Autologous patient-derived exhausted nano T-cells exploit tumor immune evasion to engage an effective cancer therapy.

BACKGROUND: Active targeting by surface-modified nanoplatforms enables a more precise and elevated accumulation of nanoparticles within the tumor, thereby enhancing drug delivery and efficacy for a successful cancer treatment. However, surface functionalization involves complex procedures that increase costs and timelines, presenting challenges for clinical implementation. Biomimetic nanoparticles (BNPs) have emerged as unique drug delivery platforms that overcome the limitations of actively targeted nanoparticles. Nevertheless, BNPs coated with unmodified cells show reduced functionalities such as specific tumor targeting, decreasing the therapeutic efficacy. Those challenges can be overcome by engineering non-patient-derived cells for BNP coating, but these are complex and cost-effective approaches that hinder their wider clinical application. Here we present an immune-driven strategy to improve nanotherapeutic delivery to tumors. Our unique perspective harnesses T-cell exhaustion and tumor immune evasion to develop a groundbreaking new class of BNPs crafted from exhausted T-cells (NExT) of triple-negative breast cancer (TNBC) patients by specific culture methods without sophisticated engineering. METHODS: NExT were generated by coating PLGA (poly(lactic-co-glycolic acid)) nanoparticles with TNBC-derived T-cells exhausted in vitro by acute activation. Physicochemical characterization of NExT was made by dynamic light scattering, electrophoretic light scattering and transmission electron microscopy, and preservation and orientation of immune checkpoint receptors by flow cytometry. The efficacy of chemotherapy-loaded NExT was assessed in TNBC cell lines in vitro. In vivo toxicity was made in CD1 mice. Biodistribution and therapeutic activity of NExT were determined in cell-line- and autologous patient-derived xenografts in immunodeficient mice. RESULTS: We report a cost-effective approach with a good performance that provides NExT naturally endowed with immune checkpoint receptors (PD1, LAG3, TIM3), augmenting specific tumor targeting by engaging cognate ligands, enhancing the therapeutic efficacy of chemotherapy, and disrupting the PD1/PDL1 axis in an immunotherapy-like way. Autologous patient-derived NExT revealed exceptional intratumor accumulation, heightened chemotherapeutic index and efficiency, and targeted the tumor stroma in a PDL1+ patient-derived xenograft model of triple-negative breast cancer. CONCLUSIONS: These advantages underline the potential of autologous patient-derived NExT to revolutionize tailored adoptive cancer nanotherapy and chemoimmunotherapy, which endorses their widespread clinical application of autologous patient-derived NExT.

Unlocking the effective alliance of ß-lapachone and hydroxytyrosol against triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) is characterised by its aggressiveness and resistance to chemotherapy, demanding the development of effective strategies against its unique characteristics. Derived from lapacho tree bark, ß-lapachone (ß-LP) selectively targets cancer cells with elevated levels of the detoxifying enzyme NQO1. Hydroxytyrosol (HT) is a phenolic compound derived from olive trees with important anticancer properties that include the inhibition of cancer stem cells (CSCs) and metastatic features in TNBC, as well as relevant antioxidant activities by mechanisms such as the induction of NQO1. We aimed to study whether these compounds could have synergistic anticancer activity in TNBC cells and the possible role of NQO1. For this pourpose, we assessed the impact of ß-LP (0.5 or 1.5 µM) and HT (50 and 100 µM) on five TNBC cell lines. We demonstrated that the combination of ß-LP and HT exhibits anti-proliferative, pro-apoptotic, and cell cycle arrest effects in several TNBC cells, including docetaxel-resistant TNBC cells. Additionally, it effectively inhibits the self-renewal and clonogenicity of CSCs, modifying their aggressive phenotype. However, the notable impact of the ß-LP-HT combination does not appear to be solely associated with the levels of the NQO1 protein and ROS. RNA-Seq analysis revealed that the combination's anticancer activity is linked to a strong induction of endoplasmic reticulum stress and apoptosis through the unfolded protein response. In conclusion, in this study, we demonstrated how the combination of ß-LP and HT could offer an affordable, safe, and effective approach against TNBC.

TGFß Governs the Pleiotropic Activity of NDRG1 in Triple-Negative Breast Cancer Progression.

