The MITF/mir-579-3p regulatory axis dictates BRAF-mutated melanoma cell fate in response to MAPK inhibitors.

Therapy of melanoma has improved dramatically over the last years thanks to the development of targeted therapies (MAPKi) and immunotherapies. However, drug resistance continues to limit the efficacy of these therapies. Our research group has provided robust evidence as to the involvement of a set of microRNAs in the development of resistance to target therapy in BRAF-mutated melanomas. Among them, a pivotal role is played by the oncosuppressor miR-579-3p. Here we show that miR-579-3p and the microphthalmia-associated transcription factor (MITF) influence reciprocally their expression through positive feedback regulatory loops. In particular we show that miR-579-3p is specifically deregulated in BRAF-mutant melanomas and that its expression levels mirror those of MITF. Luciferase and ChIP studies show that MITF is a positive regulator of miR-579-3p, which is located in the intron 11 of the human gene ZFR (Zink-finger recombinase) and is co-transcribed with its host gene. Moreover, miR-579-3p, by targeting BRAF, is able to stabilize MITF protein thus inducing its own transcription. From biological points of view, early exposure to MAPKi or, alternatively miR-579-3p transfection, induce block of proliferation and trigger senescence programs in BRAF-mutant melanoma cells. Finally, the long-term development of resistance to MAPKi is able to select cells characterized by the loss of both miR-579-3p and MITF and the same down-regulation is also present in patients relapsing after treatments. Altogether these findings suggest that miR-579-3p/MITF interplay potentially governs the balance between proliferation, senescence and resistance to therapies in BRAF-mutant melanomas.

Upregulated expression of miR-4443 and miR-4488 in drug resistant melanomas promotes migratory and invasive phenotypes through downregulation of intermediate filament nestin.

BACKGROUND: BRAF-mutant melanoma patients benefit from the combinatorial treatments with BRAF and MEK inhibitors. However, acquired drug resistance strongly limits the efficacy of these targeted therapies in time. Recently, many findings have underscored the involvement of microRNAs as main drivers of drug resistance. In this context, we previously identified a subset of oncomiRs strongly up-regulated in drug-resistant melanomas. In this work, we shed light on the molecular role of two as yet poorly characterized oncomiRs, miR-4443 and miR-4488. METHODS: Invasion and migration have been determined by wound healing, transwell migration/invasion assays and Real Time Cell Analysis (RTCA) technology. miR-4488 and miR-4443 have been measured by qRT-PCR. Nestin levels have been tested by western blot, confocal immunofluorescence, immunohistochemical and flow cytometry analyses. RESULTS: We demonstrate that the two oncomiRs are responsible for the enhanced migratory and invasive phenotypes, that are a hallmark of drug resistant melanoma cells. Moreover, miR-4443 and miR-4488 promote an aberrant cytoskeletal reorganization witnessed by the increased number of stress fibers and cellular protrusions-like cancer cell invadopodia. Mechanistically, we identified the intermediate filament nestin as a molecular target of both oncomiRs. Finally, we have shown that nestin levels are able to predict response to treatments in melanoma patients. CONCLUSIONS: Altogether these findings have profound translational implications in the attempt i) to develop miRNA-targeting therapies to mitigate the metastatic phenotypes of BRAF-mutant melanomas and ii) to identify novel biomarkers able to guide clinical decisions.

ERK Signaling Pathway Is Constitutively Active in NT2D1 Non-Seminoma Cells and Its Inhibition Impairs Basal and HGF-Activated Cell Proliferation.

