Nicotinamide (niacin) supplement increases lipid metabolism and ROS-induced energy disruption in triple-negative breast cancer: potential for drug repositioning as an anti-tumor agent.

Metabolic dysregulation is an important hallmark of cancer. Nicotinamide (NAM), a water-soluble amide form of niacin (vitamin B3), is currently available as a supplement for maintaining general physiologic functions. NAM is a crucial regulator of mitochondrial metabolism and redox reactions. In this study, we aimed to identify the mechanistic link between NAM-induced metabolic regulation and the therapeutic efficacy of NAM in triple-negative breast cancer (TNBC). The combined analysis using multiomics systems biology showed that NAM decreased mitochondrial membrane potential and ATP production, but increased the activities of reverse electron transport (RET), fatty acid ß-oxidation and glycerophospholipid/sphingolipid metabolic pathways in TNBC, collectively leading to an increase in the levels of reactive oxygen species (ROS). The increased ROS levels triggered apoptosis and suppressed tumour growth and metastasis of TNBC in both human organoids and xenograft mouse models. Our results showed that NAM treatment leads to cancer cell death in TNBC via mitochondrial dysfunction and activation of ROS by bifurcating metabolic pathways (RET and lipid metabolism); this provides insights into the repositioning of NAM supplement as a next-generation anti-metabolic agent for TNBC treatment.

Combined the SMAC mimetic and BCL2 inhibitor sensitizes neoadjuvant chemotherapy by targeting necrosome complexes in tyrosine aminoacyl-tRNA synthase-positive breast cancer.

BACKGROUND: Chemotherapy is the standard treatment for breast cancer; however, the response to chemotherapy is disappointingly low. Here, we investigated the alternative therapeutic efficacy of novel combination treatment with necroptosis-inducing small molecules to overcome chemotherapeutic resistance in tyrosine aminoacyl-tRNA synthetase (YARS)-positive breast cancer. METHODS: Pre-chemotherapeutic needle biopsy of 143 invasive ductal carcinomas undergoing the same chemotherapeutic regimen was subjected to proteomic analysis. Four different machine learning algorithms were employed to determine signature protein combinations. Immunoreactive markers were selected using three common candidate proteins from the machine-learning algorithms and verified by immunohistochemistry using 123 cases of independent needle biopsy FFPE samples. The regulation of chemotherapeutic response and necroptotic cell death was assessed using lentiviral YARS overexpression and depletion 3D spheroid formation assay, viability assays, LDH release assay, flow cytometry analysis, and transmission electron microscopy. The ROS-induced metabolic dysregulation and phosphorylation of necrosome complex by YARS were assessed using oxygen consumption rate analysis, flow cytometry analysis, and 3D cell viability assay. The therapeutic roles of SMAC mimetics (LCL161) and a pan-BCL2 inhibitor (ABT-263) were determined by 3D cell viability assay and flow cytometry analysis. Additional biologic process and protein-protein interaction pathway analysis were performed using Gene Ontology annotation and Cytoscape databases. RESULTS: YARS was selected as a potential biomarker by proteomics-based machine-learning algorithms and was exclusively associated with good response to chemotherapy by subsequent immunohistochemical validation. In 3D spheroid models of breast cancer cell lines, YARS overexpression significantly improved chemotherapy response via phosphorylation of the necrosome complex. YARS-induced necroptosis sequentially mediated mitochondrial dysfunction through the overproduction of ROS in breast cancer cell lines. Combination treatment with necroptosis-inducing small molecules, including a SMAC mimetic (LCL161) and a pan-BCL2 inhibitor (ABT-263), showed therapeutic efficacy in YARS-overexpressing breast cancer cells. CONCLUSIONS: Our results indicate that, before chemotherapy, an initial screening of YARS protein expression should be performed, and YARS-positive breast cancer patients might consider the combined treatment with LCL161 and ABT-263; this could be a novel stepwise clinical approach to apply new targeted therapy in breast cancer patients in the future.

Tryptophanyl-tRNA Synthetase Sensitizes Hormone Receptor-Positive Breast Cancer to Docetaxel-Based Chemotherapy.

