypropyl-ß-Cyclodextrin Eye Drops in an Endotoxin-Induced Uveitis Model.

BACKGROUND: Uveitis is an infrequent disease which constitutes a major cause of ocular morbidity. Correct management is essential, being corticosteroids its cornerstone. In case of contraindication to corticosteroids or treatment failure, the use of topical tacrolimus (TAC) could be an alternative which has already demonstrated safety and effectiveness in other ocular pathologies. However, TAC eye drops are not marketed, thus their elaboration must be carried out in Hospital Pharmacy Departments (HPDs). METHODS: 32 Sprague-Dawley rats were divided into 4 groups of 8 rats each: (a) untreated healthy rats (Healthy); (b) untreated Endotoxin-Induced Uveitis model-rats (EIU); (c) EIU-rats treated with standard treatment of dexamethasone ophthalmic drops (DXM) and (d) EIU-rats treated with TAC-hydroxypropyl-ß-cyclodextrin eye drops previously developed by our group (TAC-HPßCD). The mRNA expression levels of IL-6, IL-8, MIP-1α and TNF-α, quantitative analysis of leucocytes in aqueous humor and histological evaluation were performed. RESULTS: TAC-HPßCD eye drops demonstrated to reduce ocular inflammation, expression of IL-6, TNF-α, MIP-1α and leukocyte infiltration in aqueous humor. CONCLUSIONS: TAC-HPßCD eye drops showed beneficial effect in EIU model in rats, positioning as an alternative for uveitis treatment in case of corticosteroids resistance or intolerance.

Tailored Hydrogels as Delivery Platforms for Conditioned Medium from Mesenchymal Stem Cells in a Model of Acute Colitis in Mice.

Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), is increasingly prevalent and current therapies are not completely effective. Mesenchymal stem cells are emerging as a promising therapeutic option. Here, the effect of local hydrogel application loaded with conditioned medium (CM) from human uterine cervical stem cells (hUCESC-CM) in an experimental acute colitis mice model has been evaluated. Colitis induction was carried out in C57BL/6 mice by dissolving dextran sulfate sodium (DSS) in drinking water for nine days. Ulcers were treated by rectal administration of either mesalazine (as positive control) or a mucoadhesive and thermosensitive hydrogel loaded with hUCESC-CM (H-hUCESC-CM). Body weight changes, colon length, and histopathological analysis were evaluated. In addition, pro-inflammatory TNF-α, IL-6, and IFN-γ mRNA levels were measured by qPCR. Treatment with H-hUCESC-CM inhibited body weight loss and colon shortening and induced a significant decrease in colon mucosa degeneration, as well as TNF-α, IFN-γ, and IL-6 mRNA levels. Results indicate that H-hUCESC-CM effectively alleviated DSS-induced colitis in mice, suggesting that H-hUCESC-CM may represent an attractive cell-free therapy for local treatment of IBD.

O-GlcNAcylated p53 in the liver modulates hepatic glucose production.

p53 regulates several signaling pathways to maintain the metabolic homeostasis of cells and modulates the cellular response to stress. Deficiency or excess of nutrients causes cellular metabolic stress, and we hypothesized that p53 could be linked to glucose maintenance. We show here that upon starvation hepatic p53 is stabilized by O-GlcNAcylation and plays an essential role in the physiological regulation of glucose homeostasis. More specifically, p53 binds to PCK1 promoter and regulates its transcriptional activation, thereby controlling hepatic glucose production. Mice lacking p53 in the liver show a reduced gluconeogenic response during calorie restriction. Glucagon, adrenaline and glucocorticoids augment protein levels of p53, and administration of these hormones to p53 deficient human hepatocytes and to liver-specific p53 deficient mice fails to increase glucose levels. Moreover, insulin decreases p53 levels, and over-expression of p53 impairs insulin sensitivity. Finally, protein levels of p53, as well as genes responsible of O-GlcNAcylation are elevated in the liver of type 2 diabetic patients and positively correlate with glucose and HOMA-IR. Overall these results indicate that the O-GlcNAcylation of p53 plays an unsuspected key role regulating in vivo glucose homeostasis.

LIPG endothelial lipase and breast cancer risk by subtypes.

