Two Triple-negative Breast Cancer Research Articles

Passage dependence of NADH redox status and reactive oxygen species level in vitro in triple-negative breast cancer cell lines with different invasiveness.

The redox status of nicotinamide adenine dinucleotide (NAD; including oxidized form NAD+ and reduced form NADH) plays key roles in both health and disease and has been actively studied to develop cancer biomarkers and therapeutic strategies. With the optical redox imaging (ORI) technique, we have been investigating the relationship of NADH redox status, reactive oxygen species (ROS), and invasiveness in breast cancer cell cultures, and have associated higher invasiveness with more oxidized NADH redox state. However, the cell cultures may have phenotypic drift and metabolic change with increased passage numbers. We investigated the passage-dependence of NADH redox status and ROS levels in two triple-negative breast cancer (TNBC) cell cultures: the more invasive/metastatic MDA-MB-231 and the less invasive/metastatic HCC1806 cell lines. We measured the NADH redox status, redox plasticity, and cytoplasmic and mitochondrial ROS levels under the basal condition and metabolic perturbations of the mitochondrial electron transport chain. We evaluated the dependence of redox and ROS profiles on the cell passage number by comparing the early (<20 passages) with the late (>60 passages) passage cells. (I) NADH redox and ROS baselines are stable and independent of cell passage number, but can vary with passage number under metabolic perturbations depending on specific perturbation and cell line; (II) NADH redox status and intracellular ROS levels can change discordantly in cancer cells; (III) under both basal and metabolically perturbed conditions, the more invasive cell line has a more oxidized NADH redox status with a higher basal cytoplasmic ROS level than the less invasive line, regardless of passage number. The general correlation between redox, ROS, and invasiveness in studied TNBC cells is not very sensitive to passage number. These results indicate that NADH redox and basal ROS status in TNBC likely reflect the intrinsic progressive nature of TNBC cells.

Synthesis, anticancer activity and molecular modeling study of novel substituted triazole linked tetrafluoronaphthalene hybrid derivatives

To create some novel anticancer molecules, a library of novel series of various triazoles linked to the hydroxyl group of 5,6,7,8-tetrafluoronaphthalen-1-ol (3) was designed and synthesized via CuAAC reaction ‘Click Chemistry’ of tetrafluoronaphthalene based terminal alkyne with substituted organic azides. The structural characterizations of the targeted Click products 9–18 were confirmed by FTIR, 1H NMR, 19F NMR, 13C NMR and HRMS spectroscopy. Synthesized compounds were tested in two triple negative breast cancer (TNBC) cell lines to understand their anticancer potentials. According to our findings, compounds 14 and 13 showed high cytotoxicity in BT549 cells at 20 μM and 30 μM, respectively. Moreover, these compounds blocked the migration of BT549 cells. In the MDA-MB-231 cell line, compound 18 exhibited high cytotoxicity and can block cell migration for 24 h. Molecular docking study with synthesized novel compounds was performed by Glide/SP method against SphK1 drug target. Furthermore, molecular dynamics (MD) simulation was carried out for the compounds 12–14 and 18. The compounds 13 and 14 may be potential inhibitor candidates in place of a reference inhibitor. A pharmacophore model was generated with the most potent compound 14, and the approved drugs were screened using the modules of Discovery Studio to find similar drugs. Consequently, this comprehensive study encompassing design, synthesis, in vitro and in silico analyses were correlated with the structure-activity relationship between compounds. The findings have the potential to unveil promising drug candidates for future studies. Communicated by Ramaswamy H. Sarma

Ent-kaurane-type Diterpenes Induce ROS-mediated Mitochondrial Dysfunction and Apoptosis by Suppress the Homologous Recombination DNA Repair in Triple-negative Breast Cancer Cells.

Six ent-kaurane-type diterpenes were isolated from the roots of Isodon ternifolia. Previous studies have shown that compounds 1 and 2 exhibited cytotoxicity against three human cancer cell lines (MCF-7, A549, and HCT116), but its molecular mechanism has not been studied yet. In the present study, the inhibited proliferation of compounds 1 and 2 of two triple-negative breast cancer (TNBC) cell lines (4T1 and MDA-MB-231) have been demonstrated by MTT and colony formation assay. Flow cytometry, western blotting, and qPCR were used to further demonstrate the apoptosis process in TNBCs. Importantly, the following mitochondrial membrane potential (MMP) decrease during apoptosis was demonstrated to correlate to reactive oxygen species (ROS) production. In addition, DNA damage induced by compounds 1 and 2 was illustrated by detect of homologous recombination (HR) DNA repair genes and proteins expression, such as RAD51. These results indicated that compounps 1 and 2 could trigger the TNBCs apoptosis mediated by ROS-induced mitochondrial dysfunction and induce DNA double-strand breaks (DSBs) by down regulating HR DNA repair. Furthermore, this research reveals that the mechanism between mitochondria dysfunction and DNA damage is deserved to be investigated for elucidating the dynamic signal transduction between the nucleus and the cellular matrix during apoptosis.