c-MET/hepatocyte growth factor (HGF) system deregulation is a well-known feature of malignancy in several solid tumors, and for this reason this system and its pathway have been considered as potential targets for therapeutic purposes. In previous manuscripts we reported c-MET/HGF expression and the role in testicular germ cell tumors (TGCTs) derived cell lines. We demonstrated the key role of c-Src and phosphatidylinositol 3-kinase (PI3K)/AKT adaptors in the HGF-dependent malignant behavior of the embryonal carcinoma cell line NT2D1, finding that the inhibition of these onco-adaptor proteins abrogates HGF triggered responses such as proliferation, migration, and invasion. Expanding on these previous studies, herein we investigated the role of mitogen-activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK) pathways in the HGF-dependent and HGF-independent NT2D1 cells biological responses. To inhibit MAPK/ERK pathways we chose a pharmacological approach, by using U0126 inhibitor, and we analyzed cell proliferation, collective migration, and chemotaxis. The administration of U0126 together with HGF reverts the HGF-dependent activation of cell proliferation but, surprisingly, does not exert the same effect on NT2D1 cell migration. In addition, we found that the use of U0126 alone significantly promotes the acquisition of NT2D1 «migrating phenotype¼, while collective migration of NT2D1 cells was stimulated. Notably, the inhibition of ERK activation in the absence of HGF stimulation resulted in the activation of the AKT-mediated pathway, and this let us speculate that the paradoxical effects obtained by using U0126, which are the increase of collective migration and the acquisition of partial epithelium-mesenchyme transition (pEMT), are the result of compensatory pathways activation. These data highlight how the specific response to pathway inhibitors, should be investigated in depth before setting up therapy.

Human Platelet-Rich Plasma Regulates Canine Mesenchymal Stem Cell Migration through Aquaporins.

Platelet products are commonly used in regenerative medicine due to their effects on the acceleration and promotion of wound healing, reduction of bleeding, synthesis of new connective tissue, and revascularization. Furthermore, a novel approach for the treatment of damaged tissues, following trauma or other pathological damages, is represented by the use of mesenchymal stem cells (MSCs). In dogs, both platelet-rich plasma (PRP) and MSCs have been suggested to be promising options for subacute skin wounds. However, the collection of canine PRP is not always feasible. In this study, we investigated the effect of human PRP (hPRP) on canine MSCs (cMSCs). We isolated cMSCs and observed that hPRP did not modify the expression levels of the primary class of major histocompatibility complex genes. However, hPRP was able to increase cMSC viability and migration by at least 1.5-fold. hPRP treatment enhanced both Aquaporin (AQP) 1 and AQP5 protein levels, and their inhibition by tetraethylammonium chloride led to a reduction of PRP-induced migration of cMSCs. In conclusion, we have provided evidence that hPRP supports cMSC survival and may promote cell migration, at least through AQP activation. Thus, hPRP may be useful in canine tissue regeneration and repair, placing as a promising tool for veterinary therapeutic approaches.

Oncosuppressive miRNAs loaded in lipid nanoparticles potentiate targeted therapies in BRAF-mutant melanoma by inhibiting core escape pathways of resistance.

BRAF-mutated melanoma relapsing after targeted therapies is an aggressive disease with unmet clinical need. Hence the need to identify novel combination therapies able to overcome drug resistance. miRNAs have emerged as orchestrators of non-genetic mechanisms adopted by melanoma cells to challenge therapies. In this context we previously identified a subset of oncosuppressor miRNAs downregulated in drug-resistant melanomas. Here we demonstrate that lipid nanoparticles co-encapsulating two of them, miR-199-5p and miR-204-5p, inhibit tumor growth both in vitro and in vivo in combination with target therapy and block the development of drug resistance. Mechanistically they act by directly reducing melanoma cell growth and also indirectly by hampering the recruitment and reprogramming of pro-tumoral macrophages. Molecularly, we demonstrate that the effects on macrophages are mediated by the dysregulation of a newly identified miR-204-5p-miR-199b-5p/CCL5 axis. Finally, we unveiled that M2 macrophages programs are molecular signatures of resistance and predict response to therapy in patients. Overall, these findings have strong translational implications to propose new combination therapies making use of RNA therapeutics for metastatic melanoma patients.

Radioresistance Mechanisms in Prostate Cancer Cell Lines Surviving Ultra-Hypo-Fractionated EBRT: Implications and Possible Clinical Applications.