Experimental data showed that endothelial lipase (LIPG) is a crucial player in breast cancer. However, very limited data exists on the role of LIPG on the risk of breast cancer in humans. We examined the LIPG-breast cancer association within our population-based case-control study from Galicia, Spain, BREOGAN (BREast Oncology GAlicia Network). Plasma LIPG and/or OxLDL were measured on 114 breast cancer cases and 82 controls from our case-control study, and were included in the present study. The risk of breast cancer increased with increasing levels of LIPG (multivariable OR for the highest category (95% CI) 2.52 (1.11-5.81), P-trend = 0.037). The LIPG-breast cancer association was restricted to Pre-menopausal breast cancer (Multivariable OR for the highest LIPG category (95% CI) 4.76 (0.94-28.77), P-trend = 0.06, and 1.79 (0.61-5.29), P-trend = 0.372, for Pre-menopausal and Post-menopausal breast cancer, respectively). The LIPG-breast cancer association was restricted to Luminal A breast cancers (Multivariable OR for the highest LIPG category (95% CI) 3.70 (1.42-10.16), P-trend = 0.015, and 2.05 (0.63-7.22), P-trend = 0.311, for Luminal A and non-Luminal A breast cancers, respectively). Subset analysis only based on HER2 receptor indicated that the LIPG-breast cancer relationship was restricted to HER2-negative breast cancers (Multivariable OR for the highest LIPG category (95% CI) 4.39 (1.70-12.03), P-trend = 0.012, and 1.10 (0.28-4.32), P-trend = 0.745, for HER2-negative and HER2-positive tumors, respectively). The LIPG-breast cancer association was restricted to women with high total cholesterol levels (Multivariable OR for the highest LIPG category (95% CI) 6.30 (2.13-20.05), P-trend = 0.018, and 0.65 (0.11-3.28), P-trend = 0.786, among women with high and low cholesterol levels, respectively). The LIPG-breast cancer association was also restricted to non-postpartum breast cancer (Multivariable OR for the highest LIPG category (95% CI) 3.83 (1.37-11.39), P-trend = 0.003, and 2.35 (0.16-63.65), P-trend = 0.396, for non-postpartum and postpartum breast cancer, respectively), although we lacked precision. The LIPG-breast cancer association was more pronounced among grades II and III than grade I breast cancers (Multivariable ORs for the highest category of LIPG (95% CI) 2.73 (1.02-7.69), P-trend = 0.057, and 1.90 (0.61-6.21), P-trend = 0.170, for grades II and III, and grade I breast cancers, respectively). No association was detected for OxLDL levels and breast cancer (Multivariable OR for the highest versus the lowest category (95% CI) 1.56 (0.56-4.32), P-trend = 0.457).

POU1F1 transcription factor induces metabolic reprogramming and breast cancer progression via LDHA regulation.

Metabolic reprogramming is considered hallmarks of cancer. Aerobic glycolysis in tumors cells has been well-known for almost a century, but specific factors that regulate lactate generation and the effects of lactate in both cancer cells and stroma are not yet well understood. In the present study using breast cancer cell lines, human primary cultures of breast tumors, and immune deficient murine models, we demonstrate that the POU1F1 transcription factor is functionally and clinically related to both metabolic reprogramming in breast cancer cells and fibroblasts activation. Mechanistically, we demonstrate that POU1F1 transcriptionally regulates the lactate dehydrogenase A (LDHA) gene. LDHA catalyzes pyruvate into lactate instead of leading into the tricarboxylic acid cycle. Lactate increases breast cancer cell proliferation, migration, and invasion. In addition, it activates normal-associated fibroblasts (NAFs) into cancer-associated fibroblasts (CAFs). Conversely, LDHA knockdown in breast cancer cells that overexpress POU1F1 decreases tumor volume and [18F]FDG uptake in tumor xenografts of mice. Clinically, POU1F1 and LDHA expression correlate with relapse- and metastasis-free survival. Our data indicate that POU1F1 induces a metabolic reprogramming through LDHA regulation in human breast tumor cells, modifying the phenotype of both cancer cells and fibroblasts to promote cancer progression.

Mesenchymal Stem Cells in Homeostasis and Systemic Diseases: Hypothesis, Evidences, and Therapeutic Opportunities.

Mesenchymal stem cells (MSCs) are present in all organs and tissues, playing a well-known function in tissue regeneration. However, there is also evidence indicating a broader role of MSCs in tissue homeostasis. In vivo studies have shown MSC paracrine mechanisms displaying proliferative, immunoregulatory, anti-oxidative, or angiogenic activity. In addition, recent studies also demonstrate that depletion and/or dysfunction of MSCs are associated with several systemic diseases, such as lupus, diabetes, psoriasis, and rheumatoid arthritis, as well as with aging and frailty syndrome. In this review, we hypothesize about the role of MSCs as keepers of tissue homeostasis as well as modulators in a variety of inflammatory and degenerative systemic diseases. This scenario opens the possibility for the use of secretome-derived products from MSCs as new therapeutic agents in order to restore tissue homeostasis, instead of the classical paradigm "one disease, one drug".

POU1F1 transcription factor promotes breast cancer metastasis via recruitment and polarization of macrophages.

