Roseovarius bejariae sp. nov., a moderately halophilic bacterium isolated from a hypersaline steep-sided river bed.

An aerobic, Gram-stain-negative ovoid, designated as strain A21T, was isolated using the dilution-to-extinction method from a soil sample taken from Rambla Salada, an athalassohaline habitat located in Murcia (south-eastern Spain). Strain A21T is non-motile, has a respiratory metabolism and grows at NaCl concentrations within the range 0.5-15 % (w/v) [optimum, 5 % (w/v)], at 5-35 °C (optimum, 28 °C) and at pH 6-8 (optimum, pH 7.0). This strain is positive for catalase activity, oxidase activity and nitrate reduction. The 16S rRNA gene sequence indicates that it belongs to the genus Roseovarius in the class Alphaproteobacteria. The most closely related species are Roseovarius pacificus and Roseovarius halotolerans to which the strain A21T shows 16S rRNA gene sequence similarity values of 98.06 and 97.7 %, respectively. The average nucleotide identity in blast and digital DNA-DNA hybridization values between strain A21T and R. pacificus LMG 24575T are 76.8 and 21 %, respectively. The DNA G+C content based on the genome is 61.28 mol%. The major fatty acids (>5 % of the total fatty acids) of strain A21T are C18 : 1 ω7c/C18 : 1 ω6c and C16 : 0. The only detected isoprenoid quinone in strain A21T is ubiquinone 10 (Q-10). The polar lipid profile contains phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and three unidentified polar lipids. Based on the phylogenetic, genotypic, phenotypic and chemotaxonomic data, the strain represents a novel species of the genus Roseovarius, for which the name Roseovarius bejariae sp. nov. is proposed. Strain A21T (=CECT 9817T=LMG 31311T) is the type strain.

Draft genome sequence of Lactobacillus plantarum C4 (CECT 9567), a potential probiotic strain isolated from kefir.

Lactobacillus plantarum C4 (CECT 9567) was isolated from kefir and has been extensively studied because of its probiotic properties. Here we report the genome sequence of this strain. The genome consists of 3,221,350 bp, and contains 3058 CDSs with an average G + C content of 44.5%. The genome harbors genes encoding the AraC-family transcription regulator, the penicillin-binding protein Pbp2A, and the Na+/H+ antiporter NapA3, which have important roles in the survival of lactobacilli in the gastrointestinal tract. Also, the genome encodes the catalase KatE, NADH peroxidase and glutathione peroxidase, which enable anaerobic respiration, and a nitrate reductase complex, which enable anaerobic respiration. Additionally, genes encoding plantaricins and sactipeptides, and genes involved in the use of fructooligosaccharides and in the production of butyric acid were also identified. BLASTn analysis revealed that 91.4% of CDSs in C4 genome aligned with those of the reference strain L. plantarum WCFS1, with a mean identity of 98.96%. The genome information of L. plantarum C4 provides the basis for understanding the probiotic properties of C4 and to consider its use as a potential component of functional foods.

Roseovarius ramblicola sp. nov., a moderately halophilic bacterium isolated from saline soil in Spain.

Strain D15T was isolated from a soil sample taken from Rambla Salada (Murcia), south-eastern Spain, by using the dilution-to-extinction method. The strain, a Gram-stain-negative aerobic bacteria, is non-motile, ovoid- or rod-shaped, catalase- and oxidase-positive, and grows at NaCl concentrations within the range 0.5-10 % (w/v) [optimum 3 % (w/v)], at 5-30 °C (optimum 28 °C) and at pH 6-9 (optimum pH 7.0). The 16S rRNA gene sequence indicates that it belongs to the genus Roseovarius in the class Alphaproteobacteria. Its closest relatives are Roseovarius tolerans EL-172T and Roseovarius azorensis SSW084T, to which the strain shows 16S rRNA gene-sequence similarity values of 96.1 and 95.3 %, respectively. The DNA G+C content is 63 mol%. The major fatty acids (>5 % of the total fatty acids) of strain D15T are C18 : 1ω7c, C16 : 0 and C12 : 0. The only detected isoprenoid quinone of strain D15T is ubiquinone 10 (Q-10). The polar lipid profile contains phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, aminolipid and three polar lipids. Based on the phylogenetic, genotypic, phenotypic and chemotaxonomic data, the strain represents a novel species of the genus Roseovarius, for which the name Roseovarius ramblicola sp. nov. is proposed. Strain D15T (=CECT 9424=LMG 30322) is the type strain.