Fibronectin Functions as a Selective Agonist for Distinct Toll-like Receptors in Triple-Negative Breast Cancer.

The microenvironment of tumors is characterized by structural changes in the fibronectin matrix, which include increased deposition of the EDA isoform of fibronectin and the unfolding of the fibronectin Type III domains. The impact of these structural changes on tumor progression is not well understood. The fibronectin EDA (FnEDA) domain and the partially unfolded first Type III domain of fibronectin (FnIII-1c) have been identified as endogenous damage-associated molecular pattern molecules (DAMPs), which induce innate immune responses by serving as agonists for Toll-Like Receptors (TLRs). Using two triple-negative breast cancer (TNBC) cell lines MDA-MB-468 and MDA-MB-231, we show that FnEDA and FnIII-1c induce the pro-tumorigenic cytokine, IL-8, by serving as agonists for TLR5 and TLR2, the canonical receptors for bacterial flagellin and lipoprotein, respectively. We also find that FnIII-1c is not recognized by MDA-MB-468 cells but is recognized by MDA-MB-231 cells, suggesting a cell type rather than ligand specific utilization of TLRs. As IL-8 plays a major role in the progression of TNBC, these studies suggest that tumor-induced structural changes in the fibronectin matrix promote an inflammatory microenvironment conducive to metastatic progression.

Parathyroid Hormone-Related Protein Inhibition Blocks Triple-Negative Breast Cancer Expansion in Bone Through Epithelial to Mesenchymal Transition Reversal.

ABSTRACTParathyroid hormone‐related protein (PTHrP) plays a major role in skeletal metastasis but its action mechanism has not been fully defined. We previously demonstrated the crucial importance of PTHrP in promoting mammary tumor initiation, growth, and metastasis in a mouse model with a mammary epithelium‐targeted Pthlh gene ablation. We demonstrate here a novel mechanism for bone invasion involving PTHrP induction of epithelial to mesenchymal transition (EMT) and cancer stem cells (CSCs) regulation. Clustered regularly interspaced short palindromic repeats (CRISPR)‐mediated Pthlh gene ablation was used to study EMT markers, phenotype, and invasiveness in two triple‐negative breast cancer (TNBC) cell types (established MDA‐MB‐231 and patient‐derived PT‐TNBC cells). In vitro, Pthlh ablation in TNBC cells reduced EMT markers, mammosphere‐forming ability, and CD44high/CD24low cells ratio. In vivo, cells were injected intratibially into athymic nude mice, and therapeutic treatment with our anti‐PTHrP blocking antibody was started 2 weeks after skeletal tumors were established. In vivo, compared to control, lytic bone lesion from Pthlh ‐ablated cells decreased significantly over 2 weeks by 27% for MDA‐MB‐231 and by 75% for PT‐TNBC‐injected mice (p < 0.001). Micro‐CT (μCT) analyses also showed that antibody therapy reduced bone lytic volume loss by 52% and 48% for non‐ablated MDA‐MB‐231 and PT‐TNBC, respectively (p < 0.05). Antibody therapy reduced skeletal tumor burden by 45% and 87% for non‐ablated MDA‐MB‐231 and PT‐TNBC, respectively (p < 0.002) and caused a significant decrease of CSC/EMT markers ALDH1, vimentin, and Slug, and an increase in E‐cadherin in bone lesions. We conclude that PTHrP is a targetable EMT molecular driver and suggest that its pharmacological blockade can provide a potential therapeutic approach against established TNBC‐derived skeletal lesions. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Identification of a Potent Cytotoxic Pyrazole with Anti-Breast Cancer Activity That Alters Multiple Pathways.

In this study, we identified a novel pyrazole-based derivative (P3C) that displayed potent cytotoxicity against 27 human cancer cell lines derived from different tissue origins with 50% cytotoxic concentrations (CC50) in the low micromolar and nanomolar range, particularly in two triple-negative breast cancer (TNBC) cell lines (from 0.25 to 0.49 µM). In vitro assays revealed that P3C induces reactive oxygen species (ROS) accumulation leading to mitochondrial depolarization and caspase-3/7 and -8 activation, suggesting the participation of both the intrinsic and extrinsic apoptotic pathways. P3C caused microtubule disruption, phosphatidylserine externalization, PARP cleavage, DNA fragmentation, and cell cycle arrest on TNBC cells. In addition, P3C triggered dephosphorylation of CREB, p38, ERK, STAT3, and Fyn, and hyperphosphorylation of JNK and NF-kB in TNBC cells, indicating the inactivation of both p38MAPK/STAT3 and ERK1/2/CREB signaling pathways. In support of our in vitro assays, transcriptome analyses of two distinct TNBC cell lines (MDA-MB-231 and MDA-MB-468 cells) treated with P3C revealed 28 genes similarly affected by the treatment implicated in apoptosis, oxidative stress, protein kinase modulation, and microtubule stability.