Cancer progression requires cells surrounding tumors be reeducated and activated to support tumor growth. Oncogenic signals from malignant cells directly influence stromal composition and activation, but the factors mediating this communication are still not well understood. We have previously shown that the transcription factor POU class 1 homeobox 1 (POU1F1), also known as Pit-1, induces profound changes on neoplastic cell-autonomous processes favoring metastasis in human breast cancer. Here we describe for the first time Pit-1-mediated paracrine actions on macrophages in the tumor microenvironment by using cell lines in vitro, zebrafish and mouse models in vivo, and samples from human breast cancer patients. Through the release of CXCL12, Pit-1 in tumor cells was found to mediate the recruitment and polarization of macrophages into tumor-associated macrophages (TAMs). In turn, TAMs collaborated with tumor cells to increase tumor growth, angiogenesis, extravasation and metastasis to lung. Our data reveal a new mechanism of cooperation between tumor cells and macrophages favoring metastasis and poor clinical outcome in human breast cancer, which suggests that Pit-1 and CXCL12 should be further studied as potential prognostic and therapeutic indicators. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Corrigendum: Antifungal Activity of the Human Uterine Cervical Stem Cells Conditioned Medium (hUCESC-CM) Against Candida albicans and Other Medically Relevant Species of Candida.

[This corrects the article DOI: 10.3389/fmicb.2018.02818.].

Corneal regeneration by conditioned medium of human uterine cervical stem cells is mediated by TIMP-1 and TIMP-2.

The aim of the present study was to evaluate the effect and the mechanism of action of the conditioned medium from human uterine cervical stem cells (CM-hUCESC) on corneal wound healing in a rabbit dry eye model. To do this, dry eye and corneal epithelial injuries were induced in rabbits by topical administration of atropine sulfate and NaOH. Hematoxylin-Eosin (H&E) and Ki-67 immunostaining were carried out to evaluate corneal damage and cell proliferation, and real-time PCR was used to evaluate proinflammatory cytokines in the cornea. In addition, in order to investigate possible factors involved in corneal regeneration, primary cultures of rat corneal epithelial cells (rCECs) were used to evaluate cell migration, proliferation, and apoptosis before and after immunoprecipitation of specific factors from the CM-hUCESC. Results showed that CM-hUCESC treatment significantly improved epithelial regeneration in rabbits with dry eye induced by atropine and reduced corneal pro-inflammatory TNF-α, MCP-1, MIP-1α and IL-6 cytokines. In addition, metalloproteinase inhibitors TIMP-1 and TIMP-2, which are present at high levels in CM-hUCESC, mediated corneal regenerative effects by both inducing corneal epithelial cell proliferation and inhibiting apoptosis. In summary, CM-hUCESC induces faster corneal regeneration in a rabbit model of dry eye induced by atropine than conventional treatments, being TIMP-1 and TIMP-2 mediators in this process. The results indicate that an alternative CM-based treatment for some corneal conditions is achievable, although future studies would be necessary to investigate other factors involved in the multiple observed effects of CM-hUCESC.

Antifungal Activity of the Human Uterine Cervical Stem Cells Conditioned Medium (hUCESC-CM) Against Candida albicans and Other Medically Relevant Species of Candida.

Background: Candidiasis is a major cause of human morbidity and mortality. Human uterine cervical stem cells conditioned medium (hUCESC-CM) is obtained from stromal stem cells of the cervical transformation zone, which are in permanent contact with a wide array of potential vaginal pathogens. In previous reports we have found that hUCESC-CM has antitumor and antibacterial potential. Since Candida is the most prevalent yeast in the human vagina, it seems plausible that hUCESC-CM might also show activity against it. Methods: In a preliminary step, to evaluate if hUCESC-CM showed any activity at all on Candida growth, in vitro activities of hUCESC-CM against fluconazole-susceptible reference strains of Candida albicans, Candida glabrata, Candida krusei, and Candida parapsilosis were studied with a microdilution method on RPMI 1640, using the BioScreen C microbiological incubator. Each measurement was repeated five times. The same methodology was used subsequently on fluconazole-susceptible and fluconazole-resistant Candida isolates from blood and vagina of those species corresponding to the reference strains of Candida against which activity had been detected in the previous study. Moreover, two fluconazole-resistant clinical isolates of Candida auris from blood and urine were also included. Findings: In vitro inhibitory activity of hUCESC-CM ranged from 57.5 to 96.6% growth-reduction against fluconazole-susceptible reference strains of Candida albicans, Candida glabrata, and Candida parapsilosis. hUCESC-CM also reduced the growth of all fluconazole-susceptible tested vaginal isolates by more than 50%. For fluconazole-resistant isolates, growth-reduction was higher than 67% for Candida albicans, regardless of its origin (vagina or blood). The isolate of Candida auris from urine with a MIC > 128 µ/ml for fluconazole was also significantly inhibited. However, hUCESC-CM was almost inactive against any of the fluconazole-resistant blood isolates of Candida glabrata, Candida parapsilosis, and Candida auris tested. Interpretation: This is the first report about the growth-inhibiting properties of conditioned medium from human stromal stem cells against different species of Candida. Antifungal activity of stromal stem cells depends on their site of origin, being most effective against Candida species most prevalent at that particular location. If confirmed in further studies, these findings might result in a completely new therapeutic approach against superficial and invasive candidiasis.

