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Introduction: Breast cancer is the most diagnosed neoplasia and the second leading cause of malignancy death in women. Drug resistance is still the major challenge in the clinical management of breast cancer patients. Indeed, despite the improvements in the early diagnosis and in the therapeutic approaches, many breast cancer patients experience disease relapse due to de novo or acquired drug resistance. Growing evidence recognized a small population of breast cancer cells, named Breast Cancer Stem Cells (BCSCs), as the leading cause of tumor progression, metastasis formation and resistance against conventional therapy. BCSCs have some specific properties such as self-renewal, differentiation into different cell types, migration, tumorsphere formation, antioxidative activity, that make tumors more aggressive. Tumor microenvironment plays a crucial role in the regulation of stem cell proliferation and resistance to apoptosis through the secretion of cytokines and growth factors. Adipocytes represent the most abundant cellular component of mammary microenvironment. The excessive fat accumulation in obesity leads to the development of a dysfunctional adipose tissue, producing an unbalance in adipokines secretion. Among the secreted factors, adiponectin plays a crucial role in breast cancer development and progression. The aim of the present study was to investigate the effects of low adiponectin level (5 μl/ml), hallmark of obese status, on BCSCs activity in hormone-resistant cells. Methods: We tested the ability of MCF-7 wild type (WT) and tamoxifen-resistant (TR) to grow as mammospheres (Mammospheres Forming Efficiency, MFE), measuring the ability to maintain cell viability (self-renewal) upon serial non-adherent passages. mRNA levels of stemness, EMT and cell cycle markers were evaluated by qRT-PCR. CD44+/24- cell ratio, ALDH expression, ROS production, cell cycle, were analyzed by flow cytometry. Results: Adiponectin treatment significantly enhanced MFE and self-renewal capacity in TR-MCF-7 cells compared to MCF-7 WT cells. To identify the presence of BCSCs into mammospheres it has been evaluated the CD44+/CD24– biomarker signature. Flow cytometry revealed an enrichment of CD44, a trans-membrane glycoprotein which regulates growth signals in stem cells, in TR-MCF-7 mammospheres, whereas expression levels of the differentiation marker CD24 were decreased. The gene expression of these biomarkers was also analyzed by qRT-PCR. The increased BCSCs subpopulation in adiponectin-treated TR-MCF-7 mammospheres was also confirmed by the enhanced ALDH-expressing cells. qRT-PCR revealed that adiponectin increased the mRNA levels of stemness and EMT markers in TR-MCF-7 cells mammospheres. Interestingly, cell cycle analysis showed in adiponectin-treated TR-MCF-7 mammospheres a reduction of apoptosis. Moreover, flow cytometry analysis displayed a reduction of ROS levels, which generally are assumed as cues determining DNA damage-induced cell death, in adiponectin-treated TR-MCF-7 mammospheres. Conclusions: Our results demonstrated that low adiponectin level, as it occurs in obese breast cancer microenvironment, driving EMT, enhances stem-like features in TR-MCF-7 cells to sustain tumor progression. Citation Format: Giuseppina Daniela Naimo, Martina Forestiero, Alessandro Paolì, Francesca Giordano, Maria Luisa Panno, Loredana Mauro, Sebastiano Andò. Adiponectin regulates stem cell activity in tamoxifen-resistant breast cancer cells [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-26-18.

Over the last two decades, obesity has reached pandemic proportions in several countries, and expanding evidence is showing its contribution to several types of malignancies, including breast cancer (BC). The conditioned medium (CM) from mature adipocytes contains a complex of secretes that may mimic the obesity condition in studies on BC cell lines conducted in vitro. Here, we report a transcriptomic analysis on MCF-7 BC cells exposed to adipocyte-derived CM and focus on the predictive functional relevance that CM-affected pathways/processes and related biomarkers (BMs) may have in BC response to obesity. CM was demonstrated to increase cell proliferation, motility and invasion as well as broadly alter the transcript profiles of MCF-7 cells by significantly modulating 364 genes. Bioinformatic functional analyses unraveled the presence of five highly relevant central hubs in the direct interaction networks (DIN), and Kaplan–Meier analysis sorted the CCAAT/enhancer binding protein beta (CEBP-β) and serine/threonine-protein kinase PLK1 (PLK1) as clinically significant biomarkers in BC. Indeed, CEBP-β and PLK1 negatively correlated with BC overall survival and were up-regulated by adipocyte-derived CM. In addition to their known involvement in cell proliferation and tumor progression, our work suggests them as a possible “deus ex machina” in BC response to fat tissue humoral products in obese women.