PURPOSE: A relatively low response to chemotherapy has been reported for hormone receptor (HR)-positive breast cancer. In this study, we investigated the role of tryptophanyl-transfer RNA synthetase (WARS) in the chemotherapeutic response of HR-positive breast cancer. METHODS: Pre-chemotherapeutic needle biopsy samples of 45 HR-positive breast cancer patients undergoing the same chemotherapeutic regimen were subjected to immunohistochemistry. To investigate the biological functions of WARS in HR-positive breast cancer, we conducted cell viability assay, flow cytometry analysis, caspase activity assay, Quantitative real-time polymerase chain reaction, and western blotting using WARS gene-modulated HR-positive breast cancer cells (T47D, ZR-75-1, and MCF7). RESULTS: WARS overexpression in HR-positive breast cancer patients showed a significant correlation with favorable chemotherapy response. Downregulation of WARS increased cell viability following docetaxel treatment in tumor cell lines. On the other hand, WARS overexpression sensitized the therapeutic response to docetaxel. Additionally, downregulation of WARS caused a decrease in the number of apoptotic cell populations by docetaxel. Poly (ADP-ribose) polymerase cleavage and caspase 3/7 activity were increased in docetaxel-treated tumor cells with WARS overexpression. CONCLUSION: Our results suggest that WARS might be a potential predictor for chemotherapy response in patients with HR-positive breast cancer as well as a novel molecular target to improve chemosensitivity.

IL-6-mediated cross-talk between human preadipocytes and ductal carcinoma in situ in breast cancer progression.

BACKGROUND: The function of preadipocytes in the progression of early stage breast cancer has not been fully elucidated at the molecular level. To delineate the role of preadipocytes in breast cancer progression, we investigated the cross-talk between human breast ductal carcinoma in situ (DCIS) cells and preadipocytes with both an in vitro culture and xenograft tumor model. METHODS: GFP or RFP was transduced into human DCIS cell line MCF10DCIS.com cells or preadipocytes using lentivirus. Cell sorter was used to separate pure, viable populations of GFP- or RFP-transduced cells. Cell viability and proliferation was assessed by crystal violet assays and cell migration and invasion capability was assayed by the transwell strategy. Gene and protein levels were measured by western blot, RT-PCR and immunostaining. Adipokines and cytokines were quantified using ELISA. Human tumor xenografts in a nude mice model were used. Ultrasound imaging of tumors was performed to evaluate the therapeutic potential of a IL-6 neutralizing antibody. RESULTS: In the co-culture system with the MCF10DCIS.com and preadipocytes, MCF10DCIS.com proliferation, migration and invasion were enhanced by preadipocytes. Preadipocytes exhibited in an increased IL-6 secretion and cancer-associated fibroblast markers expression, FSP1 and α-SMC in co-culture with MCF10DCIS.com or in MCF10DCIS.com conditioned media, whereas the adipocyte differentiation capacity was suppressed by co-culture with MCF10DCIS.com. A neutralizing antibody of IL-6 or IL-6R suppressed the promotion of MCF10DCIS.com proliferation and migration by co-culture with preadipocytes. In the xenograft tumor model, the tumor growth of MCF10DCIS.com was enhanced by the co-injection of preadipocytes, and the administration of IL-6 neutralizing antibodies resulted in potent effects on tumor inhibition. CONCLUSIONS: Our findings suggest that IL-6-mediated cross-talk between preadipocytes and breast DCIS cells can promote the progression of early stage breast cancer. Therefore, blocking IL-6 signaling might be a potential therapeutic strategy for breast DCIS characterized by pathological IL-6 overproduction.

Breast cancer cell-derived exosomes and macrophage polarization are associated with lymph node metastasis.

