Bmi1 regulates self-renewal and epithelial to mesenchymal transition in breast cancer cells through Nanog

Background: The Bmi1 polycomb ring finger oncogene, a transcriptional repressor belonging to the Polycomb group of proteins plays an important role in the regulation of stem cell self-renewal and is elevated in several cancers. In the current study, we have explored the role of Bmi1 in regulating the stemness and drug resistance of breast cancer cells. Methods: Using real time PCR and immunohistochemistry primary breast tissues were analyzed. Retro-and lentiviruses were utilized to overexpress and knockdown Bmi1, RT-PCR and Western blot was performed to evaluate mRNA and protein expression. Stemness properties were analyzed by flow cytometry and sphere-formation and tumor formation was determined by mouse xenograft experiments. Dual luciferase assay was employed to assess promoter activity and MTT assay was used to analyze drug response. Results: We found Bmi1 overexpression in 64% of grade III invasive ductal breast adenocarcinomas compared to normal breast tissues. Bmi1 overexpression in immortalized and transformed breast epithelial cells increased their sphere-forming efficiency, induced epithelial to mesenchymal transition ( EMT) with an increase in the expression of stemness-related genes. Knockdown of Bmi1 in tumorigenic breast cells induced epithelial morphology, reduced expression of stemness-related genes, decreased the IC50 values of doxorubicin and abrogated tumor-formation. Bmi1-high tumors showed elevated Nanog expression whereas the tumors with lower Bmi1 showed reduced Nanog levels. Overexpression of Bmi1 increased Nanog levels whereas knockdown of Bmi1 reduced its expression. Dual luciferase promoter-reporter assay revealed Bmi1 positively regulated the Nanog and NF kappa B promoter activity. RT-PCR analysis showed that Bmi1 overexpression activated the NF kappa B pathway whereas Bmi1 knockdown reduced the expression of NF kappa B target genes, suggesting that Bmi1 might regulate Nanog expression through the NF kappa B pathway. Conclusions: Our study showed that Bmi1 is overexpressed in several high-grade, invasive ductal breast adenocarcinomas, thus supporting its role as a prognostic marker. While Bmi1 overexpression increased self-renewal and promoted EMT, its knockdown reversed EMT, reduced stemness, and rendered cells drug sensitive, thus highlighting a crucial role for Bmi1 in regulating the stemness and drug response of breast cancer cells. Bmi1 may control self-renewal through the regulation of Nanog expression via the NF kappa B pathway.

Coordinate Hyperactivation of Notch1 and Ras/MAPK Pathways Correlates with Poor Patient Survival: Novel Therapeutic Strategy for Aggressive Breast Cancers

Aberrant activation of Notch and Ras pathways has been detected in breast cancers. A synergy between these two pathways has also been shown in breast cell transformation in culture. Yet, the clinical relevance of Notch-Ras cooperation in breast cancer progression remains unexplored. In this study, we show that coordinate hyperactivation of Notch1 and Ras/MAPK pathways in breast cancer patient specimens, as assessed by IHC for cleaved Notch1 and pErk1/2, respectively, correlated with early relapse to vital organs and poor overall survival. Interestingly, majority of such Notch1 (high)Erk(high) cases encompassed the highly aggressive triple-negative breast cancers (TNBC), and were enriched in stem cell markers. We further show that combinatorial inhibition of Notch1 and Ras/MAPK pathways, using a novel mAb against Notch1 and a MEK inhibitor, respectively, led to a significant reduction in proliferation and survival of breast cancer cells compared with individual inhibition. Combined inhibition also abrogated sphere-forming potential, and depleted the putative cancer stem-like cell subpopulation. Most importantly, combinatorial inhibition of Notch1 and Ras/MAPK pathways completely blocked tumor growth in a panel of breast cancer xenografts, including the TNBCs. Thus, our study identifies coordinate hyperactivation of Notch1 and Ras/MAPK pathways as novel biomarkers for poor breast cancer outcome. Furthermore, based on our preclinical data, we propose combinatorial targeting of these two pathways as a treatment strategy for highly aggressive breast cancers, particularly the TNBCs that currently lack any targeted therapeutic module. (C) 2014 AACR.