In triple-negative breast cancer (TNBC), the pleiotropic NDRG1 (N-Myc downstream regulated gene 1) promotes progression and worse survival, yet contradictory results were documented, and the mechanisms remain unknown. Phosphorylation and localization could drive NDRG1 pleiotropy, nonetheless, their role in TNBC progression and clinical outcome was not investigated. We found enhanced p-NDRG1 (Thr346) by TGFß1 and explored whether it drives NDRG1 pleiotropy and TNBC progression. In tissue microarrays of 81 TNBC patients, we identified that staining and localization of NDRG1 and p-NDRG1 (Thr346) are biomarkers and risk factors associated with shorter overall survival. We found that TGFß1 leads NDRG1, downstream of GSK3ß, and upstream of NF-κB, to differentially regulate migration, invasion, epithelial-mesenchymal transition, tumor initiation, and maintenance of different populations of cancer stem cells (CSCs), depending on the progression stage of tumor cells, and the combination of TGFß and GSK3ß inhibitors impaired CSCs. The present study revealed the striking importance to assess both total NDRG1 and p-NDRG1 (Thr346) positiveness and subcellular localization to evaluate patient prognosis and their stratification. NDRG1 pleiotropy is driven by TGFß to differentially promote metastasis and/or maintenance of CSCs at different stages of tumor progression, which could be abrogated by the inhibition of TGFß and GSK3ß.

EZH2 endorses cell plasticity to non-small cell lung cancer cells facilitating mesenchymal to epithelial transition and tumour colonization.

Reversible transition between the epithelial and mesenchymal states are key aspects of carcinoma cell dissemination and the metastatic disease, and thus, characterizing the molecular basis of the epithelial to mesenchymal transition (EMT) is crucial to find druggable targets and more effective therapeutic approaches in cancer. Emerging studies suggest that epigenetic regulators might endorse cancer cells with the cell plasticity required to conduct dynamic changes in cell state during EMT. However, epigenetic mechanisms involved remain mostly unknown. Polycomb Repressive Complexes (PRCs) proteins are well-established epigenetic regulators of development and stem cell differentiation, but their role in different cancer systems is inconsistent and sometimes paradoxical. In this study, we have analysed the role of the PRC2 protein EZH2 in lung carcinoma cells. We found that besides its described role in CDKN2A-dependent cell proliferation, EZH2 upholds the epithelial state of cancer cells by repressing the transcription of hundreds of mesenchymal genes. Chemical inhibition or genetic removal of EZH2 promotes the residence of cancer cells in the mesenchymal state during reversible epithelial-mesenchymal transition. In fitting, analysis of human patient samples and tumour xenograft models indicate that EZH2 is required to efficiently repress mesenchymal genes and facilitate tumour colonization in vivo. Overall, this study discloses a novel role of PRC2 as a master regulator of EMT in carcinoma cells. This finding has important implications for the design of therapies based on EZH2 inhibitors in human cancer patients.

Nanomedicine as a Promising Tool to Overcome Immune Escape in Breast Cancer.

Breast cancer is the most common type of malignancy and leading cause of cancer death among women worldwide. Despite the current revolutionary advances in the field of cancer immunotherapy, clinical response in breast cancer is frequently below expectations, in part due to various mechanisms of cancer immune escape that produce tumor variants that are resistant to treatment. Thus, a further understanding of the molecular events underlying immune evasion in breast cancer may guarantee a significant improvement in the clinical success of immunotherapy. Furthermore, nanomedicine provides a promising opportunity to enhance the efficacy of cancer immunotherapy by improving the delivery, retention and release of immunostimulatory agents in targeted cells and tumor tissues. Hence, it can be used to overcome tumor immune escape and increase tumor rejection in numerous malignancies, including breast cancer. In this review, we summarize the current status and emerging trends in nanomedicine-based strategies targeting cancer immune evasion and modulating the immunosuppressive tumor microenvironment, including the inhibition of immunosuppressive cells in the tumor area, the activation of dendritic cells and the stimulation of the specific antitumor T-cell response.

Antioxidants for the Treatment of Breast Cancer: Are We There Yet?