Glioblastoma multiforme (GBM) is one of the most aggressive types of cancer characterized by poor patient outcomes. To date, it is believed that the major cause of its recurrence and chemoresistance is represented by the enrichment of GBM stem cells (GSCs) sustained by the abnormal activation of a number of signaling pathways. In this study, we found that in GBM cells, treatment with low toxicity doses of the γ-secretase inhibitor RO4929097 (GSI), blocking the Notch pathway activity, in combination with resveratrol (RSV) was able to reverse the basal mesenchymal phenotype to an epithelial-like phenotype, affecting invasion and stemness interplay. The mechanism was dependent on cyclin D1 and cyclin-dependent kinase (CDK4), leading to a reduction of paxillin (Pxn) phosphorylation. Consequently, we discovered the reduced interaction of Pxn with vinculin (Vcl), which, during cell migration, transmits the intracellular forces to the extracellular matrix. The exogenous expression of a constitutively active Cdk4 mutant prevented the RSV + GSI inhibitory effects in GBM cell motility/invasion and augmented the expression of stemness-specific markers, as well as the neurosphere sizes/forming abilities in untreated cells. In conclusion, we propose that Cdk4 is an important regulator of GBM stem-like phenotypes and invasive capacity, highlighting how the combined treatment of Notch inhibitors and RSV could be prospectively implemented in the novel therapeutic strategies to target Cdk4 for these aggressive brain tumors.

LTG activity depends on PTEN expression.

Supplementary Methods, Figure Legends

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, p27Kip1, and p21Waf1/Cip1), 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 in vivo, increasing FoxO3a expression.Implications: The anticonvulsant drug lamotrigine shows strong antiproliferative activity on breast cancer, both in vitro and in vivo. Thus, drug repurposing could represent a valuable option for a molecularly targeted therapy in breast cancer patients. Mol Cancer Res; 16(6); 923–34. ©2018 AACR.