Gene Expression Alterations Associated with Sanguinarine‐Induced Antiproliferative effects and Apoptosis in Triple‐Negative Breast Cancer Cells

Sanguinarine (SAN) is a plant alkaloid found in various plants, mainly Sanguinaria canadensis. SAN has various pharmacological properties, including anti-bacterial, anti-fungal, anti-inflammatory, and anti-cancer effects. The current study investigated the mechanisms that mediated the anti-cancer effects of SAN in two triple-negative breast cancer (TNBC) cell lines; MDA-MB-231 and MDA-MB-468 cells. The data obtained suggested SAN is a potent compound in reducing cell viability and proliferation. Moreover, the results show that MDA-MB-468 cells were more sensitive than MDA-MB-231 cells to SAN. Also, SAN induced apoptosis in both cell lines by arresting cell growth. The compound arrested MDA-MB-231 cells at the Sub-G1 phase and MDA-MB-468 cells at the G2/M phase. Gene expression profiling indicated that MDA-MB-468 cells had significant transcription activation (2-4-fold) in eighteen genes. The genes activated include two members of the caspase family (caspase 1 and caspase 10), seven members of the TNF receptor superfamily (TNFRSF) such as TNFRSF10A, TNFRSF10B, TNFRSF11B, TNFRSF21, and TNFRSF25), in addition to TNFRSF1A Associated Via Death Domain (TRADD) and Fas-associated via death domain (FADD). On the other hand, in MDA-MB-231 cells, SAN was found to alter twelve five genes. The upregulated genes include Lymphotoxin Alpha (LTA; 15-fold), growth arrest and DNA damage-inducible 45 alpha (GADD45A), BCL2 Related Protein A1 (BCL2A1), BCL2-Like 11 (BCL2L11), and Bcl-2-Binding Protein Bis (BAG3). These results provide evidence that genetically different cells may respond differently to treatments, explaining the inadequate therapeutic response of some patients with advanced TNBC. The data also suggest that this plant alkaloid SAN could have therapeutic potential for TNBC patients.

Resibufogenin Suppresses Triple-Negative Breast Cancer Angiogenesis by Blocking VEGFR2-Mediated Signaling Pathway.

Resibufogenin (RBF), an active compound from Bufo bufonis, has been used for the treatment of multiple malignant cancers, including pancreatic cancer, colorectal cancer, and breast cancer. However, whether RBF could exert its antitumor effect by inhibiting angiogenesis remains unknown. Here, we aimed to explore the antiangiogenic activity of RBF and its underlying mechanism on human umbilical vein endothelial cell (HUVEC), and the therapeutic efficacy with regard to antiangiogenesis in vivo using two triple-negative breast cancer (TNBC) models. Our results demonstrated that RBF can inhibit the proliferation, migration, and tube formation of HUVECs in a dose-dependent manner. Spheroid sprouts were thinner and shorter after RBF treatment in vitro 3D spheroid sprouting assay. RBF also significantly suppressed VEGF-mediated vascular network formation in vivo Matrigel plug assay. In addition, Western blot analysis was used to reveal that RBF inhibited the phosphorylation of VEGFR2 and its downstream protein kinases FAK and Src in endothelial cells (ECs). Molecular docking simulations showed that RBF affected the phosphorylation of VEGFR2 by competitively binding to the ATP-bound VEGFR2 kinase domain, thus preventing ATP from providing phosphate groups. Finally, we found that RBF exhibited promising antitumor effect through antiangiogenesis in vivo without obvious toxicity. The present study first revealed the high antiangiogenic activity and the underlying molecular basis of RBF, suggesting that RBF could be a potential antiangiogenic agent for angiogenesis-related diseases.

Synthesis of Novel Methyl 3-(hetero)arylthieno[3,2-b]pyridine-2-carboxylates and Antitumor Activity Evaluation: Studies In Vitro and In Ovo Grafts of Chick Chorioallantoic Membrane (CAM) with a Triple Negative Breast Cancer Cell Line.