Blastomonas quesadae sp. nov., isolated from a saline soil by dilution-to-extinction cultivation.

We isolated a Gram-stain-negative, aerobic bacterial strain, 912T, from a soil sample taken from Rambla Salada (Murcia), south-eastern Spain, by using the dilution-to-extinction method. Cells of the strain were motile with a polar flagellum, short rod-shaped, catalase- and oxidase-positive and grew at NaCl concentrations within the range 0-5 % (w/v) (optimum 3 %, w/v), at 4-32 °C (optimum 30 °C) and at pH 6-9 (optimum pH 7); bacteriochlorophyll a was produced. Analysis of the 16S rRNA gene sequence indicated that this strain belonged to the genus Blastomonas in the class Alphaproteobacteria. Its closest relatives were Blastomonas natatoria EY 4220T, Blastomonas ursincola KR-99T and Blastomonas aquatica PE 4-5T, to which the strain showed 16S rRNA gene sequence similarity values of 95.9, 95.8 and 95.1 %, respectively. The DNA G+C content was 63 mol%. The major fatty acids of strain 912T were C18 : 1ω7c/C18 : 1ω6c, C16 : 1ω7c/C16 : 1ω6c, C16 : 0 and C17 : 1ω6c. The predominant isoprenoid quinone was ubiquinone 10 (Q-10). The polar lipids contained diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, sphingoglycolipid, phosphoglycolipid, one unidentified phospholipid and two unidentified polar lipids. Based on the phylogenetic, genotypic, phenotypic and chemotaxonomic data, the strain represents a novel species of the genus Blastomonas, for which the name Blastomonas quesadae sp. nov. is proposed. Strain 912T (=CECT 9186T=LMG 29921T) is the type strain.

Cationic Polymer Modified Mesoporous Silica Nanoparticles for Targeted SiRNA Delivery to HER2+ Breast Cancer.

In vivo delivery of siRNAs designed to inhibit genes important in cancer and other diseases continues to be an important biomedical goal. We now describe a new nanoparticle construct that has been engineered for efficient delivery of siRNA to tumors. The construct is comprised of a 47-nm mesoporous silica nanoparticle (MSNP) core coated with a cross-linked PEI-PEG copolymer, carrying siRNA against the HER2 oncogene, and coupled to the anti-HER2 monoclonal antibody (trastuzumab). The construct has been engineered to increase siRNA blood half-life, enhance tumor-specific cellular uptake, and maximize siRNA knockdown efficacy. The optimized anti-HER2-nanoparticles produced apoptotic death in HER2 positive (HER2+) breast cancer cells grown in vitro, but not in HER2 negative (HER2-) cells. One dose of the siHER2-nanoparticles reduced HER2 protein levels by 60% in trastuzumab-resistant HCC1954 xenografts. Multiple doses administered intravenously over 3 weeks significantly inhibited tumor growth (p < 0.004). The siHER2-nanoparticles have an excellent safety profile in terms of blood compatibility and low cytokine induction, when exposed to human peripheral blood mononuclear cells. The construct can be produced with high batch-to-batch reproducibility and the production methods are suitable for large-scale production. These results suggest that this siHER2-nanoparticle is ready for clinical evaluation.

ROCK1 inhibition promotes the self-renewal of a novel mouse mammary cancer stem cell.

The differentiation of stem-like tumor cells may contribute to the cellular heterogeneity of breast cancers. We report the propagation of highly enriched mouse mammary cancer stem cells that retain the potential to differentiate both in vivo and in culture and their use to identify chemical compounds that influence both self-renewal and differentiation. We identify epithelial tumor-initiating cells (ETICs) that express lineage markers of both basal and luminal mammary cell lineages and retain the potential, from even single cells, to generate heterogeneous tumors similar to the tumor of origin. ETICs can progress through a Rho-associated coiled-coil containing protein kinase 1 dependent, epithelial to mesenchymal transition to generate mesenchymal tumor-initiating cells capable of initiating tumors of limited heterogeneity. The propagation of ETICs may allow for the identification of new therapeutic compounds that may inhibit or prevent progression of some types of breast cancer.

Induction of Aryl Hydrocarbon Receptor-Mediated Cancer Cell-Selective Apoptosis in Triple-Negative Breast Cancer Cells by a High-Affinity Benzimidazoisoquinoline.