The use of a higher dose per fraction to overcome the high radioresistance of prostate cancer cells has been unsuccessfully proposed. Herein, we present PC3 and DU-145, castration-resistant prostate cancer cell lines that survived a clinically used ultra-higher dose per fraction, namely, radioresistant PC3 and DU-145 cells (PC3RR and DU-145RR). Compared to PC3, PC3RR showed a higher level of aggressive behaviour, with enhanced clonogenic potential, DNA damage repair, migration ability and cancer stem cell features. Furthermore, compared to PC3, PC3RR more efficiently survived further radiation by increasing proliferation and down-regulating pro-apoptotic proteins. No significant changes of the above parameters were described in DU-145RR, suggesting that different prostate cancer cell lines that survive ultra-higher dose per fraction do not display the same grade of aggressive phenotype. Furthermore, both PC3RR and DU-145RR increased antioxidant enzymes and mesenchymal markers. Our data suggest that different molecular mechanisms could be potential targets for future treatments plans based on sequential strategies and synergistic effects of different modalities, possibly in a patient-tailored fashion. Moreover, PC3RR cells displayed an increase in specific markers involved in bone remodeling, indicating that radiotherapy selects a PC3 population capable of migrating to secondary metastatic sites. Finally, PC3RR cells showed a better sensitivity to Docetaxel as compared to native PC3 cells. This suggests that a subset of patients with castration-resistant metastatic disease could benefit from upfront Docetaxel treatment after the failure of radiotherapy.

Identification of a miRNA-based non-invasive predictive biomarker of response to target therapy in BRAF-mutant melanoma.

Rationale: Metastatic melanoma is the most aggressive and dangerous form of skin cancer. The introduction of immunotherapy with Immune checkpoint Inhibitors (ICI) and of targeted therapy with BRAF and MEK inhibitors for BRAF mutated melanoma, has greatly improved the clinical outcome of these patients. Nevertheless, response to therapy remains highly variable and the development of drug resistance continues to be a daunting challenge. Within this context there is a need to develop diagnostic tools capable of predicting response or resistance to therapy in order to select the best therapeutic approach. Over the years, accumulating evidence brought to light the role of microRNAs (miRNAs) as disease biomarkers. Methods: In particular, the detection of miRNAs in whole blood or specific blood components such as serum or plasma, allows these molecules to be good candidates for diagnosis, prognosis and for monitoring response to anticancer therapy. In this paper, we evaluated circulating basal levels of 6 previously identified miRNAs in serum samples of 70 BRAF-mutant melanoma patients before starting targeted therapy. Results: Results show that the circulating levels of the oncosuppressor miR-579-3p and of the oncomiR miR-4488 are able to predict progression free survival (PFS) but not overall survival (OS). Most importantly, we observed that the best predictor of disease outcome is represented by the ratio of circulating miR-4488 vs. miR-579-3p (miRatio). Finally, the combination of the Lactate dehydrogenase (LDH) blood levels with the two circulating miRNAs alone or together did not produce any improvement in predicting PFS indicating that miR-579-3p and miR-4488 are independent predictors of PFS as compared to LDH. Conclusions: All together these data underscored the relevance of circulating miRNAs as suitable tools to predict therapy response in melanoma and maybe further developed as companion diagnostics in the clinic.

Glucose Enhances Pro-Tumorigenic Functions of Mammary Adipose-Derived Mesenchymal Stromal/Stem Cells on Breast Cancer Cell Lines.

Adiposity and diabetes affect breast cancer (BC) progression. We addressed whether glucose may affect the interaction between mammary adipose tissue-derived mesenchymal stromal/stem cells (MAT-MSCs) and BC cells. Two-dimensional co-cultures and spheroids were established in 25 mM or 5.5 mM glucose (High Glucose-HG or Low Glucose-LG) by using MAT-MSCs and MCF7 or MDA-MB231 BC cells. Gene expression was measured by qPCR, while protein levels were measured by cytofluorimetry and ELISA. CD44high/CD24low BC stem-like sub-population was quantified by cytofluorimetry. An in vivo zebrafish model was assessed by injecting spheroid-derived labeled cells. MAT-MSCs co-cultured with BC cells showed an inflammatory/senescent phenotype with increased abundance of IL-6, IL-8, VEGF and p16INK4a, accompanied by altered levels of CDKN2A and LMNB1. BC cells reduced multipotency and increased fibrotic features modulating OCT4, SOX2, NANOG, αSMA and FAP in MAT-MSCs. Of note, these co-culture-mediated changes in MAT-MSCs were partially reverted in LG. Only in HG, MAT-MSCs increased CD44high/CD24low MCF7 sub-population and promoted their ability to form mammospheres. Injection in zebrafish embryos of HG spheroid-derived MCF7 and MAT-MSCs was followed by a significant cellular migration and caudal dissemination. Thus, MAT-MSCs enhance the aggressiveness of BC cells in a HG environment.

miR-125a-5p impairs the metastatic potential in breast cancer via IP6K1 targeting.