Breast cancer is the most frequent cancer and the leading cause of cancer death in women. Oxidative stress and the generation of reactive oxygen species (ROS) have been related to cancer progression. Compared to their normal counterparts, tumor cells show higher ROS levels and tight regulation of REDOX homeostasis to maintain a low degree of oxidative stress. Traditionally antioxidants have been extensively investigated to counteract breast carcinogenesis and tumor progression as chemopreventive agents; however, there is growing evidence indicating their potential as adjuvants for the treatment of breast cancer. Aimed to elucidate whether antioxidants could be a reality in the management of breast cancer patients, this review focuses on the latest investigations regarding the ambivalent role of antioxidants in the development of breast cancer, with special attention to the results derived from clinical trials, as well as their potential use as plausible agents in combination therapy and their power to ameliorate the side effects attributed to standard therapeutics. Data retrieved herein suggest that antioxidants play an important role in breast cancer prevention and the improvement of therapeutic efficacy; nevertheless, appropriate patient stratification based on "redoxidomics" or tumor subtype is mandatory in order to define the dosage for future standardized and personalized treatments of patients.

Drug Repurposing for Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is the most aggressive type of breast cancer which presents a high rate of relapse, metastasis, and mortality. Nowadays, the absence of approved specific targeted therapies to eradicate TNBC remains one of the main challenges in clinical practice. Drug discovery is a long and costly process that can be dramatically improved by drug repurposing, which identifies new uses for existing drugs, both approved and investigational. Drug repositioning benefits from improvements in computational methods related to chemoinformatics, genomics, and systems biology. To the best of our knowledge, we propose a novel and inclusive classification of those approaches whereby drug repurposing can be achieved in silico: structure-based, transcriptional signatures-based, biological networks-based, and data-mining-based drug repositioning. This review specially emphasizes the most relevant research, both at preclinical and clinical settings, aimed at repurposing pre-existing drugs to treat TNBC on the basis of molecular mechanisms and signaling pathways such as androgen receptor, adrenergic receptor, STAT3, nitric oxide synthase, or AXL. Finally, because of the ability and relevance of cancer stem cells (CSCs) to drive tumor aggressiveness and poor clinical outcome, we also focus on those molecules repurposed to specifically target this cell population to tackle recurrence and metastases associated with the progression of TNBC.

GARP is a key molecule for mesenchymal stromal cell responses to TGF-ß and fundamental to control mitochondrial ROS levels.

Multipotent mesenchymal stromal cells (MSCs) have emerged as a promising cell therapy in regenerative medicine and for autoimmune/inflammatory diseases. However, a main hurdle for MSCs-based therapies is the loss of their proliferative potential in vitro. Here we report that glycoprotein A repetitions predominant (GARP) is required for the proliferation and survival of adipose-derived MSCs (ASCs) via its regulation of transforming growth factor-ß (TGF-ß) activation. Silencing of GARP in human ASCs increased their activation of TGF-ß which augmented the levels of mitochondrial reactive oxygen species (mtROS), resulting in DNA damage, a block in proliferation and apoptosis. Inhibition of TGF-ß signaling reduced the levels of mtROS and DNA damage and restored the ability of GARP-/low ASCs to proliferate. In contrast, overexpression of GARP in ASCs increased their proliferative capacity and rendered them more resistant to etoposide-induced DNA damage and apoptosis, in a TGF-ß-dependent manner. In summary, our data show that the presence or absence of GARP on ASCs gives rise to distinct TGF-ß responses with diametrically opposing effects on ASC proliferation and survival.

Heart Histopathology and Mitochondrial Ultrastructure in Aged Rats Fed for 24 Months on Different Unsaturated Fats (Virgin Olive Oil, Sunflower Oil or Fish Oil) and Affected by Different Longevity.

Diet plays a decisive role in heart physiology, with lipids having especial importance in pathology prevention and development. This study aimed to investigate how dietary lipids varying in lipid profile (virgin olive oil, sunflower oil or fish oil) affected the heart of rats during aging. Heart histopathology, mitochondrial morphometry, and oxidative status were assessed. Typical histopathological features associated with aging, such as valvular lesions, endomyocardical hyperplasia, or papillary muscle calcification, were found at a low extent in all the experimental groups. The most relevant finding was that inflammation registered by fish oil group was lower compared to the other treatments. At the ultrastructural level, heart mitochondrial area, perimeter, and aspect ratio were higher in fish oil-fed rats than in those fed on sunflower oil. Concerning oxidative stress markers, there were differences only in coenzyme Q levels and catalase activity, lower in sunflower oil-fed animals compared with those fed on fish oil. In summary, dietary intake for a long period on dietary fats with different fatty acids profile led to differences in some aspects associated with the aging process at the heart. Fish oil seems to be the fat most protective of heart during aging.