Introduction: EZH2 is the main enzymatic subunit of the Polycomb Repressive Complex 2 (PRC2) that promotes transcriptional repression by trimethylating histone H3 at lysine 27 (H3K27me3). Several reports evidenced EZH2 overexpression in many types of cancers, including breast cancer. In invasive breast carcinoma high levels of EZH2 are associated with triple-negative breast cancer (TNBC) status, presence of metastasis and poor clinical outcome. Despite an oncogenic role of EZH2 as a transcriptional silencer is well known, recent studies showed the association between high levels of EZH2 and low H3K27me3 in TNBC, suggesting that EZH2 may act via non-canonical mechanisms in breast cancer initiation, development, and progression. Interestingly, emerging data highlight that EZH2 can methylate non-histone substrates, regulating numerous cellular functions in a PRC2-dependent or -independent manner. We have established a critical role of p38-mediated EZH2 T367 phosphorylation in the cytoplasmic compartment in promoting cell migration, invasion, and metastasis in TNBC. Here, we tested the hypothesis that EZH2 phosphorylation at T367 induces EZH2 binding to PRC2 in the cytoplasmic compartment of triple-negative breast cancer cells to methylate non-histone proteins. Methods: In vitro and in vivo studies were carried out by using MDA-MB-231 cells. Stable knockdown followed by rescue of EZH2 was attained using lentiviral transduction of EZH2 with pBabe-myc-EZH2 (wild-type), phospho-deficient pBabe-myc-EZH2 (T367A) or HMTase-deficient p-Babe-myc-EZH2 (H694A) in MDA-MB-231 cells. To better evaluate the relevance of cytoplasmic EZH2 in breast cancer progression, we transiently overexpressed EZH2 full length (WT-EZH2) and EZH2 mutant lacking the nuclear localization signaling (ΔNLS-EZH2) adenoviral constructs in MDA-MB-231 shEZH2 cells. The EZH2-catalytic activity inhibition, in vitro and in vivo, was performed through two different drugs, GSK126 and EPZ6438, which exhibit high selectivity for EZH2 lysine N-methyltransferase (KMTase) activity maintaining PRC2 complex integrity. To investigate the interaction between p-EZH2 T367 and PRC2 complex components we carried out Co-immunoprecipitation assay (Co-IP) in the nuclear and cytoplasmic compartments of MDA-MB-231 cells. The interaction was validated by Proximity ligation assays (PLA). We evaluated EZH2 KMTase in the cytoplasmic and nuclear compartments using MTase-Glo Methyltransferase assay in vitro and in vivo. Xenografts obtained from NOD/SCID mice orthotopically injected with MDA-MB-231 cells and treated 5 days/weeks by intraperitoneal injection with 4% DMSO-30% PEG 300-5% Tween 80 (control), or EPZ-6438 (10mg/kg/day) were used to test EZH2 KMTase in vivo. Results: Co-IP and PLA revealed the binding of p-EZH2 T367 to PRC2-complex members in the cytoplasmic compartment of MDA-MB-231 cells expressing WT-EZH2, H694A-EZH2 and ΔNLS-EZH2. In contrast, T367A-EZH2 showed reduced binding to PRC2 members in the cytoplasm. To test the biological relevance of this interaction we measured the KMTase of WT-EZH2 and mutants-EZH2 in the cytoplasmic fraction of MDA-MB-231 cells. Methyltransferase assays using p38 and histone H3 as substrates, showed that WT-EZH2, H694A-EZH2 and ΔNLS-EZH2 are able to methylate p38 protein in the cytoplasmic fraction of MDA-MB-231 cells, and that this requires T367 phosphorylation of EZH2. Similar results were obtained in xenografts of MDA-MB-231 cells in vivo. Conclusion: Our results provide evidence that EZH2 phosphorylation at T376 induces EZH2 binding to PRC2-complex members in the cytoplasm of triple-negative breast cancer cells to methylate non-histone proteins in vitro and in vivo. These data deepen our understanding of the mechanism(s) by which pEZH2 T367 functions in TNBC progression Citation Format: Giuseppina Daniela Naimo, Maria Elena Gonzalez, Shilpa Reddy Tekula, Jessica Camille Gauss, Loredana Mauro, Sebastiano Andò, Celina Graciela Kleer. Novel pEZH2 T367-PRC2 interaction and methyltransferase activity in the nuclear and cytoplasmic fractions of breast cancer cells [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-12-05.