The deregulation of PI3K/Akt signaling is among the most causes in inducing the acquisition of a metastatic phenotype in breast cancer cells, leading to Epithelial-Mesenchymal Transition (EMT). Inhibition of the PI3K/Akt pathway is known to be beneficial in the clinical setting. However, the activation of secondary pathways and toxicity profiles of available inhibitors, hindering optimal therapeutic results. Preliminary studies showed that myo-Inositol inhibits the PI3K/Akt pathway by exerting a pleiotropic anti-tumor action. Herein, we demonstrate that myo-Inositol triggers a prompt and profound remodeling of delineated expression pattern in triple-negative breast cancer cells (MDA-MB-231). Consequently, it inhibits metastasis and tumor progression through miR-125a-5p transcription and the subsequent inhibition of IP6K1. In contrast, hormone-responsive breast cancer cells (MCF-7) are insensitive to myo-Inositol. This is due to the persistence of MDM2 synthesis promoted by estrogen-dependent pathways. Conversely, the counteraction of estrogen effects recovered the sensitivity to myo-Inositol in the hormone-responsive model. Overall, these results identify a novel axis primed by miR-125a-5p to downregulate IP6K1 gene that inhibits metastasis. Thus, administration of myo-Inositol can activate this axis as a molecular target therapy in breast cancer.

In severe obesity, subcutaneous adipose tissue cell-derived cytokines are early markers of impaired glucose tolerance and are modulated by quercetin.

BACKGROUND: Excessive adiposity provides an inflammatory environment. However, in people with severe obesity, how systemic and local adipose tissue (AT)-derived cytokines contribute to worsening glucose tolerance is not clear. METHODS: Ninty-two severely obese (SO) individuals undergoing bariatric surgery were enrolled and subjected to detailed clinical phenotyping. Following an oral glucose tolerance test, participants were included in three groups, based on the presence of normal glucose tolerance (NGT), impaired glucose tolerance (IGT), or type 2 diabetes (T2D). Serum and subcutaneous AT (SAT) biopsies were obtained and mesenchymal stem cells (MSCs) were isolated, characterized, and differentiated in adipocytes in vitro. TNFA and PPARG mRNA levels were determined by qRT-PCR. Circulating, adipocyte- and MSC-released cytokines, chemokines, and growth factors were assessed by multiplex ELISA. RESULTS: Serum levels of IL-9, IL-13, and MIP-1ß were increased in SO individuals with T2D, as compared with those with either IGT or NGT. At variance, SAT samples obtained from SO individuals with IGT displayed levels of TNFA which were threefold higher compared to those with NGT, but not different from those with T2D. Elevated levels of TNFα were also found in differentiated adipocytes, isolated from the SAT specimens of individuals with IGT and T2D, compared to those with NGT. Consistent with the pro-inflammatory milieu, IL-1ß and IP-10 secretion was significantly higher in adipocytes from individuals with IGT and T2D. Moreover, increased levels of TNFα, both mRNA and secreted protein were detected in MSCs obtained from IGT and T2D, compared to NGT SO individuals. Exposure of T2D and IGT-derived MSCs to the anti-inflammatory flavonoid quercetin reduced TNFα levels and was paralleled by a significant decrease of the secretion of inflammatory cytokines. CONCLUSION: In severe obesity, enhanced SAT-derived inflammatory phenotype is an early step in the progression toward T2D and maybe, at least in part, attenuated by quercetin.

The Promise of Liquid Biopsy to Predict Response to Immunotherapy in Metastatic Melanoma.

Metastatic melanoma is the deadliest form of skin cancer whose incidence has been rising dramatically over the last few decades. Nowadays, the most successful approach in treating advanced melanoma is immunotherapy which encompasses the use of immune checkpoint blockers able to unleash the immune system's activity against tumor cells. Immunotherapy has dramatically changed clinical practice by contributing to increasing long term overall survival. Despite these striking therapeutic effects, the clinical benefits are strongly mitigated by innate or acquired resistance. In this context, it is of utmost importance to develop methods capable of predicting patient response to immunotherapy. To this purpose, one major step forward may be provided by measuring non-invasive biomarkers in human fluids, namely Liquid Biopsies (LBs). Several LB approaches have been developed over the last few years thanks to technological breakthroughs that have allowed to evaluate circulating components also when they are present in low abundance. The elements of this so-called "circulome" mostly encompass: tumor DNA, tumor and immune cells, soluble factors and non-coding RNAs. Here, we review the current knowledge of these molecules as predictors of response to immunotherapy in metastatic melanoma and predict that LB will soon enter into routine practice in order to guide clinical decisions for cancer immunotherapy.

