Hypoxia-inducible factor-1␣ (HIF-1␣The hypoxic response is mainly regulated by the hypoxiainducible factor-1 (HIF-1), 2 a basic helix-loop-helix transcription factor composed of two subunits HIF-1␣ and HIF-1␤ (1). HIF-1␣ forms heterodimers with HIF-1␤, and this complex binds to hypoxia-responsive element (HRE: 5Ј-RCGTG-3Ј) within the promoter regions of target genes. Multiple studies of HIF-1␣ and breast cancer have shown a significant association between HIF-1␣ overexpression and poor prognosis coupled to increased patient mortality (2-6). The levels of HIF-1␣ in human primary breast tumors increased with the progression of the pathologic stage (7). In a large retrospective study of 745 patients with high levels of HIF-1␣ at diagnosis, early relapse and metastatic disease were predicted (5). HIF-1␣ expression is closely linked to an aggressive phenotype in breast cancer, and HIF-1␣ expression enhanced osteolytic bone metastasis of breast cancer (8, 9). After prolonged treatment hormone-sensitive breast tumors frequently become resistant to hormonal therapy, and it was hypothesized that hypoxia may promote estrogen-independent growth. Deletion of HIF-1␣ in the mammary epithelium resulted in delayed tumor onset and retarded tumor growth as well as decreased pulmonary metastasis (10). These results suggest that HIF-1␣ is a negative prognostic factor in breast cancer progression. The HIF-1␤ subunit is constitutively expressed, whereas expression of HIF-1␣ is regulated by oxygen tension. HIF-1␣ protein is not detected in cells under normoxic conditions (20 -22% O 2 ) and is rapidly induced by hypoxic conditions (1-2% O 2 ). However, in the invasive carcinoma cells, including breast, steady-state HIF-1␣ expression can be detected even under normoxia. The synthesis of HIF-1␣ protein has been shown to be regulated in an O 2 -independent fashion, for example, through activation of the receptor tyrosine kinase pathways (11,12). The molecular targets of HIF-1␣ that contribute to breast tumorigenesis are under active investigation.Macrophage-stimulating protein (MSP) is the only known ligand for recepteur d'origine nantais (RON), a tyrosine kinase receptor. MSP is an 80-kDa heterodimer consisting of a 53-kDa ␣-chain and a 30-kDa ␤-chain linked by a disulfide bond. The ␤-chain of MSP binds to RON (13). RON is initially synthesized as a single chain precursor, 170-kDa pro-RON, which is subsequently cleaved into 40-kDa alpha chain and 150-kDa beta chain. The alpha chain is completely extracellular, whereas the beta chain traverses the cell membrane and contains the intracellular tyrosine kinase (13). The RON receptor also participates in cross-talk with other receptor tyrosine kinases such as MET and epidermal growth factor receptor. Several human tumor tissues show increased RON expression, including tumors of the breast, colon, lung, liver, kidney, ovary, stomach, pancreas, bladder, and prostate (14). Gene expression analyses indicated increase in RON expression is associated with metastatic disease. Transgenic mice that ov...

Macrophage-stimulating protein (MSP)2 is the only known ligand for RON (recepteur d'origine nantais). MSP is an 80 kDa heterodimer consisting of a 53 kDa ␣ chain and a 30-kDa ␤ chain linked by a disulfide bond. The ␤ chain of MSP binds to RON. MSP belongs to the plasminogen-prothrombin gene family (1, 2). MSP gene knock-out in mice is not lethal, indicating that MSP is not required for embryonic development and growth (3). Besides macrophages, MSP is expressed in a variety of epithelial cells. RON is initially synthesized as a single chain precursor, 170-kDa pro-RON, which is subsequently cleaved into 40-kDa ␣ chain and 150-kDa ␤ chain. The ␣ chain is completely extracellular, whereas the ␤ chain traverses the cell membrane and contains the intracellular tyrosine kinase (1). The C-terminal of RON regulates its kinase activity (4). RON forms either homodimers or heterodimers with other receptors such as c-Met and epidermal growth factor receptor (5-7). In addition to macrophages, RON is also expressed in multiple epithelial cells both malignant and nonmalignant. Homozygous deletion of RON was embryonically lethal. However, RON heterozygous mice mature normally except for an inappropriate inflammatory response (8, 9). The RON protein is regulated through c-Cbl ubiquitin ligase binding to phosphorylated RON leading to endocytosis and the subsequent degradation of RON (10).Abnormal expression of RON was reported in various cancers of epithelial origin. However, fibroblasts do not express RON. RON is moderately expressed in normal colorectal mucosa but significantly elevated in a majority of primary human colorectal adenocarcinoma samples (11). RON protein accumulation was reported to induce autophosphorylation of RON tyrosine kinase receptor and transduces signals that regulate tumorigenic activities of colon cancer cells (12). In nonsmall cell lung cancer cell lines, RON overexpression was reported in a majority of the cell lines examined. In addition, these cell lines expressed high levels of MSP ligand (13). The combination of RON overexpression and activation by MSP leads to increased invasion and resistance to apoptosis. These tumors supported by either autocrine or paracrine effects may acquire a survival advantage because of increased activation of the RON receptor by the local secretion of MSP.Altered RON expression was noticed in bladder and ovarian cancers. RON expression was positively associated with tumor size, stage, and grade in bladder carcinomas (14). The majority of ovarian carcinoma samples showed up-regulation in RON expression with a mix of cytoplasmic and membrane staining (5). Co-expression of MSP with RON was observed in ovarian carcinomas, providing a selective growth advantage and subsequent tumor progression. RON overexpression and not mutations is associated with head and neck squamous cell carcinomas (15). Normal breast cells and benign lesions (adenomas and papillomas) express relatively low levels of RON. However, RON is highly expressed in tumor specimens (1). Further, increased RON e...