Chemical Profile and Safety Assessment of a Food-Grade Acetogenin-Enriched Antimicrobial Extract from Avocado Seed.

Acetogenins are bioactive fatty acid derivatives found in avocado tissues. Their efficacy as antimicrobials has been documented and initiated interest to use them as replacements of synthetic food additives. The present work focused on evaluation of multiple analytical methodologies for detection and quantification of organic solids present in a food-grade acetogenin-enriched extract (Avosafe®), and on its safety evaluations using bacterial reverse mutation (AMES) tests and acute oral toxicity to rat assays. Results confirmed chemical structures of two acetogenins as present in Avosafe® (AcO-avocadyne-(0) and AcO-avocadiene B-(3)), and together with seven other previously known compounds, quantified 94.74 ± 5.77% w/w of its solids as acetogenins. Safety evaluations indicated that Avosafe® was non-mutagenic and had an acute median lethal oral dose (LD50) to rats higher than the maximum concentration tested (>2000 mg·kg-1), with no signs of macroscopic abnormalities in organs. Mean body weight and hematological and biochemical parameters were normal after 14 days of a single oral dose of 2000 mg·kg-1. The results advance scientific information on the safety of avocado seed acetogenins and also generate new knowledge on profiles and concentrations of individual acetogenins found in avocado tissues (seed, pulp, and leaves) and in Avosafe®.

Modulation by hydroxytyrosol of oxidative stress and antitumor activities of paclitaxel in breast cancer.

PURPOSE: The main objective of this study was to test the therapeutic potential of hydroxytyrosol and its combination with paclitaxel in breast cancer on oxidative stress status. METHODS: Impact on proliferation rates of different chemotherapy administration patterns was assayed in MCF-7 and MDA-MB-231 breast cancer cell lines. Breast tumor-bearing rats were randomly assigned to Control, Hydroxytyrosol, Paclitaxel and Paclitaxel plus hydroxytyrosol groups, for 6 weeks. Tumor volume, cell proliferation and several systemic oxidative stress parameters were measured. Anti-proliferative activity in vitro experiments was correlated with in vivo experiments. RESULTS: Combination group did significantly reduce tumor volume when compared with paclitaxel alone. Additionally, the combination improved the antioxidant status without compromising the antitumor activity of standard chemotherapy. CONCLUSION: These findings reveal for the first time that hydroxytyrosol is an active partner in combined therapies with paclitaxel against breast cancer. Combination with hydroxytyrosol would also ensure a less oxidative impact of chemotherapeutic drugs that could potentially improve patient wellness.

Hydroxytyrosol inhibits cancer stem cells and the metastatic capacity of triple-negative breast cancer cell lines by the simultaneous targeting of epithelial-to-mesenchymal transition, Wnt/ß-catenin and TGFß signaling pathways.

PURPOSE: This study was aimed to determine the impact of hydroxytyrosol (HT), a minor compound found in olive oil, on breast cancer stem cells (BCSCs) and the migration capacity of triple-negative breast cancer (TNBC) cell lines through the alteration of epithelial-to-mesenchymal transition (EMT) and embryonic signaling pathways. METHODS: BCSCs self-renewal was determined by the mammosphere-forming efficiency in SUM159PT, BT549, MDA-MB-231 and Hs578T TNBC cell lines. Flow cytometric analysis of CD44+/CD24-/low and aldehyde dehydrogenase positive (ALDH+) subpopulations, migration by the "wound healing assay", invasion and Western blot of EMT markers and TGFß signaling were investigated in SUM159PT, BT549 and MDA-MB-231 cell lines. Wnt/ß-catenin signaling was assessed by Western blot in BT549 cells expressing WNT1 and MDA-MB-231 cells. Changes in TGFß activity was determined by SMAD Binding Element (SBE) reporter assay. RESULTS: HT reduced BCSCs self-renewal, ALDH+ (aldehyde dehydrogenase) and CD44+/CD24-/low subpopulations, tumor cell migration and invasion. Consistently, HT suppressed Wnt/ß-catenin signaling by decreasing p-LRP6, LRP6, ß-catenin and cyclin D1 protein expression and the EMT markers SLUG, ZEB1, SNAIL and VIMENTIN. Finally, HT inhibited p-SMAD2/3 and SMAD2/3 in SUM159PT, BT549 and MDA-MB-231 cells, what was correlated with a less TGFß activity. CONCLUSION: In conclusion, we report for the first time the inhibitory role of HT on BCSCs and tumor cell migration by targeting EMT, Wnt/ß-catenin and TGFß signaling pathways. Our findings highlight the importance of the chemopreventive compound HT as a novel candidate to be investigated as an alternative targeted therapy for TNBC.