Drug tolerance to target therapy in melanoma revealed at single cell level: What next?

Drug resistance strongly impairs the efficacy of virtually every kind of anticancer therapy. This phenomenon is commonly fueled by intrinsic or acquired mechanisms. In this mini-review, focusing on BRAF-mutated melanoma as prototypical example, we analyze how recent studies that make use of single cell analysis identify the involvement of distinct transcriptional trajectories as the common thread at the basis of drug tolerance. The identification of these transcriptional trajectories provide a mechanistic basis for the development of both intrinsic and acquired drug resistance. These studies also suggest that hitting these transcriptional trajectories through personalized adaptive treatments can delay or abrogate the onset of drug resistance.

In Vitro Biophysical and Biological Characterization of Lipid Nanoparticles Co-Encapsulating Oncosuppressors miR-199b-5p and miR-204-5p as Potentiators of Target Therapy in Metastatic Melanoma.

Uncontrolled MAPK signaling is the main oncogenic driver in metastatic melanomas bearing mutations in BRAF kinase. These tumors are currently treated with the combination of BRAF/MEK inhibitors (MAPKi), but this therapy is plagued by drug resistance. In this context we recently discovered that several microRNAs are involved in the development of drug resistance. In particular miR-204-5p and miR-199b-5p were found to function as antagonists of resistance because their enforced overexpression is able to inhibit melanoma cell growth in vitro either alone or in combination with MAPKi. However, the use of miRNAs in therapy is hampered by their rapid degradation in serum and biological fluids, as well as by the poor intracellular uptake. Here, we developed lipid nanoparticles (LNPs) encapsulating miR-204-5p, miR-199b-5p individually or in combination. We obtained LNPs with mean diameters < 200 nm and high miRNA encapsulation efficiency. These formulations were tested in vitro on several melanoma cell lines sensitive to MAPKi or rendered drug resistant. Our results show that LNPs encapsulating combinations of the two oncosuppressor miRNAs are highly efficient in impairing melanoma cell proliferation and viability, affect key signaling pathways involved in melanoma cell survival, and potentiate the efficacy of drugs inhibiting BRAF and MEK. These results warrant further assessment of the anti-tumor efficacy of oncosuppressor miRNAs encapsulating LNPs in in vivo tumor models.

Single cell analysis to dissect molecular heterogeneity and disease evolution in metastatic melanoma.

Originally described as interpatient variability, tumour heterogeneity has now been demonstrated to occur intrapatiently, within the same lesion, or in different lesions of the same patient. Tumour heterogeneity involves both genetic and epigenetic changes. Intrapatient heterogeneity is responsible for generating subpopulations of cancer cells which undergo clonal evolution with time. Tumour heterogeneity develops also as a consequence of the selective pressure imposed by the immune system. It has been demonstrated that tumour heterogeneity and different spatiotemporal interactions between all the cellular compontents within the tumour microenvironment lead to cancer adaptation and to therapeutic pressure. In this context, the recent advent of single cell analysis approaches which are able to better study tumour heterogeneity from the genomic, transcriptomic and proteomic standpoint represent a major technological breakthrough. In this review, using metastatic melanoma as a prototypical example, we will focus on applying single cell analyses to the study of clonal trajectories which guide the evolution of drug resistance to targeted therapy.

Low-dose Bisphenol-A regulates inflammatory cytokines through GPR30 in mammary adipose cells.