Crosstalk between breast cancer and macrophages has potential implications for tumor metastasis. This study investigates macrophage polarization induced by triple-negative breast cancer (TNBC) cell-derived exosomes that promote lymph node (LN) metastasis in orthotopic TNBC models. The MDA-MB-231 cancer cell line expressing the exosomal CD63-red fluorescence (RFP) fusion protein was generated to noninvasively visualize exosome transfer into cancer cells and macrophages. Administration of RFP-tagged exosomes enhanced migration of macrophages and induced macrophage polarization in vitro. In orthotopic TNBC models, noninvasive bioluminescent imaging, ultrasound-guided photoacoustic imaging, and histological analysis revealed that intravenous injection of RFP-tagged exosomes promoted primary tumor growth and axillary LN metastasis in which expression of CD206, a marker or alternatively activated type 2 (M2) macrophages, was significantly higher than expression of NOS2, a marker of classically activated type 1 (M1) macrophages. These results suggest breast cancer cell-derived exosomes stimulate macrophage polarization that creates favorable conditions for LN metastatic processes in TNBC.

Overexpression of the miR-141/200c cluster promotes the migratory and invasive ability of triple-negative breast cancer cells through the activation of the FAK and PI3K/AKT signaling pathways by secreting VEGF-A.

BACKGROUND: The role of microRNA-200 (miR-200) family members in the migration and invasion of breast cancer is controversial. This study investigated the mechanisms by which the miR-200 family members modulated the migratory and invasive abilities of an aggressive triple-negative breast cancer (TNBC) cell line, MDA-MB-231. METHODS: The miR-200 family (miR-200b/200a/429 and miR-141/200c clusters) and green fluorescence protein (GFP) were transduced into MDA-MB-231 cells using a lentiviral system. Stable cells highly expressing the miR-200 family and GFP were isolated by puromycin selection and fluorescence-activated cell sorting. Gene expression was evaluated using real-time polymerase chain reaction (PCR) and reverse transcriptase-PCR (RT-PCR). The migratory and invasive abilities were assessed using trans-well and wound-healing assays. The secreted cytokines and growth factors in cultured media were quantified using a Bio-Plex200 multiplex array system. Western blot assays and immunofluorescence staining were conducted to investigate miR-200 family-regulated signaling pathways. The entire dataset obtained in this study was statistically evaluated using a one-way ANOVA followed by a t-test. RESULTS: The stable overexpression of the miR-200b/200a/429 or miR-141/200c cluster suppressed cell growth and significantly increased migration and invasion of MDA-MB-231 cells. miR-141/200c overexpression was more effective in decreasing cell growth and promoting migration and invasion of MDA-MB-231 cells than was miR-200b/200a/429 overexpression. In addition, the overexpression of the miR-200b/200a/429 or miR-141/200c cluster led to an increase in the phosphorylation of focal adhesion kinase (FAK) and protein kinase B (AKT). Chemical inhibitors of FAK and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT suppressed the migration and invasion of MDA-MB-231 cells that was enhanced by the overexpression of the miR-200b/200a/429 or miR-141/200c cluster. Compared to the miR-200b/200a/429 cluster-transduced MDA-MB-231 cells, the miR-141/200c cluster-transduced MDA-MB-231 cells exhibited a significant increase in vascular endothelial growth factor (VEGF)-A secretion and integrin-alphaV (integrin-αV) expression. Treatment with an anti-VEGF-A-neutralizing antibody inhibited the increase in migration and invasion in both the miR-200b/200a/429- and miR-141/200c-transduced MDA-MB-231 cells but significantly reduced the phosphorylation of FAK and AKT in only the miR-141/200c cluster-transduced MDA-MB-231 cells. CONCLUSIONS: Taken together, our data demonstrate a mechanism in which the miR-141/200c cluster, through FAK- and PI3K/AKT-mediated signaling by means of increased VEGF-A secretion, promotes the migratory and invasive abilities of MDA-MB-231 cells.

Noninvasive MRI and multilineage differentiation capability of ferritin-transduced human mesenchymal stem cells.