Activating Transcription Factor 4 Modulates TGFß-Induced Aggressiveness in Triple-Negative Breast Cancer via SMAD2/3/4 and mTORC2 Signaling.

Purpose: On the basis of the identified stress-independent cellular functions of activating transcription factor 4 (ATF4), we reported enhanced ATF4 levels in MCF10A cells treated with TGFß1. ATF4 is overexpressed in patients with triple-negative breast cancer (TNBC), but its impact on patient survival and the underlying mechanisms remain unknown. We aimed to determine ATF4 effects on patients with breast cancer survival and TNBC aggressiveness, and the relationships between TGFß and ATF4. Defining the signaling pathways may help us identify a cell signaling-tailored gene signature.Experimental Design: Patient survival data were determined by Kaplan-Meier analysis. Relationship between TGFß and ATF4, their effects on aggressiveness (tumor proliferation, metastasis, and stemness), and the underlying pathways were analyzed in three TNBC cell lines and in vivo using patient-derived xenografts (PDX).Results: ATF4 overexpression correlated with TNBC patient survival decrease and a SMAD-dependent crosstalk between ATF4 and TGFß was identified. ATF4 expression inhibition reduced migration, invasiveness, mammosphere-forming efficiency, proliferation, epithelial-mesenchymal transition, and antiapoptotic and stemness marker levels. In PDX models, ATF4 silencing decreased metastases, tumor growth, and relapse after chemotherapy. ATF4 was shown to be active downstream of SMAD2/3/4 and mTORC2, regulating TGFß/SMAD and mTOR/RAC1-RHOA pathways independently of stress. We defined an eight-gene signature with prognostic potential, altered in 45% of 2,509 patients with breast cancer.Conclusions: ATF4 may represent a valuable prognostic biomarker and therapeutic target in patients with TNBC, and we identified a cell signaling pathway-based gene signature that may contribute to the development of combinatorial targeted therapies for breast cancer. Clin Cancer Res; 24(22); 5697-709. ©2018 AACR.

The ERß4 variant induces transformation of the normal breast mammary epithelial cell line MCF-10A; the ERß variants ERß2 and ERß5 increase aggressiveness of TNBC by regulation of hypoxic signaling.

Triple negative breast cancer (TNBC) still remains a challenge to treat in the clinic due to a lack of good targets for treatment. Although TNBC lacks expression of ERα, the expression of ERß and its variants are detected quite frequently in this cancer type and can represent an avenue for treatment. We show that two of the variants of ERß, namely ERß2 and ERß5, control aggressiveness of TNBC by regulating hypoxic signaling through stabilization of HIF-1α. RNA-seq of patient derived xenografts (PDX) from TNBC shows expression of ERß2, ERß4 and ERß5 variants in more than half of the samples. Furthermore, expression of ERß4 in the immortalized, normal mammary epithelial cell line MCF-10A that is resistant to tumorsphere formation caused transformation and development of tumorspheres. By contrast, ERß1, ERß2 or ERß5 were unable to support tumorsphere formation. We have previously shown that all variants except ERß1 stabilize HIF-1α but only ERß4 appears to have the ability to transform normal mammary epithelial cells, pointing towards a unique property of ERß4. We propose that ERß variants may be good diagnostic tools and also serve as novel targets for treatment of breast cancer.

Pharmacological Inhibition of NOS Activates ASK1/JNK Pathway Augmenting Docetaxel-Mediated Apoptosis in Triple-Negative Breast Cancer.