The dramatic rise in obesity and metabolic syndrome can be related, at least in part, to environmental chemical factors such as Bisphenol-A (BPA). In this study, we aimed to understand the effects of low-dose Bisphenol-A on the human mature adipocytes and stromal vascular fraction (SVF) cells, obtained from subcutaneous mammary adipose tissue of overweight female patients, undergoing surgical mammary reduction. 24 and/or 48-h exposure to BPA 0.1 nM elicited significant increase of the inflammatory molecules interleukin-6 (IL-6), interleukin-8 (IL-8), monocyte chemo-attractant protein 1α (MCP1α) and induced G protein-coupled estrogen receptor 30 (GPR30) levels more than two-fold both in mature adipocytes and SVF cells. These effects were similar to that obtained in the presence of GPR30-specific agonist G1 (100 nM) and were reverted by G15 (1 µM), a GPR30-selective antagonist. As a result of BPA-GPR30 signaling activation, fatty acid synthase (FAS) and leptin mRNA levels were significantly higher upon BPA exposure (P < 0.05) in mature adipocytes, with an opposite effect on adiponectin (ADIPOQ). In addition, an increase in SVF cell proliferation and ERK1/2 phosphorylation, was observed, compared to untreated cells. G15 reverted all of these effects. Interestingly, the action of BPA on SVF cell growth was mimicked by IL-8 treatment and was reverted by incubation with anti-IL8 antibodies. All these data suggest that BPA at 0.1 nM, a ten times lower concentration than environmental exposure, increases the expression of pro-inflammatory cytokines via GPR30 both in mature mammary adipocytes and in SVF cells with a possible involvement of IL-8.

ErbB3 Phosphorylation as Central Event in Adaptive Resistance to Targeted Therapy in Metastatic Melanoma: Early Detection in CTCs during Therapy and Insights into Regulation by Autocrine Neuregulin.

In recent years the introduction of target therapies with BRAF and MEK inhibitors (MAPKi) and of immunotherapy with anti-CTLA-4 and anti-PD-1 monoclonal antibodies have dramatically improved survival of metastatic melanoma patients. Despite these changes drug resistance remains a major hurdle. Several mechanisms are at the basis of drug resistance. Particular attention has been devoted over the last years to unravel mechanisms at the basis of adaptive/non genetic resistance occurring in BRAF mutated melanomas upon treatment with to MAPKi. In this paper we focus on the involvement of activation of ErbB3 receptor following early exposure of melanoma cells to BRAF or MEK inhibitors, and the following induction of PI3K/AKT pathway. Although different mechanisms have been invoked in the past at the basis of this activation we show here with a combination of approaches that autocrine production of neuregulin by melanoma cells is a major factor responsible for ErbB3 phosphorylation and downstream AKT activation. Interestingly the kinetic of neuregulin production and of the ensuing ErbB3 phosphorylation is different in different melanoma cell lines which underscores the high degree of tumor heterogeneity. Moreover, heterogeneity is further highlighted by the evidence that in different cell lines neuregulin upregulation can occur at the transcriptional or at the post-transcritpional level. Finally we complement our study by showing with a liquid biopsy assay that circulating tumor cells (CTCs) from melanoma patients undergo upregulation of ErbB3 phosphorylation in vivo shortly after initiation of therapy.

Reprogramming miRNAs global expression orchestrates development of drug resistance in BRAF mutated melanoma.

Drug resistance imposes severe limitations to the efficacy of targeted therapy in BRAF-mutated metastatic melanoma. Although this issue has been mitigated by the development of combination therapies with BRAF plus MEK inhibitors, drug resistance inevitably occurs with time and results in clinical recurrences and untreatable disease. Hence, there is strong need of developing new combination therapies and non-invasive diagnostics for the early identification of drug-resistant patients. We report here that the development of drug resistance to BRAFi is dominated by a dynamic deregulation of a large population of miRNAs, leading to the alteration of cell intrinsic proliferation and survival pathways, as well as of proinflammatory and proangiogenic cues, where a prominent role is played by the miR-199b-5p/VEGF axis. Significant alterations of miRNA expression levels are detectable in tumor biopsies and plasma from patients after disease recurrence. Targeting these alterations blunts the development of drug resistance.

Glucose impairs tamoxifen responsiveness modulating connective tissue growth factor in breast cancer cells.