Triple-negative breast cancer (TNBC) remains a disease with a paucity of targeted treatment opportunities. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is involved in a wide range of physiological processes, including the sensing of xenobiotics, immune function, development, and differentiation. Different small-molecule AhR ligands drive strikingly varied cellular and organismal responses. In certain cancers, AhR activation by select small molecules induces cell cycle arrest or apoptosis via activation of tumor-suppressive transcriptional programs. AhR is expressed in triple-negative breast cancers, presenting a tractable therapeutic opportunity. Here, we identify a novel ligand of the aryl hydrocarbon receptor that potently and selectively induces cell death in triple-negative breast cancer cells and TNBC stem cells via the AhR. Importantly, we found that this compound, Analog 523, exhibits minimal cytotoxicity against multiple normal human primary cells. Analog 523 represents a high-affinity AhR ligand with potential for future clinical translation as an anticancer agent.

Evaluation of Phenolic Compounds and Pigments Content in Yellow Bell Pepper Wastes.

Bell peppers are one of the most important species consumed and cultivated in Spain. Peppers are a source of carotenoids and phenolic compounds widely associated with biological activities such as antimicrobial, antiseptic, anticancer, counterirritant, cardioprotective, appetite stimulator, antioxidant, and immunomodulator. However, undersized and damaged fruits are usually wasted. Thus, in order to evaluate the phenolic content, a Box-Behnken design has been carried out to optimize the extraction from Capsicum annuum yellow pepper by ultrasound-assisted extraction (UAE). The independent factors were time (min), ethanol/water (% v/v) and solvent/sample ratio (v/w). The model was validated by ANOVA and confirmed. Furthermore, the whole pepper and the pepper without peduncles and seeds were extracted using optimal conditions and characterized by HPLC-ESI-TOF-MS. Moreover, their antioxidant activities, measured by three different methods (DPPH, ABTS, and FRAP), carotenoid composition, assessed by HPLC-MS, and chlorophyll content, assessed by a spectrophotometric method, were compared. A total of 38 polar compounds were found of which seven have been identified in pepper fruit extracts for the first time. According to the results, whole pepper (WP) samples presented higher content in phenolic acids; meanwhile, the edible portion (EP) was higher in flavonoids. No differences were found in the antioxidant activity except for the FRAP assay where the WP sample showed higher radical scavenging activity. EP samples showed the highest content of carotenoids and WP ones in chlorophylls.

Taxogenomic and Metabolic Insights into Marinobacterium ramblicola sp. nov., a New Slightly Halophilic Bacterium Isolated from Rambla Salada, Murcia.

A Gram-negative, motile, rod-shaped bacteria, designated D7T, was isolated by using the dilution-to-extinction method, from a soil sample taken from Rambla Salada (Murcia, Spain). Growth of strain D7T was observed at 15-40 °C (optimum, 37 °C), pH 5-9 (optimum, 7) and 0-7.5% (w/v) NaCl (optimum, 3%). It is facultatively anaerobic. Phylogenetic analysis based on 16S rRNA gene sequence showed it belongs to the genus Marinobacterium. The in silico DDH and ANI against closest Marinobacterium relatives support its placement as a new species within this genus. The major fatty acids of strain D7T were C16:0, summed feature 3 (C16:1 ω7c/C16:1 ω6c) and summed feature 8 (C18:1 ω7c/C18:1 ω6c). The polar lipid profile consists of phosphatidylethanolamine, phosphatidylglycerol and two uncharacterized lipids. Ubiquinone 8 was the unique isoprenoid quinone detected. The DNA G + C content was 59.2 mol%. On the basis of the phylogenetic, phenotypic, chemotaxonomic and genomic characterization, strain D7T (= CECT 9818T = LMG 31312T) represents a novel species of the genus Marinobacterium for which the name Marinobacterium ramblicola sp. nov. is proposed. Genome-based metabolic reconstructions of strain D7T suggested a heterotrophic and chemolitotrophic lifestyle, as well as the capacity to biosynthetize and catabolize compatible solutes, and to degrade hydrocarbon aromatic compounds.

Targeting drug-metabolizing enzymes for effective chemoprevention and chemotherapy.