Purpose: Chemoresistance in triple-negative breast cancer (TNBC) is associated with the activation of a survival mechanism orchestrated by the endoplasmic reticulum (EnR) stress response and by inducible nitric oxide synthase (iNOS). Our aim was to determine the effects of pharmacologic NOS inhibition on TNBC.Experimental Design: TNBC cell lines, SUM-159PT, MDA-MB-436, and MDA-MB-468, were treated with docetaxel and NOS inhibitor (L-NMMA) for 24, 48, and 72 hours. Apoptosis was assessed by flow cytometry using Annexin-V and propidium iodide. Western blot was used to assess ER stress and apoptosis, and rtPCR was used to evaluate s-XBP1. TNBC patient-derived xenografts (PDX) were treated either with vehicle, docetaxel, or combination therapy (NOS inhibition + docetaxel). Mouse weight and tumor volumes were recorded twice weekly. Docetaxel concentration was determined using mass spectrometry. To quantify proliferation and apoptosis, PDX tumor samples were stained using Ki67 and TUNEL assay.Results:In vitro, L-NMMA ameliorated the iNOS upregulation associated with docetaxel. Apoptosis increased when TNBC cells were treated with combination therapy. In TNBC PDXs, combination therapy significantly reduced tumor volume growth and increased survival proportions. In the BCM-5998 PDX model, intratumoral docetaxel concentration was higher in mice receiving combination therapy. Coupling docetaxel with NOS inhibition increased EnR-stress response via coactivation of ATF4 and CHOP, which triggered the pASK1/JNK proapoptotic pathway, promoting cleavage of caspases 3 and 9.Conclusions: iNOS is a critical target for docetaxel resistance in TNBC. Pharmacologic inhibition of NOS enhanced chemotherapy response in TNBC PDX models. Combination therapy may improve prognosis and prevent relapse in TNBC patients who have failed conventional chemotherapy. Clin Cancer Res; 24(5); 1152-62. ©2018 AACR.

HN1L Promotes Triple-Negative Breast Cancer Stem Cells through LEPR-STAT3 Pathway.

Here, we show that HEMATOLOGICAL AND NEUROLOGICAL EXPRESSED 1-LIKE (HN1L) is a targetable breast cancer stem cell (BCSC) gene that is altered in 25% of whole breast cancer and significantly correlated with shorter overall or relapse-free survival in triple-negative breast cancer (TNBC) patients. HN1L silencing reduced the population of BCSCs, inhibited tumor initiation, resensitized chemoresistant tumors to docetaxel, and hindered cancer progression in multiple TNBC cell line-derived xenografts. Additionally, gene signatures associated with HN1L correlated with shorter disease-free survival of TNBC patients. We defined HN1L as a BCSC transcription regulator for genes involved in the LEPR-STAT3 signaling axis as HN1L binds to a putative consensus upstream sequence of STAT3, LEPTIN RECEPTOR, and MIR-150. Our data reveal that BCSCs in TNBC depend on the transcription regulator HN1L for the sustained activation of the LEPR-STAT3 pathway, which makes it a potentially important target for both prognosis and BCSC therapy.

Gene pathways associated with mitochondrial function, oxidative stress and telomere length are differentially expressed in the liver of rats fed lifelong on virgin olive, sunflower or fish oils.

This study investigates the effect of lifelong intake of different fat sources rich in monounsaturated (virgin olive oil), n6 polyunsaturated (sunflower oil) or n3 polyunsaturated (fish oil) fatty acids in the aged liver. Male Wistar rats fed lifelong on diets differing in the fat source were killed at 6 and at 24 months of age. Liver histopathology, mitochondrial ultrastructure, biogenesis, oxidative stress, mitochondrial electron transport chain, relative telomere length and gene expression profiles were studied. Aging led to lipid accumulation in the liver. Virgin olive oil led to the lowest oxidation and ultrastructural alterations. Sunflower oil induced fibrosis, ultrastructural alterations and high oxidation. Fish oil intensified oxidation associated with age, lowered electron transport chain activity and enhanced the relative telomere length. Gene expression changes associated with age in animals fed virgin olive oil and fish oil were related mostly to mitochondrial function and oxidative stress pathways, followed by cell cycle and telomere length control. Sunflower oil avoided gene expression changes related to age. According to the results, virgin olive oil might be considered the dietary fat source that best preserves the liver during the aging process.