Type 2 diabetes and obesity are negative prognostic factors in patients with breast cancer (BC). We found that sensitivity to tamoxifen was reduced by 2-fold by 25 mM glucose (High Glucose; HG) compared to 5.5 mM glucose (Low Glucose; LG) in MCF7 BC cells. Shifting from HG to LG ameliorated MCF7 cell responsiveness to tamoxifen. RNA-Sequencing of MCF7 BC cells revealed that cell cycle-related genes were mainly affected by glucose. Connective Tissue Growth Factor (CTGF) was identified as a glucose-induced modulator of cell sensitivity to tamoxifen. Co-culturing MCF7 cells with human adipocytes exposed to HG, enhanced CTGF mRNA levels and reduced tamoxifen responsiveness of BC cells. Inhibition of adipocyte-released IL8 reverted these effects. Interestingly, CTGF immuno-detection in bioptic specimens from women with estrogen receptor positive (ER+) BC correlated with hormone therapy resistance, distant metastases, reduced overall and disease-free survival. Thus, glucose affects tamoxifen responsiveness directly modulating CTGF in BC cells, and indirectly promoting IL8 release by adipocytes.

Low-Dose Bisphenol-A Impairs Adipogenesis and Generates Dysfunctional 3T3-L1 Adipocytes.

Environmental endocrine disruptors (EDCs), including bisphenol-A (BPA), have been recently involved in obesity and diabetes by dysregulating adipose tissue function. Our aim was to examine whether prolonged exposure to low doses of BPA could affect adipogenesis and adipocyte metabolic functions. Therefore, 3T3-L1 pre-adipocytes were cultured for three weeks with BPA 1 nM to mimic human environmental exposure. We evaluated BPA effect on cell proliferation, differentiation, gene expression and adipocyte metabolic function. BPA significantly increased pre-adipocyte proliferation (p<0.01). In 3T3-L1 adipocytes differentiated in the presence of BPA, the expression of Peroxisome proliferator-activated receptor gamma (PPARγ), Fatty Acid Binding Protein 4/Adipocyte Protein 2 (FABP4/AP2) and CCAAT/enhancer binding protein (C/EBPα) was increased by 3.5, 1.5 and 3 folds, respectively. Mature adipocytes also showed a significant increase in lipid accumulation (p<0.05) and alterations of insulin action, with significant reduction in insulin-stimulated glucose utilization (p<0.001). Moreover, in mature adipocytes, mRNA levels of Leptin, interleukin-6 (IL6) and interferon-γ (IFNγ) were significantly increased (p<0.05). In conclusion, BPA prolonged exposure at low doses, consistent with those found in the environment, may affect adipocyte differentiation program, enhancing pre-adipocyte proliferation and anticipating the expression of the master genes involved in lipid/glucose metabolism. The resulting adipocytes are hypertrophic, with impaired insulin signaling, reduced glucose utilization and increased pro-inflammatory cytokine expression. Thus, these data supported the hypothesis that BPA exposure, during critical stages of adipose tissue development, may cause adipocyte metabolic dysfunction and inflammation, thereby increasing the risk of developing obesity-related diseases.

Adipose microenvironment promotes triple negative breast cancer cell invasiveness and dissemination by producing CCL5.

Growing evidence indicates that adiposity is associated with raised cancer incidence, morbidity and mortality. In a subset of tumors, cancer cell growth and/or metastasis predominantly occur in adipocyte-rich microenvironment. Indeed, adipocytes represent the most abundant cell types surrounding breast cancer cells. We have studied the mechanisms by which peritumoral human adipose tissue contributes to Triple Negative Breast Cancer (TNBC) cell invasiveness and dissemination.Co-culture with human adipocytes enhanced MDA-MB231 cancer cell invasiveness. Adipocytes cultured in high glucose were 2-fold more active in promoting cell invasion and motility compared to those cultured in low glucose. This effect is induced, at least in part, by the CC-chemokine ligand 5 (CCL5). Indeed, CCL5 inhibition by specific peptides and antibodies reduced adipocyte-induced breast cancer cell migration and invasion. CCL5 immuno-detection in peritumoral adipose tissue of women with TNBC correlated with lymph node (p-value = 0.04) and distant metastases (p-value = 0.001). A positive trend was also observed between CCL5 expression and glycaemia. Finally, Kaplan-Meier curves showed a negative correlation between CCL5 staining in the peritumoral adipose tissue and overall survival of patients (p-value = 0.039).Thus, inhibition of CCL5 in adipose microenvironment may represent a novel approach for the therapy of highly malignant TNBC.