Introduction: Triple negative breast cancer (TNBC) represents ~15% of all breast cancers. The TNBC subtype is characterized by the absence of estrogen and progesterone receptors and of ErbB2. Biologically, it is well recognized that TNBC has a more aggressive phenotype compared to other subtypes, offering limited treatment options and a worse clinical outcome. Tumor cells, through production of paracrine and endocrine molecules, can recruit mesenchymal stem/stromal cells (MSCs) from the bone marrow and adipose tissue to the tumor microenvironment. MSCs, reside mainly in the bone marrow and in adipose tissue. MSCs may exert a crucial role in tumor development and progression, by increasing stemness of tumor cells and promoting extracellular matrix remodeling, cell migration and invasion, neo-angiogenesis, and drug resistance. We have shown that breast cancer cells (BCCs) can engulf MSCs and induce an aggressive phenotype and increased metastasis. The mechanisms through which MSC engulfment occurs and the generated phenotypes need to be investigated. In this study, we tested the hypothesis that MSC engulfment by BCC cells may result in a hybrid tumor cell population with features of cell senescence, a well-recognized mechanism of tumor progression and recurrence. Methods: We employed MDA-MB-231 TNBC cells and patient-derived MSCs developed and validated in our lab. MDA-MB-231 were labeled with DsRED (231- DsRED) and MSCs were not labeled. Single culture of MDA-MB-231, MSCs, and MDA-MB-231/MSC co-cultures (1:1 ratio) were subjected to SA-β-galactosidase activity assay, flow cytometry, and immunofluorescence to detect and quantify the percentage of hybrid and senescence cells. SA-β-galactosidase activity assay was assessed using Senescence-associated β-galactosidase Staining Kit. For each sample of the single cultures and co-cultures, 10 randomly chosen fields were photographed using a camera-equipped bright field microscope (Olympus). Data was plotted as the percentage of cells with β-galactosidase activity (blue cells). We performed flow cytometry to detect β-galactosidase activity in MSCs and in DsRED-231 in single cultures or co-cultures. Immunofluorescence assay was performed to evaluate β-galactosidase activity in the same conditions previously described. Results: Our studies identified the emergence of a hybrid cell population in the co-cultures of MDA-MB-231 and MSCs cells. The hybrid population contained a significantly higher percentage of senenscent cells compared to 231 and MSCs (44%, 10.23%, and 14%, respectively, t test 0.001). These results were confirmed by immunofluorescence analysis for β-galactosidase, DsRed and DAPI in situ. Conclusion: These results highlight that MSCs engulfment by TNBC cells induces the formation of senescent hybrid breast cancer cells, which may explain their survival advantage. Studies are under way to investigate if the senescent phenotype of hybrid cells is influenced by EMT-MET states and its role in TNBC chemoresistance. Citation Format: Alessandro Paolì, Maria Elena Gonzalez, Loredana Mauro, Sebastiano Andò, Celina Graciela Kleer. Msc engulfing by triple negative breast cancer cells induces a hybrid cell population with senescence [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-12-06.

We present an innovative approach allowing the identification, isolation, and molecular characterization of disease-related exosomes based on their different antigenic reactivities. The designed strategy could be immediately translated into any disease in which exosomes are involved. The identification of specific markers and their subsequent association with exosome subtypes, together with the possibility to engineer target-guided exosome-like particles, could represent the key for the effective adoption of exosomes in clinical practice.

Molecule activity predictor analysis of Ingenuity Pathway Analysis performed on Cdc42 signaling and Rac signaling in PDE5-overexpressing MCF-7 cells compared to vector-transfected cells.

Differentially expressed genes identified by RNA sequencing in PDE5-overexpressing MCF-7 cells compared to empty-vector transfected cells.

Effects of PDE5 overexpression on c-Myc and NF-κB Activation.

Detail description of microarray analysis and comparison with cancer stem cell dataset, and TMA of prostate cancer patients

RNA library preparation and sequencing. Classification of molecular subtypes. Immunohistochemical analysis.

Supplemental Figure 1. Trop2 ICD abundance is maintained by Src kinase. Supplemental Figure 2. Canertinib, a tyrosine kinase inhibitor targeting EGFR reduces cell proliferation but does not affect Trop2 ICD abundance. Supplemental Figure 3. Schematic representation of the mechanism by which Src induction of cyclin D1 enhances cleavage of the membrane associated Trop2 complex to thereby enhance Trop2 ICD. Supplemental Figure 4. Human prostate cancer Tissue Microarray (TMA) IHC staining.

Characteristic of breast cancer cohort.

Supplemental Figure 1. Trop2 ICD abundance is maintained by Src kinase. Supplemental Figure 2. Canertinib, a tyrosine kinase inhibitor targeting EGFR reduces cell proliferation but does not affect Trop2 ICD abundance. Supplemental Figure 3. Schematic representation of the mechanism by which Src induction of cyclin D1 enhances cleavage of the membrane associated Trop2 complex to thereby enhance Trop2 ICD. Supplemental Figure 4. Human prostate cancer Tissue Microarray (TMA) IHC staining.