A plasma metabolomic signature discloses human breast cancer.

PURPOSE: Metabolomics is the comprehensive global study of metabolites in biological samples. In this retrospective pilot study we explored whether serum metabolomic profile can discriminate the presence of human breast cancer irrespective of the cancer subtype. METHODS: Plasma samples were analyzed from healthy women (n = 20) and patients with breast cancer after diagnosis (n = 91) using a liquid chromatography-mass spectrometry platform. Multivariate statistics and a Random Forest (RF) classifier were used to create a metabolomics panel for the diagnosis of human breast cancer. RESULTS: Metabolomics correctly distinguished between breast cancer patients and healthy control subjects. In the RF supervised class prediction analysis comparing breast cancer and healthy control groups, RF accurately classified 100% both samples of the breast cancer patients and healthy controls. So, the class error for both group in and the out-of-bag error were 0. We also found 1269 metabolites with different concentration in plasma from healthy controls and cancer patients; and basing on exact mass, retention time and isotopic distribution we identified 35 metabolites. These metabolites mostly support cell growth by providing energy and building stones for the synthesis of essential biomolecules, and function as signal transduction molecules. The collective results of RF, significance testing, and false discovery rate analysis identified several metabolites that were strongly associated with breast cancer. CONCLUSIONS: In breast cancer a metabolomics signature of cancer exists and can be detected in patient plasma irrespectively of the breast cancer type.

Inhibition of iNOS as a novel effective targeted therapy against triple-negative breast cancer.

INTRODUCTION: Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer with no effective targeted therapy. Inducible nitric oxide synthase (iNOS) is associated with poor survival in patients with breast cancer by increasing tumor aggressiveness. This work aimed to investigate the potential of iNOS inhibitors as a targeted therapy for TNBC. We hypothesized that inhibition of endogenous iNOS would decrease TNBC aggressiveness by reducing tumor initiation and metastasis through modulation of epithelial-mesenchymal transition (EMT)-inducing factors. METHODS: iNOS protein levels were determined in 83 human TNBC tissues and correlated with clinical outcome. Proliferation, mammosphere-forming efficiency, migration, and EMT transcription factors were assessed in vitro after iNOS inhibition. Endogenous iNOS targeting was evaluated as a potential therapy in TNBC mouse models. RESULTS: High endogenous iNOS expression was associated with worse prognosis in patients with TNBC by gene expression as well as immunohistochemical analysis. Selective iNOS (1400 W) and pan-NOS (L-NMMA and L-NAME) inhibitors diminished cell proliferation, cancer stem cell self-renewal, and cell migration in vitro, together with inhibition of EMT transcription factors (Snail, Slug, Twist1, and Zeb1). Impairment of hypoxia-inducible factor 1α, endoplasmic reticulum stress (IRE1α/XBP1), and the crosstalk between activating transcription factor 3/activating transcription factor 4 and transforming growth factor ß was observed. iNOS inhibition significantly reduced tumor growth, the number of lung metastases, tumor initiation, and self-renewal. CONCLUSIONS: Considering the effectiveness of L-NMMA in decreasing tumor growth and enhancing survival rate in TNBC, we propose a targeted therapeutic clinical trial by re-purposing the pan-NOS inhibitor L-NMMA, which has been extensively investigated for cardiogenic shock as an anti-cancer therapeutic.

Targeting RPL39 and MLF2 reduces tumor initiation and metastasis in breast cancer by inhibiting nitric oxide synthase signaling.

We previously described a gene signature for breast cancer stem cells (BCSCs) derived from patient biopsies. Selective shRNA knockdown identified ribosomal protein L39 (RPL39) and myeloid leukemia factor 2 (MLF2) as the top candidates that affect BCSC self-renewal. Knockdown of RPL39 and MLF2 by specific siRNA nanoparticles in patient-derived and human cancer xenografts reduced tumor volume and lung metastases with a concomitant decrease in BCSCs. RNA deep sequencing identified damaging mutations in both genes. These mutations were confirmed in patient lung metastases (n = 53) and were statistically associated with shorter median time to pulmonary metastasis. Both genes affect the nitric oxide synthase pathway and are altered by hypoxia. These findings support that extensive tumor heterogeneity exists within primary cancers; distinct subpopulations associated with stem-like properties have increased metastatic potential.