Rrp1B gene polymorphism (1307T > C) in metastatic progression of breast cancer

Rrp1B (ribosomal RNA processing1 homolog B) is a novel candidate metastasis modifier gene in breast cancer. Functional gene assays demonstrated that a physical and functional interaction existing between Rrp1b and metastasis modifier gene SIPA1 causes reduction in the tumor growth and metastatic potential. Ectopic expression of Rrp1B modulates various metastasis predictive extra cellular matrix (ECM) genes associated with tumor suppression. The aim of this study is to determine the functional significance of single nucleotide polymorphism (SNP) in human Rrp1B gene (1307 T > C; rs9306160) with breast cancer development and progression. The study consists of 493 breast cancer cases recruited from Nizam's Institute of Medical Sciences, Hyderabad, and 558 age-matched healthy female controls from rural and urban areas. Genomic DNA was isolated by non-enzymatic method. Genotyping was done by amplification refractory mutation system (ARMS-PCR) method. Genotypes were reconfirmed by sequencing and results were analyzed statistically. We have performed Insilco analysis to know the RNA secondary structure by using online tool m fold. The TT genotype and T allele frequencies of Rrp1B1307 T > C polymorphism were significantly elevated in breast cancer (chi (2); p = < 0.008) cases compared to controls under different genetic models. The presence of T allele had conferred 1.75-fold risk for breast cancer development (OR = 1.75; 95 % CI = 1.15-2.67). The frequency of TT genotype of Rrp1b 1307T > C polymorphism was significantly elevated in obese patients (chi (2); p = 0.008) and patients with advanced disease (chi (2); p = 0.01) and with increased tumor size (chi (2); p = 0.01). Moreover, elevated frequency of T allele was also associated with positive lymph node status (chi (2); p = 0.04) and Her2 negative receptor status (chi (2); p = 0.006). Presence of Rrp1b1307TT genotype and T allele confer strong risk for breast cancer development and progression.

HIF-1 alpha (1772C > T) polymorphism as marker for breast cancer development

Hypoxia-inducible factor 1 alpha (HIF-1 alpha) is an important transcription factor that regulates different cellular responses to hypoxia. HIF-1 alpha is rapidly degraded by von Hippel-Lindau (VHL) protein under normoxic conditions and stabilized under hypoxia. A common variant of HIF-1 alpha (1772C > T) (rs 11549465) polymorphism, corresponding to an amino acid change from proline to serine at 582 position within the oxygen-dependent degradation domain, results in increased stability of the protein and altered transactivation of its target genes. The present study was aimed to find the association between HIF-1 alpha (1772C > T) (rs 11549465) polymorphism and breast cancer development. For this purpose, 348 primary breast cancer patients and 320 healthy and age-matched controls were genotyped through PCR-RFLP method. The genotype frequencies were compared between patients and controls, and their influence on clinical characteristics of breast cancer patients was analyzed. Our study revealed a significant increase of TT genotype in breast cancer patients compared to controls (p = 0.038). Further, TT genotype and T allele were found to be associated with progesterone receptor (PR)-negative status (p < 0.09). None of the clinical variables revealed significant association with HIF-1 alpha (1772C > T) (rs 11549465) polymorphism.

Lesion Detection in Breast Ultrasound Images Using Tissue Transition Analysis

Breast cancer is one of the leading cause of cancer related deaths in women and early detection is crucial for reducing mortality rates. In this paper, we present a novel and fully automated approach based on tissue transition analysis for lesion detection in breast ultrasound images. Every candidate pixel is classified as belonging to the lesion boundary, lesion interior or normal tissue based on its descriptor value. The tissue transitions are modeled using a Markov chain to estimate the likelihood of a candidate lesion region. Experimental evaluation on a clinical dataset of 135 images show that the proposed approach can achieve high sensitivity (95 %) with modest (3) false positives per image. The approach achieves very similar results (94 % for 3 false positives) on a completely different clinical dataset of 159 images without retraining, highlighting the robustness of the approach.