A series of novel functionalized methyl 3-(hetero)arylthieno[3,2-b]pyridine-2-carboxylates 2a–2h were synthesized by C-C Pd-catalyzed Suzuki-Miyaura cross-coupling of methyl 3-bromothieno[3,2-b]pyridine-2-carboxylate with (hetero)aryl pinacol boranes, trifluoro potassium boronate salts or boronic acids. Their antitumoral potential was evaluated in two triple negative breast cancer (TNBC) cell lines—MDA-MB-231 and MDA-MB-468, by sulforhodamine B assay. Their effects on the non-tumorigenic MCF-12A cells were also evaluated. The results demonstrated that three compounds caused growth inhibition in both TNBC cell lines, with little or no effect against the non-tumorigenic cells. The most promising compound was further studied concerning possible effects on cell viability (by trypan blue exclusion assay), cell proliferation (by bromodeoxyuridine assay) and cell cycle profile (by flow cytometry). The results demonstrated that the GI50 concentration of compound 2e (13 μM) caused a decreased in MDA-MB-231 cell number, which was correlated with a decreased in the % of proliferating cells. Moreover, this compound increased G0/G1 phase and decreased S phases, when compared to control cells (although was not statistic significant). Interestingly, compound 2e also reduced tumor size using an in ovo CAM (chick chorioallantoic membrane) model. This work highlights the potential antitumor effect of a novel methyl 3-arylthieno[3,2-b]pyridine-2-carboxylate derivative.

EMT Transcription Factor ZEB1 Represses the Mutagenic POLθ-Mediated End-Joining Pathway in Breast Cancers.

A characteristic of cancer development is the acquisition of genomic instability, which results from the inaccurate repair of DNA damage. Among double-strand break repair mechanisms induced by oncogenic stress, the highly mutagenic theta-mediated end-joining (TMEJ) pathway, which requires DNA polymerase theta (POLθ) encoded by the POLQ gene, has been shown to be overexpressed in several human cancers. However, little is known regarding the regulatory mechanisms of TMEJ and the consequence of its dysregulation. In this study, we combined a bioinformatics approach exploring both Molecular Taxonomy of Breast Cancer International Consortium and The Cancer Genome Atlas databases with CRISPR/Cas9-mediated depletion of the zinc finger E-box binding homeobox 1 (ZEB1) in claudin-low tumor cells or forced expression of ZEB1 in basal-like tumor cells, two triple-negative breast cancer (TNBC) subtypes, to demonstrate that ZEB1 represses POLQ expression. ZEB1, a master epithelial-to-mesenchymal transition-inducing transcription factor, interacted directly with the POLQ promoter. Moreover, downregulation of POLQ by ZEB1 fostered micronuclei formation in TNBC tumor cell lines. Consequently, ZEB1 expression prevented TMEJ activity, with a major impact on genome integrity. In conclusion, we showed that ZEB1 directly inhibits the expression of POLQ and, therefore, TMEJ activity, controlling both stability and integrity of breast cancer cell genomes. SIGNIFICANCE: These findings uncover an original mechanism of TMEJ regulation, highlighting ZEB1 as a key player in genome stability during cancer progression via its repression of POLQ.See related commentary by Carvajal-Maldonado and Wood, p. 1441.

Rosmarinic Acid‐Induced Apoptosis and Cell Cycle Arrest in Triple‐Negative Breast Cancer Cells

Rosmarinic acid (RA) is one of the potent polyphenolic compounds in rosemary (Rosmarinus officinalis) extract. The compound has shown various bioactive properties, including, anti‐inflammatory, immunomodulatory, anti‐mutagenic, hepatoprotective, and neuroprotective, as well as having antioxidant and anti‐cancer activity. This study evaluated the effects and mechanisms of RA in two triple‐negative breast cancer (TNBC) cell lines; MDA‐MB‐231(MM‐231) and MDA‐MB‐468 (MM‐468); which were derived from a Caucasian and an African American woman, respectively. In this study, RA significantly and similarly inhibited cell viability and proliferation in both cell lines in a dose‐ and time‐dependent manner. The results obtained show that RA differentially affected the two cell lines in cell cycle arrest‐related apoptosis and in the alteration of the expression of many apoptosis‐involved genes. In MM‐231 cells, RA arrested the cells in the G0/G1 phase. In contrast, the data suggest that RA causes S‐phase arrest in MM‐468 cells and leads to a 2‐fold greater apoptotic effect than in MM‐231 cells. In MM‐231 cells, RA significantly upregulated the mRNA expression of three genes; harakiri (HRK), tumor necrosis factor receptor superfamily 25 (TNFRSF25), and BCL2 interacting protein 3 (BNIP3). In contrast, in MM‐468 cell line, the compound induced a significant transcription activation in five genes, including (TNF), baculoviral IAP repeat‐containing 3 (BIRC3), receptor‐interacting serine/threonine‐protein kinase 2 (RIPK2), BNIP3, and growth arrest and DNA damage‐inducible 45 alpha (GADD45A). Furthermore, RA repressed the expression of TNF receptor superfamily 11B (TNFRSF11B) in MM‐231 cells in comparison to the ligand TNF superfamily member 10 (TNFSF10) in MM‐468 cells. In conclusion, the data suggest that the polyphenol RA may have therapeutic potential for TNBC. However, RA should be further evaluated for in‐vivo studies and in non‐cancer cells.Support or Funding InformationU54 RCMI