The primary focus of chemoprevention research is the prevention of cancer using pharmacological, biological, and nutritional interventions. Chemotherapeutic approaches that have been used successfully for both the prevention and treatment of a number of human malignancies have arisen from the identification of specific agents and appropriate molecular targets. Although drug-metabolizing enzymes have historically been targeted in attempts to block the initial, genotoxic events associated with the carcinogenic process, emerging evidence supports the idea that manipulating drug-metabolizing enzymes may also be an effective strategy to be used for treating tumor progression, invasion, and, perhaps, metastasis. This report summarizes a symposium that presents some recent progress in this area. One area of emphasis is the development of a CYP17 inhibitor for treatment of prostate cancer that may also have androgen-independent anticancer activity at higher concentrations. A second focus is the use of a mouse model to investigate the effects of aryl hydrocarbon receptor and Cyp1b1 status and chemopreventative agents on transplacental cancer. A third area of focus is the phytochemical manipulation of not only cytochrome P450 (P450) enzymes but also phase II inflammatory and antioxidant enzymes via the nuclear factor-erythroid 2-related factor 2 pathway to block tumor progression. A final highlight is the use of prodrugs activated by P450 enzymes to halt tumor growth and considerations of dosing schedule and targeted delivery of the P450 transgene to tumor tissue. In addition to highlighting recent successes in these areas, limitations and areas that should be targeted for further investigation are discussed.

Identifying efficacious approaches to chemoprevention with chlorophyllin, purified chlorophylls and freeze-dried spinach in a mouse model of transplacental carcinogenesis.

The carcinogenic potential of dibenzo[a,l]pyrene (DBP) has been well characterized in numerous animal models. We have previously documented that a single dose of 15 mg/Kg DBP to pregnant mice late in gestation (GD 17) produces an aggressive T-cell lymphoma as well as lung and liver cancer in offspring. The current study examines the chemopreventative properties of chlorophyllin (CHL) and chlorophyll (Chl) in this transplacental carcinogenesis model. Pregnant B6129SF1 females, bred to 129S1/SvIm males, received purified diets incorporated with either 2000 p.p.m. CHL, 2000 p.p.m. Chl or 10% freeze-dried spinach beginning at gestation day 9. Lymphoma-dependent mortality was not significantly altered by maternal consumption of any of the diet and little effect on lung tumor burden in mice surviving to 10 months of age was observed. However, coadministration of CHL at 380 mg/Kg with DBP by gavage (molar ratio of 10:1, CHL:DBP) provided significant protection against DBP-initiated carcinogenesis. Offspring born to dams receiving CHL co-gavaged with DBP exhibited markedly less lymphoma-dependent mortality (P < 0.001). The degree of protection by CHL, compared with controls dosed with DBP in tricaprylin (TCP) as the vehicle, was less marked, but still significant. Coadministration of CHL (TCP as vehicle) also reduced lung tumor multiplicity in mice by approximately 50% and this was observed throughout the study (P < 0.005). This is the first demonstration that CHL can provide potent chemoprotection in a transplacental carcinogenesis model and support a mechanism involving complex-mediated reduction of carcinogen uptake.

Lanthanide-Loaded Nanoparticles as Potential Fluorescent and Mass Probes for High-Content Protein Analysis.

Multiparametric and high-content protein analysis of single cells or tissues cannot be accomplished with the currently available flow cytometry or imaging techniques utilizing fluorophore-labelled antibodies, because the number of spectrally resolvable fluorochromes is limited. In contrast, mass cytometry can resolve more signals by exploiting lanthanide-tagged antibodies; however, only about 100 metal reporters can be attached to an antibody molecule. This makes the sensitivity of lanthanide-tagged antibodies substantially lower than fluorescent reporters. A new probe that can carry more lanthanide molecules per antibody is a desirable way to enhance the sensitivity needed for the detection of protein with low cellular abundance. Herein, we report on the development of new probes utilizing mesoporous silica nanoparticles (MSNPs) with hydroxyl, amine, or phosphonate functional groups. The phosphonated MSNPs proved to be best at loading lanthanides for up to 1.4 × 106 molecules per particle, and could be loaded with various lanthanide elements (Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Yb, and Lu) at relatively similar molar extents. The modified MSNPs can also load a fluorescent dye, allowing bimodal mass and fluorescence-based detection. We achieved specificity of antibody-conjugated nanoparticles (at 1.4 × 10³ antibodies per nanoparticle) for targeting proteins on the cell surface. The new materials can potentially be used as mass cytometry probes and provide a method for simultaneous monitoring of a large host of factors comprising the tumor microenvironment (e.g., extracellular matrix, cancer cells, and immune cells). These novel probes may also benefit personalized medicine by allowing for high-throughput analysis of multiple proteins in the same specimen.