SUMMARYPlasticity in hatching timing allows embryos to balance egg-and larval-stage risks, and depends on the ability of hatchingcompetent embryos to continue developing in the egg. Hypoxia can slow development, kill embryos and induce premature hatching. For terrestrial eggs of red-eyed treefrogs, the embryonic period can extend ~50% longer than development to hatching competence, and development is synchronous across perivitelline oxygen levels (P O 2) ranging from 0.5-16.5 kPa. Embryos maintain large external gills until hatching, then gills regress rapidly. We assessed the respiratory value of external gills using gill manipulations and closed-system respirometry. Embryos without external gills were oxygen limited in air and hatched at an external P O 2 of 17 kPa, whereas embryos with gills regulated their metabolism and remained in the egg at substantially lower P O 2. By contrast, tadpoles gained no respiratory benefit from external gills. We videotaped behavior and manipulated embryos to test if they position gills near the air-exposed portion of the egg surface, where P O 2 is highest. Active embryos remained stationary for minutes in gills-at-surface positions. After manipulations and spontaneous movements that positioned gills in the O 2 -poor region of the egg, however, they returned their gills to the air-exposed surface within seconds. Even neural tube stage embryos, capable only of ciliary rotation, positioned their developing head in the region of highest P O 2. Such behavior may be critical both to delay hatching after hatching competence and to obtain sufficient oxygen for normal, synchronous development at earlier stages.

Recepteur d' origine nantais (RON)2 , a tyrosine kinase receptor, for macrophage-stimulating protein (MSP) was reported to be overexpressed in various cancers of epithelial origin (1, 2). Activation of the RON receptor in breast cancer cells is linked to tamoxifen resistance (3). Further, gene expression analyses suggested that increases in RON expression are associated with metastatic disease. Transgenic mice that overexpress a wild type or constitutively active RON receptor in the mammary epithelium induced mammary transformation and associated with a high degree of metastasis (4). Aberrant RON expression in human breast cancer is associated with an aggressive cancer phenotype with decreased disease-free survival time in patients and an increase in breast cancer metastasis (5). These studies demonstrated that RON overexpression can be a causative factor for metastatic breast cancer. Nrf2 (NF-E2-related factor 2) belongs to the cap n collar subfamily of basic leucine zipper family of transcription factors (6). Its role in chemoprevention as the inducer of several hundred cytoprotective genes, which contain one or more antioxidant response elements (ARE: 5Ј-TGACnnnGC-3Ј) is well documented (7). Nrf2-null mice are susceptible to carcinogen-induced cancer development (8 -12). Nrf2 activity is regulated by keap1 (kelch-like ECHassociated protein 1). Keap1 serves as an adapter protein for interaction of cul3-based E3-ubiquitin ligase complex with Nrf2 leading to continuous ubiquitination of Nrf2 and its proteasomal degradation (13). Activators of Nrf2 like sulforaphane (SFN), an isothiocyanate, have been shown to modify the keap1 protein leading to the dissociation of Nrf2-keap1 complex resulting in the escape of Nrf2 from proteasomal degradation (14). Nrf2 was reported to be depleted in breast cancer cell lines (15). The data presented in this report indicate depletion of Nrf2 is prevalent in breast tumors and is associated with overexpression of RON. We also report that Nrf2 binds a closely related cis-element (5Ј-TGA(C/G)TCA-3Ј) to ARE but displays a novel function as an inhibitor of oncogene RON and invasion of carcinoma cells. SFN induced Nrf2 and blocked RON expression in invasive carcinoma cells from various tumor types. Consequently, our studies identified a novel functional role for Nrf2 as a "repressor" of an oncogene and RON kinase as one of the potential molecular targets of SFN, which mediates the antitumor effects of SFN. EXPERIMENTAL PROCEDURESCell Culture-Cell lines were obtained from American Type Culture Collection (ATCC). Sulforaphane was purchased from LKT Laboratories.Immunohistochemical Analysis-Breast cancer metastasis tumor microarray (60 samples) was purchased from IMGENEX (catalogue number: IMH-364). Immunohistochemical analysis using RON and Nrf2 antibodies was performed by our institution pathology core facility.Generation of Stable Cell Lines-Scramble shRNA and shRNA keap1 and Nrf2 expression plasmids were purchased from Oregene. We have generated keap1 knock-down clones for characteriza...