Herb-sourced emodin inhibits angiogenesis of breast cancer by targeting VEGFA transcription.

Anti-angiogenesis is an important and promising strategy in cancer therapy. However, the current methods using anti-vascular endothelial growth factor A (VEGFA) antibodies or inhibitors targeting VEGFA receptors are not as efficient as expected partly due to their low efficiencies in blocking VEGFA signaling in vivo. Until now, there is still no method to effectively block VEGFA production in cancer cells from the very beginning, i.e., from the transcriptional level. Here, we aimed to find bioactive small molecules to block VEGFA transcription.Methods: We screened our natural compound pool containing 330 small molecules derived from Chinese traditional herbs for small molecules activating the expression of seryl-tRNA synthetase (SerRS), which is a newly identified potent transcriptional repressor of VEGFA, by a cell-based screening system in MDA-MB-231 cell line. The activities of the candidate molecules on regulating SerRS and VEGFA expression were first tested in breast cancer cells. We next investigated the antiangiogenic activity in vivo by testing the effects of candidate drugs on the vascular development in zebrafish and by matrigel plug angiogenesis assay in mice. We further examined the antitumor activities of candidate drugs in two triple-negative breast cancer (TNBC)-bearing mouse models. Furthermore, streptavidin-biotin affinity pull-down assay, coimmunoprecipitation assays, docking analysis and chromatin immunoprecipitation were performed to identify the direct targets of candidate drugs.Results: We identified emodin that could greatly increase SerRS expression in TNBC cells, consequently reducing VEGFA transcription. Emodin potently inhibited vascular development of zebrafish and blocked tumor angiogenesis in TNBC-bearing mice, greatly improving the survival. We also identified nuclear receptor corepressor 2 (NCOR2) to be the direct target of emodin. Once bound by emodin, NCOR2 got released from SerRS promoter, resulting in the activation of SerRS expression and eventually the suppression of VEGFA transcription.Conclusion: We discovered a herb-sourced small molecule emodin with the potential for the therapy of TNBC by targeting transcriptional regulators NCOR2 and SerRS to suppress VEGFA transcription and tumor angiogenesis.

TGF-β inhibition combined with cytotoxic nanomedicine normalizes triple negative breast cancer microenvironment towards anti-tumor immunity.

Tumor normalization strategies aim to improve tumor blood vessel functionality (i.e., perfusion) by reducing the hyper-permeability of tumor vessels or restoring compressed vessels. Despite progress in strategies to normalize the tumor microenvironment (TME), their combinatorial antitumor effects with nanomedicine and immunotherapy remain unexplored.Methods: Here, we re-purposed the TGF-β inhibitor tranilast, an approved anti-fibrotic and antihistamine drug, and combined it with Doxil nanomedicine to normalize the TME, increase perfusion and oxygenation, and enhance anti-tumor immunity. Specifically, we employed two triple-negative breast cancer (TNBC) mouse models to primarily evaluate the therapeutic and normalization effects of tranilast combined with doxorubicin and Doxil. We demonstrated the optimized normalization effects of tranilast combined with Doxil and extended our analysis to investigate the effect of TME normalization to the efficacy of immune checkpoint inhibitors.Results: Combination of tranilast with Doxil caused a pronounced reduction in extracellular matrix components and an increase in the intratumoral vessel diameter and pericyte coverage, indicators of TME normalization. These modifications resulted in a significant increase in tumor perfusion and oxygenation and enhanced treatment efficacy as indicated by the notable reduction in tumor size. Tranilast further normalized the immune TME by restoring the infiltration of T cells and increasing the fraction of T cells that migrate away from immunosuppressive cancer-associated fibroblasts. Furthermore, we found that combining tranilast with Doxil nanomedicine, significantly improved immunostimulatory M1 macrophage content in the tumorigenic tissue and improved the efficacy of the immune checkpoint blocking antibodies anti-PD-1/anti-CTLA-4.Conclusion: Combinatorial treatment of tranilast with Doxil optimizes TME normalization, improves immunostimulation and enhances the efficacy of immunotherapy.