Chemoprevention of dibenzo[a,l]pyrene transplacental carcinogenesis in mice born to mothers administered green tea: primary role of caffeine.

Our laboratory recently developed a mouse model of transplacental induction of lymphoma, lung and liver cancer by the polycyclic aromatic hydrocarbon, dibenzo[a,l]pyrene (DBP). Pregnant B6129SF1 females, bred to 129S1/SvIm males, were treated on day 17 of gestation with an oral dose of 15 mg/kg DBP. Beginning on day 0 of gestation, dams were given (ad lib) buffered water, 0.5% green tea, 0.5% decaffeinated green tea, caffeine or epigallocatechin-3-gallate (EGCG) (both at equivalent concentrations found in tea). The concentration of the teas (and corresponding caffeine and EGCG) was increased to 1.0% upon entering the second trimester, 1.5% at onset of the third trimester and continued at 1.5% until pups were weaned at 21 days of age. Offspring were raised with normal drinking water and AIN93G diet. Beginning at 2 months of age, offspring experienced significant mortalities due to an aggressive T-cell lymphoma as seen in our previous studies. Ingestion of caffeinated, but not decaffeinated, green tea provided modest but significant protection (P = 0.03) against mortality. Caffeine provided a more robust (P = 0.006) protection, but EGCG was without effect. Offspring also developed DBP-dependent lung adenomas. All treatments significantly reduced lung tumor multiplicity relative to controls (P < 0.02). EGCG was most effective at decreasing tumor burden (P = 0.005) by on average over 40% compared with controls. Induction of Cytochrome P450 (Cyp)1b1 in maternal liver may reduce bioavailability of DBP to the fetus as a mechanism of chemoprevention. This is the first demonstration that maternal ingestion of green tea, during pregnancy and nursing, provides protection against transplacental carcinogenesis.

Lymphoma and lung cancer in offspring born to pregnant mice dosed with dibenzo[a,l]pyrene: the importance of in utero vs. lactational exposure.

The fetus and neonate cannot be viewed as "little adults"; they are highly sensitive to toxicity from environmental chemicals. This phenomenon contributes to the fetal basis of adult disease. One example is transplacental carcinogenesis. Animal models demonstrate that environmental chemicals, to which pregnant women are daily exposed, can increase susceptibility of the offspring to cancer. It is uncertain to what degree in utero vs. lactational exposure contributes to cancer, especially for hydrophobic chemicals such as polyhalogenated biphenyls, ethers, dioxins, furans, etc., which can partition into breast milk. We developed a pregnant mouse model in which exposure to the polycyclic aromatic hydrocarbon (PAH), dibenzo[a,l]pyrene (DBP), during late gestation, produces an aggressive T-cell lymphoma in offspring between 3 and 6 months of age. Survivors exhibit multiple lung and liver (males) tumors. Here, we adopt a cross-foster design with litters born to dams treated with DBP exchanged with those born to dams treated with vehicle. Exposure to DBP in utero (about 2 days) produced significantly greater mortality than residual DBP exposure only through breast milk (3 weeks of lactation). As previously observed pups in all groups with an ahr(b-1/d) ("responsive") genotype were more susceptible to lymphoma mortality than ahr(d/d) ("non-responsive") siblings. At termination of the study at 10 months, mice exposed in utero also had greater lung tumor multiplicity than mice exposed only during lactation. Our results demonstrate that short exposure to DBP during late gestation presents a greater risk to offspring than exposure to this very hydrophobic PAH following 3 weeks of nursing.

Study of Bacterial Community Composition and Correlation of Environmental Variables in Rambla Salada, a Hypersaline Environment in South-Eastern Spain.