Enhanced Metastatic Potential in a 3D Tissue Scaffold toward a Comprehensive in Vitro Model for Breast Cancer Metastasis

Metastasis is clinically the most challenging and lethal aspect of breast cancer. While animal-based xenograft models are expensive and time-consuming, conventional two-dimensional (2D) cell culture systems fail to mimic in vivo signaling. In this study we have developed a three-dimensional (3D) scaffold system that better mimics the topography and mechanical properties of the breast tumor, thus recreating the tumor microenvironment in vitro to study breast cancer metastasis. Porous poly(e-caprolactone) (PCL) scaffolds of modulus 7.0 +/- 0.5 kPa, comparable to that of breast tumor tissue were fabricated, on which MDA-MB-231 cells proliferated forming tumoroids. A comparative gene expression analysis revealed that cells growing in the scaffolds expressed increased levels of genes implicated in the three major events of metastasis, viz., initiation, progression, and the site-specific colonization compared to cells grown in conventional 2D tissue culture polystyrene (TCPS) dishes. The cells cultured in scaffolds showed increased invasiveness and sphere efficiency in vitro and increased lung metastasis in vivo. A global gene expression analysis revealed a significant increase in the expression of genes involved in cell cell and cell matrix interactions and tissue remodeling, cancer inflammation, and the PI3K/Akt, Wnt, NF-kappaB, and HIFI signaling pathways all of which are implicated in metastasis. Thus, culturing breast cancer cells in 3D scaffolds that mimic the in vivo tumor-like microenvironment enhances their metastatic potential. This system could serve as a comprehensive in vitro model to investigate the manifold mechanisms of breast cancer metastasis.

Preliminary studies on food and feeding habits of Polypterus endlicheri and Polypterus senegalus in Lake Chad

The food and feeding habits of Polyprerus cncllicheri and Polypterus senegalus was carried out in the months of September to October. The food of 33 Polypierus endlicheri as observed include Tilapia species (89.3%), Eutropius niloticus (28.6%), Mayfly nymph (39.3%), Dragon fly larva (56.6%) fish remains (21.4%) and detritus (7.1%). The food of27 Polypterus senegalus as observed include Tilapia sp (88.4%), Eutropius niloticus (27.9%), may fly nymph (23.3%), Dragonfly nymph (34.9%) remains (21.1%) detritus (23.3%). (9 page document) The percentage occurrence of food item found in the stomach of Polypterus endlieheri is 93.3% while that of Polyprerus senegalus is 67.4%. The dominance of Tilapia sp was establish in the study, and there is no significant difference between the feeding habit of Polypterus endlicheri and Polyprerus senegalus.

The food and feeding habits of the anchoveta, Cetengraulis mysticetus, in the Gulf of Panama