FRET biosensor-based kinase inhibitor screen for ERK and AKT activity reveals differential kinase dependencies for proliferation in TNBC cells

Enhanced expression and activity of protein kinases are critical in tumor cell proliferation and cancer progression. These various cancer-related kinases form intricate interdependent signaling networks. Evaluation of the effect of various kinase inhibitors on these networks is critical to understand kinase inhibitor efficacy in cancer therapy. The dynamic activation of some kinases can be monitored by fluorescence resonance energy transfer (FRET) biosensors with high temporal resolution. Here, we established a FRET biosensor-based high throughput imaging approach to determine ERK and AKT activity in two triple negative breast cancer (TNBC) cell lines HCC1806 and Hs578T. FRET functionality was systematically evaluated using EGF stimulation and different MEK and AKT inhibitors, respectively. Next, we assessed the effect of a kinase inhibitor library containing >350 different kinase inhibitors (KIs) on ERK and AKT kinase activity using a FRET high-throughput screening setting. Suppression of FRET-ERK activity was generally positively correlated with the proliferation phenotype against inhibitors targeting MAPK signaling in both cell lines containing FRET-ERK reporter. AKT inhibitor (AKTi) resistant HCC1806 showed decreased proliferation associated with downregulated dynamics of FRET-ERK when treated with KIs targeting protein receptor tyrosine kinase (RTK). Yet, MEK inhibitor (MEKi) resistant Hs578T showed positively correlated FRET-AKT and proliferative responses against different PI3K and AKT inhibitors. Altogether, our data demonstrate the feasibility to integrate high throughput imaging-based screening of intracellular kinase activity using FRET-based biosensors in assessing kinase specificity and possible signaling crosstalk in direct relation to therapeutic outcome.

Combining Glucose Units, m/z, and Collision Cross Section Values: Multiattribute Data for Increased Accuracy in Automated Glycosphingolipid Glycan Identifications and Its Application in Triple Negative Breast Cancer.

Glycan head-groups attached to glycosphingolipids (GSLs) found in the cell membrane bilayer can alter in response to external stimuli and disease, making them potential markers and/or targets for cellular disease states. To identify such markers, comprehensive analyses of glycan structures must be undertaken. Conventional analyses of fluorescently labeled glycans using hydrophilic interaction high-performance liquid chromatography (HILIC) coupled with mass spectrometry (MS) provides relative quantitation and has the ability to perform automated glycan assignments using glucose unit (GU) and mass matching. The use of ion mobility (IM) as an additional level of separation can aid the characterization of closely related or isomeric structures through the generation of glycan collision cross section (CCS) identifiers. Here, we present a workflow for the analysis of procainamide-labeled GSL glycans using HILIC-IM-MS and a new, automated glycan identification strategy whereby multiple glycan attributes are combined to increase accuracy in automated structural assignments. For glycan matching and identification, an experimental reference database of GSL glycans containing GU, mass, and CCS values for each glycan was created. To assess the accuracy of glycan assignments, a distance-based confidence metric was used. The assignment accuracy was significantly better compared to conventional HILIC-MS approaches (using mass and GU only). This workflow was applied to the study of two Triple Negative Breast Cancer (TNBC) cell lines and revealed potential GSL glycosylation signatures characteristic of different TNBC subtypes.

Plasmonic MoO3-x nanoparticles incorporated in Prussian blue frameworks exhibit highly efficient dual photothermal/photodynamic therapy.

Development of near infrared (NIR) light-responsive nanomaterials for high performance multimodal phototherapy within a single nanoplatform is still challenging in technology and biomedicine. Herein, a new phototherapeutic nanoagent based on FDA-approved Prussian blue (PB) functionalized oxygen-deficient molybdenum oxide nanoparticles (MoO3-x NPs) is strategically designed and synthesized by a facile one-pot size/morphology-controlled process. The as-prepared PB-MoO3-x nanocomposites (NCs) with a uniform particle size of ∼90 nm and high water dispersibility exhibited strong optical absorption in the first biological window, which is induced by plasmon resonance in an oxygen-deficient MoO3-x semiconductor. More importantly, PB-MoO3-x NCs not only exhibited a high photothermal conversion efficiency of ∼63.7% and photostability but also offered a further approach for the generation of reactive oxygen species (ROS) upon singular NIR light irradiation which significantly improved the therapeutic efficiency of the PB agent. Furthermore, PB-MoO3-x NCs showed a negligible cytotoxic effect in the dark, but an excellent therapeutic effect toward two triple-negative breast cancer (TNBC) cell lines at a low concentration (20 μg mL-1) of NCs and a moderate NIR laser power density. Additionally, efficient tumor ablation and metastasis inhibition in a 4T1 TNBC mouse tumor model can also be realized by synergistic photothermal/photodynamic therapy (PTT/PDT) under a single continuous NIR wave laser. Taken together, this study paved the way for the use of a single nanosystem for multifunctional therapy.