Proteomic analysis of castration-resistant prostate cancer demonstrated the enrichment of Src tyrosine kinase activity in approximately 90% of patients. Src is known to induce cyclin D1, and a cyclin D1–regulated gene expression module predicts poor outcome in human prostate cancer. The tumor-associated calcium signal transducer 2 (TACSTD2/Trop2/M1S1) is enriched in the prostate, promoting prostate stem cell self-renewal upon proteolytic activation via a γ-secretase cleavage complex (PS1, PS2) and TACE (ADAM17), which releases the Trop2 intracellular domain (Trop2 ICD). Herein, v-Src transformation of primary murine prostate epithelial cells increased the proportion of prostate cancer stem cells as characterized by gene expression, epitope characteristics, and prostatosphere formation. Cyclin D1 was induced by v-Src, and Src kinase induction of Trop2 ICD nuclear accumulation required cyclin D1. Cyclin D1 induced abundance of the Trop2 proteolytic cleavage activation components (PS2, TACE) and restrained expression of the inhibitory component of the Trop2 proteolytic complex (Numb). Patients with prostate cancer with increased nuclear Trop2 ICD and cyclin D1, and reduced Numb, had reduced recurrence-free survival probability (HR = 4.35). Cyclin D1, therefore, serves as a transducer of v-Src–mediated induction of Trop2 ICD by enhancing abundance of the Trop2 proteolytic activation complex. Cancer Res; 76(22); 6723–34. ©2016 AACR.

Purpose: By catalyzing cGMP hydrolysis, phosphodiesterase (PDE) 5 is a critical regulator of its concentration and effects in different (patho)physiologic processes, including cancers. As PDE5 is a known druggable target, we investigated the clinical significance of its expression in breast cancer and the underlying mechanisms by which it may contribute to tumor progression.Experimental Design: PDE5 expression was evaluated in seven breast cancer cell lines by RT-PCR and immunoblotting. To examine the impact of PDE5 on cancer phenotype, MCF-7 cells expressing lower levels of the enzyme were engineered to stably overexpress PDE5. Proliferation was evaluated by MTT assays, motility and invasion by wound-healing/transmigration/invasion assays, transcriptome-profiling by RNA-sequencing, and Rho GTPase signaling activation by GST-pulldown assays and immunoblotting. Clinical relevance was investigated by IHC on tissues and retrospective studies from METABRIC cohort.Results: PDE5 is differentially expressed in each molecular subtype of both breast cancer cell lines and tissues, with higher levels representing a startling feature of HER2-positive and triple-negative breast cancers. A positive correlation was established between elevated PDE5 levels and cancers of high histologic grade. Higher PDE5 expression correlated with shorter patient survival in retrospective analyses. On molecular level, stable PDE5 overexpression in Luminal-A–like MCF-7 cells resulted in enhanced motility and invasion through Rho GTPase signaling activation. Treatment of PDE5-stable clones with selective ROCK or PDE5 inhibitors completely restored the less motile and weak invasive behavior of control vector cells.Conclusions: PDE5 expression enhances breast cancer cell invasive potential, highlighting this enzyme as a novel prognostic candidate and an attractive target for future therapy in breast cancers. Clin Cancer Res; 22(9); 2271–82. ©2015 AACR.

Supplementary Figures 1-3 from Evidences that Leptin Up-regulates E-Cadherin Expression in Breast Cancer: Effects on Tumor Growth and Progression

Supplementary Figures 1-3 from Insulin-Like Growth Factor-I, Regulating Aromatase Expression through Steroidogenic Factor 1, Supports Estrogen-Dependent Tumor Leydig Cell Proliferation

Supplemental Figures S1-S12. Supplement 1. High resolution images of normal prostate and v-Src-PEC tumor Supplement 2. AR, CK5 and CK8 abundances in cultured v-Src-PECs compared with primary cultured normal PECs detected by western-blot. Supplement 3. High resolution images of v-Src-PEC bone metastasis tumor and adjacent bone marrow Supplement 4. Kegg-pathway analysis of up-regulated genes in v-Src-PEC tumor vs normal prostate Supplement 5. High resolution gene expression profile comparing normal FVB murine prostate epithelial cells Supplement 6. Validation of CCR5 expression by qtPCR in v-Src-PEC tumors derived by subcutaneous injection compared with normal prostate Supplement 7. CCR5 expression in public available database Supplement 8. FACS analysis of CCR5 positive population in v-Src-PECs Supplement 9. High resolution image of immunohistochemical staining for CCR5 in v-Src-PEC metastasis in bone which showed in figure 5 Supplement 10. High resolution image of (A-D) histological images Supplement 11. CK5 (A) and CK8 (C) immunohistochemical staining in the prostate of castrated mice vs normal mice. Supplement 12. CCR5 IHC staining in bone metastasis v-Src-PEC tumor.