We studied the bacterial community in Rambla Salada in three different sampling sites and in three different seasons and the effect of salinity, oxygen, and pH. All sites samples had high diversity and richness (Rr > 30). The diversity indexes and the analysis of dendrograms obtained by DGGE fingerprint after applying Pearson's and Dice's coefficient showed a strong influence of sampling season. The Pareto-Lorenz (PL) curves and Fo analysis indicated that the microbial communities were balanced and despite the changing environmental conditions, they can preserve their functionality. The main phyla detected by DGGE were Bacteroidetes (39.73%), Proteobacteria (28.43%), Firmicutes (8.23%), and Cyanobacteria (5.14%). The majority of the sequences corresponding to uncultured bacteria belonged to Bacteroidetes phylum. Within Proteobacteria, the main genera detected were Halothiobacillus and Roseovarius. The environmental factors which influenced the community in a higher degree were the salinity and oxygen. The bacteria belonging to Bacteroidetes and Proteobacteria were positively influenced by salinity. Nevertheless, bacteria related to Alpha- and Betaproteobacteria classes and phylum Firmicutes showed a positive correlation with oxygen and pH but negative with salinity. The phylum Cyanobacteria were less influenced by the environmental variables. The bacterial community composition of Rambla Salada was also studied by dilution-to-extinction technique. Using this method, 354 microorganisms were isolated. The 16S sequences of 61 isolates showed that the diversity was very different to those obtained by DGGE and with those obtained previously by using classic culture techniques. The taxa identified by dilution-to-extinction were Proteobacteria (81.92%), Firmicutes (11.30%), Actinobacteria (4.52%), and Bacteroidetes (2.26%) phyla with Gammaproteobacteria as predominant class (65.7%). The main genera were: Marinobacter (38.85%), Halomonas (20.2%), and Bacillus (11.2%). Nine of the 61 identified bacteria showed less than 97% sequence identity with validly described species and may well represent new taxa. The number of bacteria in different samples, locations, and seasons were calculated by CARD-FISH, ranging from 54.3 to 78.9% of the total prokaryotic population. In conclusion, the dilution-to-extinction technique could be a complementary method to classical culture based method, but neither gets to cultivate the major taxa detected by DGGE. The bacterial community was influenced significantly by the physico-chemical parameters (specially the salinity and oxygen), the location and the season of sampling.

Oxidized analogs of Di(1H-indol-3-yl)methyl-4-substituted benzenes are NR4A1-dependent UPR inducers with potent and safe anti-cancer activity.

Di(1H-indol-3-yl)(4-trifluoromethylphenyl)methane (DIM-Ph-4-CF3) is an analog of orphan nuclear receptor 4A1 (NR4A1) ligand cytosporone B. We have synthesized several oxidation products of DIM-Ph-4-CF3, focusing on analogs with electron-withdrawing or donating groups at their phenyl ring 4-positions, and examined their anti-cancer activity and mechanism-of-action. Mesylates (DIM-Ph-4-X+ OMs-s) having CF3, CO2Me and Cl groups were more effective inhibitors of cancer cell viability than their precursors. 19F NMR spectroscopy and differential scanning calorimetry strongly indicated interactions of DIM-Ph-4-CF3+ OMs- with the NR4A1 ligand binding domain, and compound-induced apoptosis of prostate cancer cells was dependent on NR4A1. DIM-Ph-4-CF3+ OMs- showed robust inhibition of LNCaP prostate cancer xenografts with no apparent toxicity. In vitro and in vivo, DIM-Ph-4-CF3+ OMs- activated proapoptotic unfolded protein response (UPR) signaling in prostate cancer cells. Independently of DIM-Ph-4-CF3+ OMs-, the bulk of NR4A1 localized to the cytoplasm in various cancer cell lines, suggesting a cytoplasmic mechanism-of-action of DIM-Ph-4-CF3+ OMs- in UPR induction and cell death. In summary, the data suggest that oxidized analogs of DIM-Ph-4-CF3 possess potent and safe anti-cancer activity which is mediated through UPR signaling downstream of NR4A1 binding.

Targeted Treatment of Metastatic Breast Cancer by PLK1 siRNA Delivered by an Antioxidant Nanoparticle Platform.