Abstract A15: DNA damage response-targeted molecules for breast cancer therapy

Abstract Breast cancer genes 1 and 2 (BRACA1 and BRACA2) are known to be linked to breast cancer risk. BRCA1 is an ATM substrate and plays critical roles in the DNA damage response (DDR) and maintenance of genomic stability. Furthermore, recent studies have shown that the aberrant expression of DNA damage repair transducer, ataxia-telangiectasia mutated (ATM), is associated with breast cancers. To develop target-specific drugs, we rationally designed a dual-function small molecule, SP-1-303, by conferring ATM activation and HDAC inhibition activity. The compound was screened against a panel of Class I and Class II HDAC enzymes in vitro and identified as a pan-HDAC inhibitor at an IC50 of 120 nM. Western analysis revealed that SP-1-303 increased acetylated histone H3/H4, α-tubulin, and ATM activation (p-ATM/S1987). Testing two ER-positive BC cell lines (MCF7 and T-47D), two triple-negative breast cancer (TNBC) cell lines (MDA-MB-231 and HCC 1937), and one normal breast epithelial cell line as a control (MCF10A), the effect of SP-1-303 on cell growth was examined by cytotoxicity assays. SP-1-303 demonstrated cytotoxicity (IC50) of ER-positive BC cells in a range of 0.2-0.3 μM, while the values for TNBC cells are in a range of 1-3 μM and 12 μM for normal breast epithelial cells, respectively. Furthermore, proteomic analysis of MCF7 cells following SP-1-303 treatment identified differential effects on expression of various DDR molecules and chromatin regulators, including ATM, BRCA1, TP53BP1, NBN, histones, and chromatin remodelers. Taken together, SP-1-303 shows specificity for ER-positive breast cancers, potentially by modulating the DDR signaling pathway. Citation Format: Alfredo Valenea, Scott Grindrod, Mira Jung. DNA damage response-targeted molecules for breast cancer therapy [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A15.

Abstract 790: Vitamin K pathway functions in triple-negative breast cancer

Abstract The vitamin K pathway comprises three major enzymes: gamma-glutamyl carboxylase (GGCX) and the vitamin K oxidoreductases VKORC1 and VKORC1L1. GGCX requires the reduced form of vitamin K as a co-factor to post-translationally generate γ-carboxylated (GLA) residues in proteins. Either VKORC1 or VKORC1L1 can reduce the oxidized form of vitamin K to maintain pathway activity. Interrogation of TCGA data indicates a subset of invasive breast cancer tumors harbor mutations in GGCX (8%), VKORC1 (9%), and/or VKORC1L1 (9%), with nearly all alterations being amplifications. As these data suggest upregulation of these genes is associated with aggressive breast cancer, we measured their expression in a variety of established breast cancer cell lines. qPCR results correlated with the TCGA data indicating the highest expression of GGCX, VKORC1 and VKORC1L1 expression in the two triple-negative breast cancer (TNBC) cell lines, Hs578T and SUM159PT. Since the function of these genes is to generate GLA residues in proteins, we used Western blotting to detect GLA-modified proteins in TNBC cells cultured in the presence or absence of vitamin K. In lysates from cells maintained in vitamin K, multiple bands representing GLA-modified proteins were observed between 40-60 kDa and 80 kDa. These bands were not present in lysates of cells grown in standard media that are typically devoid of vitamin K. Treatment with the VKOR antagonist, warfarin, decreased expression of these GLA-modified proteins in cells cultured in vitamin K. Thus, our data suggest that vitamin K acts in TNBC cells to produce GLA-modified proteins, and that these effects are abrogated by warfarin. To confirm the presence of a functional vitamin K pathway, we used a vitamin K-dependent reporter protein (FIXgla-PC) to measure γ-carboxylation activity. TNBC cells stably transfected with the FIXgla-PC reporter were cultured in the presence and absence of vitamin K, with or without warfarin, and secretion of γ-carboxylated FIX-gla was measured by ELISA. FIX-gla γ-carboxylation was enhanced in the presence of vitamin K and blocked by warfarin in both Hs578T and SUM159PT cell lines. These data confirm that the classical vitamin K pathway is activated in these representative TNBC cell lines when cultured in vitamin K-supplemented media. To assess the biologic relevance of this pathway in TNBC, mammosphere assays were performed with Hs578T and SUM159PT cells. Both cell lines formed significantly more mammospheres when cultured in the presence of vitamin K, suggesting that γ-carboxylation may enhance stemness. Further studies to identify the GLA-modified proteins generated in TNBC cells treated with vitamin K and their mechanisms of action may lead to new therapeutic targets for aggressive TNBC. Citation Format: Sarah Beaudin, Leila Kokabee, JoEllen Welsh. Vitamin K pathway functions in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 790.