Supplementary Figure 1 from Absence of the Full-Length Breast Cancer–Associated Gene-1 Leads to Increased Expression of Insulin-Like Growth Factor Signaling Axis Members

Legend for Supplementary Figures S1-S12.

The aim of this study was to investigate the role of estrogens in Leydig cell tumor proliferation. We used R2C rat Leydig tumor cells and testicular samples from Fischer rats with a developed Leydig tumor. Both experimental models express high levels of aromatase and estrogen receptor α (ERα). Treatment with exogenous 17β-estradiol (E2) induced proliferation of R2C cells and up-regulation of cell cycle regulators cyclin D1 and cyclin E, the expression of which was blocked by addition of antiestrogens. These observations led us to hypothesize an E2/ERα–dependent mechanism for Leydig cell tumor proliferation. In determining the molecular mechanism responsible for aromatase overexpression, we found that total and phosphorylated levels of transcription factors cyclic AMP–responsive element binding protein and steroidogenic factor 1 (SF-1) were higher in tumor samples. Moreover, we found that tumor Leydig cells produce high levels of insulin-like growth factor I (IGF-I), which increased aromatase mRNA, protein, and activity as a consequence of increased total and phosphorylated SF-1 levels. Specific inhibitors of IGF-I receptor, protein kinase C, and phosphatidylinositol 3-kinase determined a reduction in SF-1 expression and in IGF-I–dependent SF-1 recruitment to the aromatase PII promoter. The same inhibitors also inhibited aromatase expression and activity and, consequently, R2C cell proliferation. We can conclude that one of the molecular mechanisms determining Leydig cell tumorigenesis is an excessive estrogen production that stimulates a short autocrine loop determining cell proliferation. In addition, cell-produced IGF-I amplifies estrogen signaling through an SF-1–dependent up-regulation of aromatase expression. The identification of this molecular mechanism will be helpful in defining new therapeutic approaches for Leydig cell tumors. [Cancer Res 2007;67(17):8368–77]

The breast cancer–associated gene-1 (BRCA1) plays many important functions in multiple biological processes/pathways. Mice homozygous for a targeted deletion of full-length BRCA1 (Brca1Δ11/Δ11) display both increased tumorigenesis and premature aging, yet molecular mechanisms underlying these defects remain elusive. Here, we show that Brca1 deficiency leads to increased expression of several insulin-like growth factor (IGF) signaling axis members in multiple experimental systems, including BRCA1-deficient mice, primary mammary tumors, and cultured human cells. Furthermore, we provide evidence that activation of IGF signaling by BRCA1 deficiency can also occur in a p53-independent fashion. Our data indicate that BRCA1 interacts with the IRS-1 promoter and inhibits its activity that is associated with epigenetic modification of histone H3 and histone H4 to a transcriptional repression chromatin configuration. We further show that BRCA1-deficient mammary tumor cells exhibit high levels of IRS-1, and acute suppression of Irs-1 using RNA interference significantly inhibits growth of these cells. Those observations provide a molecular insight in understanding both fundamental and therapeutic BRCA1-associated tumorigenesis and aging. (Cancer Res 2006; 66(14): 7151-7)

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We hereby apply the approach of photoacoustic chemical imaging, performing an in vivo chemical analysis that is spatially resolved (200 µm) and in real time, to predict a given tumor’s response to therapy. Using triple negative breast cancer as a model, we took photoacoustic images of tumors’ oxygen distributions in patient-derived xenografts (PDXs) in mice using biocompatible, oxygen-sensitive tumor-targeted chemical contrast nano-elements (nanosonophores), which function as contrast agents for photoacoustic imaging. Following radiation therapy, we established a quantitatively significant correlation between the spatial distribution of the initial oxygen levels in the tumor and its spatial distribution of the therapy’s efficacy: the lower the local oxygen, the lower the local radiation therapy efficacy. We thus provide a simple, noninvasive, and inexpensive method to both predict the efficacy of radiation therapy for a given tumor and identify treatment-resistant regions within the tumor’s microenvironment.