Metastatic breast cancer is developed in about 20% to 30% of newly diagnosed patients with early-stage breast cancer despite treatments. Herein, we report a novel nanoparticle platform with intrinsic antimetastatic properties for the targeted delivery of Polo-like kinase 1 siRNA (siPLK1). We first evaluated it in a triple-negative breast cancer (TNBC) model, which shows high metastatic potential. PLK1 was identified as the top therapeutic target for TNBC cells and tumor-initiating cells in a kinome-wide screen. The platform consists of a 50-nm mesoporous silica nanoparticle (MSNP) core coated layer-by-layer with bioreducible cross-linked PEI and PEG polymers, conjugated with an antibody for selective uptake into cancer cells. siRNA is loaded last and fully protected under the PEG layer from blood enzymatic degradation. The material has net neutral charge and low nonspecific cytotoxicity. We have also shown for the first time that the MSNP itself inhibited cancer migration and invasion in TNBC cells owing to its ROS- and NOX4-modulating properties. In vivo, siPLK1 nanoconstructs (six doses of 0.5 mg/kg) knocked down about 80% of human PLK1 mRNA expression in metastatic breast cancer cells residing in mouse lungs and reduced tumor incidence and burden in lungs and other organs of an experimental metastasis mouse model. Long-term treatment significantly delayed the onset of death in mice and improved the overall survival. The platform capable of simultaneously inhibiting the proliferative and metastatic hallmarks of cancer progression is unique and has great therapeutic potential to also target other metastatic cancers beyond TNBC. Mol Cancer Ther; 16(4); 763-72. ©2017 AACR.

Current development of targeted oligonucleotide-based cancer therapies: Perspective on HER2-positive breast cancer treatment.

This Review discusses the various types of non-coding oligonucleotides, which have garnered extensive interest as new alternatives for targeted cancer therapies over small molecule inhibitors and monoclonal antibodies. These oligonucleotides can target any hallmark of cancer, no longer limited to so-called "druggable" targets. Thus, any identified gene that plays a key role in cancer progression or drug resistance can be exploited with oligonucleotides. Among them, small-interfering RNAs (siRNAs) are frequently utilized for gene silencing due to the robust and well established mechanism of RNA interference. Despite promising advantages, clinical translation of siRNAs is hindered by the lack of effective delivery platforms. This Review provides general criteria and consideration of nanoparticle development for systemic siRNA delivery. Different classes of nanoparticle candidates for siRNA delivery are discussed, and the progress in clinical trials for systemic cancer treatment is reviewed. Lastly, this Review presents HER2 (human epidermal growth factor receptor type 2)-positive breast cancer as one example that could benefit significantly from siRNA technology. How siRNA-based therapeutics can overcome cancer resistance to such therapies is discussed.

Therapeutic siRNA for drug-resistant HER2-positive breast cancer.

HER2 is overexpressed in about 20% of breast cancers and contributes to poor prognosis. Unfortunately, a large fraction of patients have primary or acquired resistance to the HER2-targeted therapy trastuzumab, thus a multi-drug combination is utilized in the clinic, putting significant burden on patients. We systematically identified an optimal HER2 siRNA from 76 potential sequences and demonstrated its utility in overcoming intrinsic and acquired resistance to trastuzumab and lapatinib in 18 HER2-positive cancer cell lines. We provided evidence that the drug-resistant cancer maintains dependence on HER2 for survival. Importantly, cell lines did not readily develop resistance following extended treatment with HER2 siRNA. Using our recently developed nanoparticle platform, systemic delivery of HER2 siRNA to trastuzumab-resistant tumors resulted in significant growth inhibition. Moreover, the optimal HER2 siRNA could also silence an exon 16 skipped HER2 splice variant reported to be highly oncogenic and linked to trastuzumab resistance.

ERN1 and ALPK1 inhibit differentiation of bi-potential tumor-initiating cells in human breast cancer.

Cancers are heterogeneous by nature. While traditional oncology screens commonly use a single endpoint of cell viability, altering the phenotype of tumor-initiating cells may reveal alternative targets that regulate cellular growth by processes other than apoptosis or cell division. We evaluated the impact of knocking down expression of 420 kinases in bi-lineage triple-negative breast cancer (TNBC) cells that express characteristics of both myoepithelial and luminal cells. Knockdown of ERN1 or ALPK1 induces bi-lineage MDA-MB-468 cells to lose the myoepithelial marker keratin 5 but not the luminal markers keratin 8 and GATA3. In addition, these cells exhibit increased ß-casein production. These changes are associated with decreased proliferation and clonogenicity in spheroid cultures and anchorage-independent growth assays. Confirmation of these assays was completed in vivo, where ERN1- or ALPK1-deficient TNBC cells are less tumorigenic. Finally, treatment with K252a, a kinase inhibitor active on ERN1, similarly impairs anchorage-independent growth of multiple breast cancer cell lines. This study supports the strategy to identify new molecular targets for types of cancer driven by cells that retain some capacity for normal differentiation to a non-tumorigenic phenotype. ERN1 and ALPK1 are potential targets for therapeutic development.