Abstract 3215: Therapeutic potential of dual-function small molecules for breast cancers

Abstract Epigenetic silencing of tumor suppressors is an important mechanism in tumor initiation and progression. A tumor suppressor gene, BRCA1, is part of complex molecules in the repair of DNA double-strand breaks (DSBs), and loss of BRCA1 contributes to both sporadic and inherited breast tumor progression. Furthermore, recent studies have shown that the ataxia-telangiectasia mutated (ATM) activity is abnormal in breast cancer. Therefore, we tested the hypothesis that the dual-functional drug that fused the HDAC inhibitor with an ATM activator may enhance killing of breast cancer (BC) cells by increasing ATM activation. In addition, the expression of ATM may enhance the synergistic antitumor effects of HDAC inhibitors in combination with radiotherapy. To determine the HDAC inhibitory potency, SP-1-303 was screened against a panel of Class I and Class II HDAC enzymes in vitro and identified as a pan-HDAC inhibitor (IC50=120 nM). Western blot confirmed that SP-1-303 increased acetylated histone H3/H4, α-tubulin, and phosphorylation of ATM within 30 min - 4 h in a time-dependent manner. Using two ER positive BC cell lines (MCF7 and T-47D), two triple negative breast cancer (TNBC) cell lines (MDA-MB-231 and HCC 1937), and one normal breast epithelial cell line as a control (MCF10A), the effects of SP-1-303 on cell growth and cell death were examined by performing cytotoxicity assays and flow cytometry. SAHA and Tamoxifen were used as controls. SP-1-303 conferred the values of cytotoxicity (IC50) for the ER positive BC cells in a range of 0.25-0.4 μM, while the values are in a range of 1-3 μM for TNBC and 12 μM for normal breast epithelial cells, respectively. Flow cytometry (FACS) analyses demonstrated significant increases in G1 arrest (81%) in MCF7 cells and a further enhancement in the G1 phase (91%) in combination with radiation. The data implies the mechanism of action from this drug—cancer cells are being arrested in the G1 phase of the cell cycle, a radiation sensitive phase, suggesting that SP-1-303 may have a synergistic effect with radiation on cell killing. Taken together, SP-1-303 had the largest impact on ER positive breast cancer over TNBC. These data imply that an increased cell death is occurring in ER positive breast cancers from these dual-action molecules through HDAC inhibition and increased ATM activation, suggesting that SP-1-303 alone or its combination with radiotherapy may improve clinical efficacy. Citation Format: Nicole Nicholas, Alfredo Velena, Scott Grindrod, Mira Jung. Therapeutic potential of dual-function small molecules for breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3215. doi:10.1158/1538-7445.AM2017-3215

Binding of galectin-1 to integrin β1 potentiates drug resistance by promoting survivin expression in breast cancer cells.

Galectin-1 is a β-galactoside binding protein secreted by many types of aggressive cancer cells. Although many studies have focused on the role of galectin-1 in cancer progression, relatively little attention has been paid to galectin-1 as an extracellular therapeutic target. To elucidate the molecular mechanisms underlying galectin-1-mediated cancer progression, we established galectin-1 knock-down cells via retroviral delivery of short hairpin RNA (shRNA) against galectin-1 in two triple-negative breast cancer (TNBC) cell lines, MDA-MB-231 and Hs578T. Ablation of galectin-1 expression decreased cell proliferation, migration, invasion, and doxorubicin resistance. We found that these effects were caused by decreased galectin-1-integrin β1 interactions and suppression of the downstream focal adhesion kinase (FAK)/c-Src pathway. We also found that silencing of galectin-1 inhibited extracellular signal-regulated kinase (ERK)/signal transducer and activator of transcription 3 (STAT3) signaling, thereby down-regulating survivin expression. This finding implicates STAT3 as a transcription factor for survivin. Finally, rescue of endogenous galectin-1 knock-down and recombinant galectin-1 treatment both recovered signaling through the FAK/c-Src/ERK/STAT3/survivin pathway. Taken together, these results suggest that extracellular galectin-1 contributes to cancer progression and doxorubicin resistance in TNBC cells. These effects appear to be mediated by galectin-1-induced up-regulation of the integrin β1/FAK/c-Src/ERK/STAT3/survivin pathway. Our results imply that extracellular galectin-1 has potential as a therapeutic target for triple-negative breast cancer.