Aromatic-Based Design of Highly Active and Noncalcemic Vitamin D Receptor Agonists.

We report the design, synthesis, biological evaluation, and structural analysis of a new class of vitamin D analogues that possess an aromatic m-phenylene D-ring and an alkyl chain replacing the C-ring. A key feature of the synthetic strategy is a stereoselective Pd-catalyzed construction of the triene system in aqueous medium that allows the rapid preparation of small amounts of VDR ligands for biological screening. Analogues with the shorter (2a) and longer (2d, 2e) side chains attached to the triene system have no calcemic activity. Compound 2a binds to VDR with the same order of magnitude than calcipotriol and oxacalcitriol. It also reduces proliferation in normal and tumor cells similarly to the natural hormone 1α,25-dihydroxyvitamin D3, calcipotriol, and oxacalcitriol, suggesting preclinical studies related to hyperproliferative disorders such as psoriasis and cancer.

Cancer-associated fibroblasts affect breast cancer cell gene expression, invasion and angiogenesis.

PURPOSE: It has been reported that stromal cell features may affect the clinical outcome of breast cancer patients. Cancer associated fibroblasts (CAFs) represent one of the most abundant cell types within the breast cancer stroma. Here, we aimed to explore the influence of CAFs on breast cancer gene expression, as well as on invasion and angiogenesis. METHODS: qRT-PCR was used to evaluate the expression of several cancer progression related genes (S100A4, TGFß, FGF2, FGF7, PDGFA, PDGFB, VEGFA, IL-6, IL-8, uPA, MMP2, MMP9, MMP11 and TIMP1) in the human breast cancer-derived cell lines MCF-7 and MDA-MB-231, before and after co-culture with CAFs. Stromal mononuclear inflammatory cell (MIC) MMP11 expression was used to stratify primary tumors. In addition, we assessed the in vitro effects of CAFs on both MDA-MB-231 breast cancer cell invasion and endothelial cell (HUVEC) tube formation. RESULTS: We found that the expression levels of most of the genes tested were significantly increased in both breast cancer-derived cell lines after co-culture with CAFs from either MMP11+ or MMP11- MIC tumors. IL-6 and IL-8 showed an increased expression in both cancer-derived cell lines after co-culture with CAFs from MMP11+ MIC tumors. We also found that the invasive and angiogenic capacities of, respectively, MDA-MB-231 and HUVEC cells were increased after co-culture with CAFs, especially those from MMP11+ MIC tumors. CONCLUSIONS: Our data indicate that tumor-derived CAFs can induce up-regulation of genes involved in breast cancer progression. Our data additionally indicate that CAFs, especially those derived from MMP11+ MIC tumors, can promote breast cancer cell invasion and angiogenesis.

Breast cancer metastasis to liver and lung is facilitated by Pit-1-CXCL12-CXCR4 axis.

Development of human tumors is driven by accumulation of alterations in tumor suppressor genes and oncogenes in cells. The POU1F1 transcription factor (also known Pit-1) is expressed in the mammary gland and its overexpression induces profound phenotypic changes in proteins involved in breast cancer progression. Patients with breast cancer and elevated expression of Pit-1 show a positive correlation with the occurrence of distant metastasis and poor overall survival. However, some mediators of Pit-1 actions are still unknown. Here, we show that CXCR4 chemokine receptor and its ligand CXCL12 play a critical role in the pro-tumoral process induced by Pit-1. We found that Pit-1 increases mRNA and protein in both CXCR4 and CXCL12. Knock-down of CXCR4 reduces tumor growth and spread of Pit-1 overexpressing cells in a zebrafish xenograft model. Furthermore, we described for the first time pro-angiogenic effects of Pit-1 through the CXCL12-CXCR4 axis, and that extravasation of Pit-1 overexpressing breast cancer cells is strongly reduced in CXCL12-deprived target tissues. Finally, in breast cancer patients, expression of Pit-1 in primary tumors was found to be positively correlated with CXCR4 and CXCL12, with specific metastasis in liver and lung, and with clinical outcome. Our results suggest that Pit-1-CXCL12-CXCR4 axis could be involved in chemotaxis guidance during the metastatic process, and may represent prognostic and/or therapeutic targets in breast tumors.