Positive reinforcement training (PRT) techniques enhance the psychological well being of nonhuman primates by increasing the animal’s control over his or her environment and desensitizing the animal to stressful stimuli. However, the literature on PRT in neotropical primates is limited. Here PRT data from owl monkeys and squirrel monkeys are presented, including the length of time to train subjects to target, present hand, and present foot, important responses that can be used to aid in health inspection and treatment. A high percentage of the squirrel and owl monkeys were successfully trained on target and present hand. Present foot, a less natural response, was harder to train and maintain. Although squirrel monkeys did learn to target significantly faster than owl monkeys, the 2 genera did not differ on time to train on subsequent behavior. These data demonstrate that although owl monkeys may require slightly more time to acclimate to a PRT program, it is still possible to establish a PRT program with neotropical primates, and once animals have been introduced to the program, they can learn new responses in a relatively few short sessions.

Strategies used in artificial grammar learning can shed light into the abilities of different species to extract regularities from the environment. In the A ( X ) n B rule, A and B items are linked, but assigned to different positional categories and separated by distractor items. Open questions are how widespread is the ability to extract positional regularities from A ( X ) n B patterns, which strategies are used to encode positional regularities and whether individuals exhibit preferences for absolute or relative position encoding. We used visual arrays to investigate whether cotton-top tamarins ( Saguinus oedipus ) can learn this rule and which strategies they use. After training on a subset of exemplars, two of the tested monkeys successfully generalized to novel combinations. These tamarins discriminated between categories of tokens with different properties ( A , B , X ) and detected a positional relationship between non-adjacent items even in the presence of novel distractors. The pattern of errors revealed that successful subjects used visual similarity with training stimuli to solve the task and that successful tamarins extracted the relative position of A s and B s rather than their absolute position, similarly to what has been observed in other species. Relative position encoding appears to be favoured in different tasks and taxa. Generalization, though, was incomplete, since we observed a failure with items that during training had always been presented in reinforced arrays, showing the limitations in grasping the underlying positional rule. These results suggest the use of local strategies in the extraction of positional rules in cotton-top tamarins. Electronic supplementary material The online version of this article (10.1007/s10071-019-01277-y) contains supplementary material, which is available to authorized users.

Theory predicts that prey behavioural responses should reflect the level of risk posed by predators. We investigated how red-eyed treefrog embryos perceive and respond to spatially variable risk during wasp attacks on their clutches. First, we spatially restricted wasp activity on clutches and compared hatching of wasp-exposed, adjacent, and protected embryos. Hatching occurred in all zones but increased with exposure, being highest in directly exposed embryos. Second, we videotaped wasps attacking clutches and compared the experiences of embryos that hatched first and those that did not hatch until later. Embryos that hatched first experienced more predatory wasp activity directed at themselves or at siblings within a 2-egg radius. Models predicting hatching indicate that cues used to assess risk originate from the behaviour of wasps, rather than other embryos. This research demonstrates that embryos can integrate information about predator behaviour and proximity to respond appropriately based on their level of risk.

Introduction: Glioblastoma is a highly vascular tumor with marked endothelial dependence. While anti-angiogenic therapy has shown transient clinical benefit for the treatment of glioblastoma, persistent vascularization is seen with a normalization of blood vessels within the tumor. VB-111 is a dual-action, anti-angiogenic and vascular disrupting agent for cancer treatment aimed specifically at destroying newly formed and existing tumor blood vessels in a safe and efficient manner. VB-111 consists of a replication incompetent adenovirus together with an endothelial angiogenic-specific promoter and a pro-apoptotic Fas chimera transgene. Methods: Intracranial xenografts were established in nude rats with a luciferase expressing U87 glioma cell line. Rats were treated intravenously with VB-111 at 1011 viral particles or placebo at 21 days post tumor cell implantation. Tumor growth was followed by in vivo bioluminescent imaging and 7T MRI. Rats were sacrificed when moribund or when marked neurologic deficit was observed. Tumors were evaluated by standard histologic methods for vessel density and necrosis. Results: Untreated rats bearing U87 tumors showed a median survival of 38 days (95% CI 35.1-40.9 days), while animals treated with VB-111 showed a median survival of 48 days (95% CI 42.1-53.9 days). Log rank analysis of survival distributions between these two groups showed a significant difference (p=0.024) in favor of treatment. Luciferase activity, used as a surrogate for tumor volume, began separating between the two groups in week two post treatment and showed a continued trend for reduced activity in the treatment group thereafter. MRI imaging confirmed decreased tumor size in representative animals from the VB-111 group. Conclusions: VB-111's Dual action therapy is promising for glioblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5400. doi:10.1158/1538-7445.AM2011-5400