Molecular imaging can be a breakthrough tool for the investigation of the behavior and ultimate feasibility of transplanted human mesenchymal stem cells (hMSCs) inside the body, and for the development of guidelines and recommendations based on the treatment and evaluation of stem cell therapy for patients. The goals of this study were to evaluate the multilineage differentiation ability of hMSCs expressing an MRI reporter, human ferritin heavy chain (FTH) and to investigate the feasibility of using FTH-based MRI to provide noninvasive imaging of transplanted hMSCs. The transduction of FTH and green fluorescence protein (GFP) did not influence the expression of the mesenchymal stem cell surface markers (CD29+/CD105+/CD34-/CD45-) or the self-renewal marker genes [octamer-binding transcription factor 4 (OCT-4) and SRY (sex determining region Y)-box 2 (Sox-2)], cell viability, migration ability and the release of cytokines [interleukin-5 (IL-5), IL-10, IL-12p70, tumor necrosis factor-α (TNF-α)]. FTH-hMSCs retained the capacity to differentiate into adipogenic, chondrogenic, osteogenic and neurogenic lineages. The transduction of FTH led to a significant enhancement in cellular iron storage capacity and caused hypointensity and a significant increase in R2 * values of FTH-hMSC-collected phantoms and FTH-hMSC-transplanted sites of the brain, as shown by in vitro and in vivo MRI performed at 9.4 T, compared with control hMSCs. This study revealed no differences in biological characteristics between hMSCs and FTH-hMSCs and, therefore, these cells could be used for noninvasive monitoring with MRI during stem cell therapy for brain injury. Our study suggests the use of FTH for in vivo long-term tracking and ultimate fate of hMSCs without alteration of their characteristics and multidifferentiation potential.

Real-time imaging of the epithelial-mesenchymal transition using microRNA-200a sequence-based molecular beacon-conjugated magnetic nanoparticles.

The epithelial-mesenchymal transition (EMT) plays important roles in tumor progression to metastasis. Thus, the development of an imaging probe that can monitor transient periods of the EMT process in live cells is required for a better understanding of metastatic process. Inspired by the fact that the mRNA expression levels of zinc finger E-box-binding homeobox 1 (ZEB1) increase when cells adopt mesenchyme characteristics and that microRNA-200a (miR-200a) can bind to ZEB1 mRNA, we conjugated molecular beacon (MB) mimicking mature miR-200a to magnetic nanoparticles (miR-200a-MB-MNPs) and devised an imaging method to observe transitional changes in the cells during EMT. Transforming growth factor-ß1 treated epithelial cells and breast cancer cell lines representing both epithelial and mesenchymal phenotypes were used for the validation of miR-200a-MB-MNPs as an EMT imaging probe. The real-time imaging of live cells acquired with the induction of EMT revealed an increase in fluorescence signals by miR-200a-MB-MNPs, cell morphology alterations, and the loss of cell-cell adhesion. Our results suggest that miR-200a-MB-MNPs can be used as an imaging probe for the real-time monitoring of the EMT process in live cells.

Hexabromocyclododecanes in crucian carp and sediment from the major rivers in Korea.

The concentration of hexabromocyclododecanes (HBCDs) was measured in crucian carp muscles and eggs and in surrounding sediments collected from the 3 major rivers in Korea. HBCDs were detected in all carp and sediment samples, indicating widespread contamination of this area by HBCD flame retardants. The ∑HBCD (sum of α-, ß-, and γ-HBCDs) concentrations ranged from 0.19 to 13 ng g(-1)dry wt in sediments, 1.7 to 7.2 ng g(-1)lipid wt in carp eggs, and 4.8 to 6.6 ng g(-1)lipid wt in the muscle of carp. The α-diastereomer predominated in the crucian carp and γ-diastereomer predominated in sediments, accounting for 76% and 77% to the ∑HBCD, respectively. The ∑HBCD concentrations in carp and sediment samples collected along the rivers were higher than those in samples collected from an isolated pond, suggesting that the rivers are likely contaminated by HBCDs from the upstream or the environment surrounding the rivers. The diastereomer ratios in carp were different from those in commercial mixtures due to the enrichment of α-diastereomer in carp. The origin of this transition, however, is yet not known, since various transformation processes can lead to a change from the diastereomer ratio in commercial mixtures to that observed in the environment.