Triple-negative breast cancer (TNBC), an aggressive breast cancer subtype lacking effective targeted therapies, is considered to feature a unique cellular microenvironment with high infiltration of tumor-associated macrophages (TAM), which contribute to worsening breast cancer patient outcomes. Previous studies have shown the antitumoral actions of the dietary omega-3 docosahexaenoic acid (DHA) in both tumor epithelial and stromal components of the breast cancer microenvironment. Particularly in breast cancer cells, DHA can be converted into its conjugate with ethanolamine, DHEA, leading to a more effective anti-oncogenic activity of the parent compound in estrogen receptor-positive breast cancer cells. Here, we investigated the ability of DHEA to attenuate the malignant phenotype of MDA-MB-231 and MDA-MB-436 TNBC cell lines, which in turn influenced TAM behaviors. Our findings revealed that DHEA reduced the viability of TNBC cells in a concentration-dependent manner and compromised cell migration and invasion. Interestingly, DHEA inhibited oxygen consumption and extracellular acidification rates, reducing respiration and the glycolytic reserve in both cell lines. In a co-culture system, TNBC cells exposed to DHEA suppressed recruitment of human THP-1 cells, reduced their viability, and the expression of genes associated with TAM phenotype. Interestingly, we unraveled that the effects of DHEA in TNCB cells were mediated by reduced C-C motif chemokine ligand 5 (CCL5) expression and secretion affecting macrophage recruitment. Overall, our data, shedding new light on the antitumoral effects of DHA ethanolamine-conjugated, address this compound as a promising option in the treatment of TNBC patients.

The traditional Mediterranean diet (MD) is characterized by a high phenolic-rich food intake, including in particular vegetables and fruits, but also legumes, whole grain cereals, nuts, and extra virgin olive oil. Evidence for beneficial effects of polyphenols in humans depends on the amount consumed and on their bioavailability. Here, we evaluated the association between the estimated polyphenol intake by fruits and vegetables food source and serum biochemical parameters in healthy adolescents, recruited into the DIMENU research project. Categorizing adolescents into three groups according to their estimated total polyphenol intake, we found that adolescents who declared high consumption of polyphenols had a higher adherence to the MD and had a better serum lipid profile than adolescents consuming low amounts of polyphenols. Moreover, using human HepG2 liver cells treated with oleic acid as an in vitro model for studying lipid accumulation, we showed that intracellular lipid accumulation is alleviated by serum from adolescents consuming a polyphenol-rich diet following MD recommendations. Our data underline the importance of promoting adherence to the typical MD foods as a superior strategy to prevent metabolic and chronic diseases and to ensure a better quality of life among adolescents.

Extracellular vesicles (EVs) are emerging key protagonists in intercellular communication between adipocytes and breast cancer (BC) cells. Here, we described a new mechanism by which EVs released by mature adipocytes promoted breast cancer cell malignancy "in vitro" and "in vivo". We found that adipocyte-derived EVs enhanced growth, motility and invasion, stem cell-like properties, as well as specific traits of epithelial-to-mesenchymal transition in both estrogen receptor positive and triple negative BC cells. Of note, adipocyte-derived EVs aid breast tumor cells in lung metastatic colonization after tail-vein injection in mice. These EV-mediated effects occur via the induction of HIF-1α activity, since they were abrogated by the use of the HIF-1α inhibitor KC7F2 or in cells silenced for HIF-1α expression. Moreover, using an “ex vivo” model of obese adipocytes we found that the depletion of EVs counteracted the ability of obese adipocytes to sustain pro-invasive phenotype in BC cells. Interestingly, EVs released by undifferentiated adipocytes failed to induce aggressiveness and HIF-1α expression. These findings shed new light on the role of adipocyte-derived EVs in breast cancer progression, suggesting the possibility to target HIF-1α activity to block the harmful adipocyte-tumor cell dialogue, especially in obese settings.