ENGLISH:The gill rakers of both juvenile and adult anchovetas are long and numerous, with many fine processes which make a very efficient straining apparatus. The stomach is modified into a gizzard. The intestine undergoes heteronomous growth, and attains about eight times the standard length in adults. The stomach contents of 39 samples of juvenile fish and 120 adult fish were examined. Diatoms were the principal food of all the sizes of fish examined, from 29 to 153 millimeters. Silicoflagellates, dinoflagellates, pollen grains, formaniferans, rotifer shells, crustaceans, and eggs, probably of crustaceans, were also found in small amounts. Coscinodiscus, a diatom, was the most important item found in the stomachs of the juvenile fish. No strong differences were observed in the feeding habits of different sizes of juveniles. Even taking into account their smaller size, the juveniles had smaller volumes of material and lesser numbers of organisms in their stomachs than did the adults. The stomachs of the adult fish, unlike those of the juveniles, usually contained considerable quantities of mud. Melosira, Coscinodiscus, and Thalassionema, all diatoms, were the most important organisms found in the stomachs of the adults. The incidence of Melosira was much higher in the stomachs of fish from the areas to the east of the entrance of the Panama Canal than from those to the west. No seasonal differences in the food were observed. The volume of material in the stomachs ranged from almost none to nearly 1.0 milliliter, with an average of a little more than 0.2 milliliter. Twenty-six bottom samples were examined; the organisms found corresponded very closely to those encountered in the stomachs of the adult fish. It is concluded that the juvenile anchovetas are chiefly or entirely filter feeders of the pelagic zone. The adults, however, are mostly iliophagous feeders, but possibly do some feeding upon plankton as well. SPANISH:Las branquispinas de las anchovetas, tanto en las juveniles como en las adultas, son largas y numerosas, can varias protuberancias finas que hacen de ellas un aparato filtrador muy eficiente. El estómago está modificado en una molleja. El intestino está sometido a un crecimiento heterónomo, llega a alcanzar unas oeho veces la longitud estandar en las adultas. Fué examinado el contenido estomacal de 39 ,muestras de peces juveniles y de 120 adultos. Las diatomeas fueron el alimento principal de todos los peces que fueron examinados cuyo tamaño varió entre los 29 y 153 milimetros. Se encontraron también en cantidades silicoflagelados, dinoflagelados, granos de polen, foraminíferos, conchas de rotiferos, crustáceos y huevos, probablemente de crustáceos. Coscinodiscus, una diatomea, fué el alimento más importante encontrado en los estómagos de los peces juveniles. No se observaron mayores diferencias en los hábitos de alimentación en los juveniles de diferentes tamaños. Aún tomando en cuenta su tamaño menor, los juveniles tenian volúmenes más pequeños de material y un número menor de organismos en sus estómagos que los adultos. Los estómagos de los peces adultos, diferentes a los de los juveniles, contenían por lo general considerables cantidades de fango. Melosira, Coscinodiscus, y Thalassionema, todas ellas diatomeas, fueron los organismos más importantes encontrados en los estómagos de los adultos. La contribuciónde Melosira fué mucho más alta en los estómagos de los peces procedentes de las áreas al este de la entrada del Canal de Panamá que la de aquellos provenientes del oeste. No se observaron diferencias estacionales en la alimentacion. El volúmen de material en los estómagos varió de casi cero a cerca de 1.0 mililitros, con un promedio de un poco mas de 0.2 mili1itros. Se examinaron 26 muestras de fonda; los organismos encontrados correspondieron muy cercanamente a los hallados en los estómagos de los peces adultos. Se ha llegado a la conclusión de que las anchovetas juveniles son principalmente ó enteramente filtradoras de alimentos de la zona pelágica. Las adultas, sin embargo, son en su mayoria iliófagas, pero posiblemente se alimentan también de plancton.

Feeding Ecology of Humpback Whales in Continental Shelf Waters near Cordell Bank, California

Daytime feeding behavior of humpback whales (Megaptera novaeangliae) in Gulf of the Farallones, California, and adjacent waters was observed during autumn of 1988 to 1990. Bodega Canyon, Cordell Bank, and the Farallon Islands were the primary sites of feeding activity. Fecal samples of whales and zooplankton tows contained euphausiids exclusively, dominated by Thysanoessa spinifera (79%), with lesser amounts of Euphausia pacifica (14%), Nyctiphanes simplex (4%), and Nematoscelis difficilis (3%). In 1988 and 1990, whales also were infrequently observed feeding on small schooling fish, presumably Pacific herring (Clupea pallasii), northern anchovy (Engraulis mordax), and juvenile rockfish (Sebastes spp.). Feeding was the most common behavior observed (52%), and less frequently traveling (23%), milling (21 %), and resting (4%). Whales used different methods to consume euphausiid prey at the surface (0-10 m), in shallow water (11-60 m), and deep water (61-140 m). Humpback whales fed at the surface 56% of time in 1988 and 32% of time in 1990, using primarily lateral lunges to capture swarms of euphausiids. In 1989, no surface feeding was observed; however, deep, long-duration dives were followed by extended surface intervals with many respirations. These 1989 observations coincided with increased prey depth as indicated by depth sounder records of diving whales and prey scattering layers. In 1989, increased prey depth and associated feeding behaviors were strongly associated with unusually high surface temperatures, calm seas, and changes in water circulation. Environmental conditions in 1989 triggered the most intense and wide-spread occurrence of red tide in this region since 1980. Red tide samples collected throughout this period contained Alexandrium (=Gonyaulax) catenella and Noctiluca scintillans. Surface feeding was observed only in 1988 and 1990, when surface prey were available and red tides were very limited in extent, duration, and intensity. Annual variations in humpback whale feeding behavior were related to prey availability which is affected by corresponding environmental conditions. (PDF contains 94 pages)

The feeding ecology of some zooplankters that are important prey items of larval fish

Diets of 76 species of fish larvae from most oceans of the world were inventoried on the basis of information in 40 published studies. Although certaln geographlc, size- and taxon-specific patterns were apparent, certain zooplankton taxa appeared in the diets of larvae of a variety of fish species in numerous localities. Included were six genera of calanoid copepods (Acartia, Calanus, Centropages, Paracalanus, Pseudocaianus, Temora), three genera of cyclopoid copepods (Corycaeus, Oilhona, Oncata), harpacticoid copepods, copepod nauplii, tintinoids, cladocerans of the genera Evadne and Podon, barnacle nauplii, gastropod larvae, pteropods of the genus Limacina, and appendicularians. Literature on feeding habits of these zooplankters reveals that most of the copepods are omnivorous, feeding upon both phytoplankton and other zooplankton. Some taxa, such as Calanus, Paracalanus, Pseudocalanus, and copepod nauplii appear to be primarily herbivorous, while others, such as Acartia, Centropages, Temora, and cyclopoids exhibit broad omnivory or carnivory. The noncopepod zooplankters are primarily filter-feeders upon pbytoplankton and/or bacterioplankton. Despite the importance of zooplankters in larval fish food webs, spectic knowledge of the feeding ecology of many taxa is poor. Further, much present knowledge comes only from laboratory investigations that may not accurately portray feeding habits of zooplankters in nature. Lack of knowledge of the feeding ecology of many abundant zooplankters, which are also important in larval fish food webs, precludes realistic understanding of pelagic ecosystem dynamics. (PDF file contains 34 pages.)

Preliminary observations on the feeding, habits and reproduction biology of Gymnarchus niloticus from Lake Chad

The preliminary results obtained from the study of stomach contents of specimens of Gymnarchus niloticus of a size ranging from 56 cm (66 grams) to 152 cm (12.0 kg.) from August to December, 1982 indicate for these sizes an exclusively piscivorous diet. The predominant prey species in Lake Chad are Tilapia/Sarotherodon , and Clarias . The prey is often cut in two parts before it is swallowed. Small prey may be swallowed whole. The implication of this prey capture method on prey-predator relationship and therefore the impact of the predator is discussed. The breeding season of Gymnarchus niloticus in Lake Chad has been deduced from observation of changes in gonad maturity stages and the results indicate that breeding takes place between August and November. Data are also presented on the fecundity, size of ripe eggs and probable size at maturity

Post-larval feeding of Clarias gariepinus (Burchell, 1802) on cultured zooplankton and artemia diets

The findings are presented of a study undertaken to provide baseline information that may be employed to develop a simplified hatchery technique for postlarval feeding of Clarias gariepinus on zooplankton cultured indoors in small aquaria. Three series of experiments were undertaken to identify the type and quantity of organic manure that would produce maximum densities of zooplankton in the shortest time in indoor cultures. Results suggest the possibility of the culture of natural zooplankton populations for use in larval feeding of C. gariepinus fry. Its effective hatchery application would bring about reduction in the unit cost per fingerling and ensure continuous hatchery operations even in the absence of Artemia cysts