Cabozantinib and dasatinib synergize to induce tumor regression in non-clear cell renal cell carcinoma.

The lack of effective treatment options for advanced non-clear cell renal cell carcinoma (NCCRCC) is a critical unmet clinical need. Applying a high-throughput drug screen to multiple human kidney cancer cells, we identify the combination of the VEGFR-MET inhibitor cabozantinib and the SRC inhibitor dasatinib acts synergistically in cells to markedly reduce cell viability. Importantly, the combination is well tolerated and causes tumor regression in vivo. Transcriptional and phosphoproteomic profiling reveals that the combination converges to downregulate the MAPK-ERK signaling pathway, a result not predicted by single-agent analysis alone. Correspondingly, the addition of a MEK inhibitor synergizes with either dasatinib or cabozantinib to increase its efficacy. This study, by using approved, clinically relevant drugs, provides the rationale for the design of effective combination treatments in NCCRCC that can be rapidly translated to the clinic.

Dietary exposure to a binary mixture of polybrominated diphenyl ethers alters innate immunity and disease susceptibility in juvenile Chinook salmon (Oncorhynchus tshawytscha).

Polybrominated diphenyl ethers (PBDEs) have been used as flame retardants in consumer products and are now found in the aquatic environment. The presence of PBDEs puts the health and survival of aquatic species at risk due to the various toxic effects associated with exposure to these compounds. The effects of a binary dietary mixture of PBDEs on innate immunity and disease susceptibility of juvenile Chinook salmon (Oncorhynchus tshawytscha) were examined in the present study. Salmon were fed roughly 1:1 mixtures of two environmentally predominant PBDE congeners, BDE-47 and BDE-99. The six resulting whole body total PBDE concentrations ranged from less than the limit of quantification to 184 ng/g, wet weight (ww). The innate immune system was assessed by using two in vitro macrophage function assays. Specifically, assays that examined the ability of head kidney macrophages to: (1) engulf sheep red blood cells (SRBCs); and (2) produce a respiratory burst, as determined by the production of a reactive oxygen species, superoxide anion. Macrophages from salmon fed the BDE-47/99 mixture diets engulfed more SRBCs and produced greater superoxide anion than salmon fed the control diet. An increase in macrophage function was observed in fish with whole body total PBDE concentrations ranging from 2.81 ng/g, ww to 184 ng/g, ww. The mechanism for this increase in macrophage function due to PBDE exposure is currently unknown, but may be due to the ability of PBDEs to act as an endocrine receptor agonist and/or antagonist. Salmon exposed to the BDE-47/99 mixture diets were also challenged with the pathogenic bacteria, Vibrio (Listonella) anguillarum to determine disease susceptibility. Kaplan-Meier survival curves of fish exposed to the BDE-47/99 mixture and control diets were significantly different. The Cox proportional hazard risk ratios of disease-induced mortality in juvenile Chinook salmon with whole body concentrations of total PBDEs of 10.9, 36.8, and 184 ng/g, ww were significantly greater than the fish fed the control diet by 1.56, 1.83 and 1.50 times, respectively. Not all concentrations of the binary mixture diets had significant hazard ratios relative to the control diet, due to a non-monotonic concentration response curve. The mixture of PBDE congeners resulted in interactive effects that were generally non-additive and dependent upon the congener concentrations and metric examined. Consequently, predicting the interactive effects in juvenile Chinook salmon exposed to mixtures of PBDE congeners on innate immunity and disease susceptibility cannot be readily determined from the adverse effects of individual PBDE congeners.

Alteration of thyroid hormone concentrations in juvenile Chinook salmon (Oncorhynchus tshawytscha) exposed to polybrominated diphenyl ethers, BDE-47 and BDE-99.

Polybrominated diphenyl ethers (PBDEs) have been used as flame-retardants in consumer products and are currently detected in salmon globally. The two most predominant PBDE congeners found in salmon are BDE-47 (2,2',4,4'-tetrabromodiphenyl ether) and BDE-99 (2,2',4,4',5-pentabromodiphenyl ether). In the present study, groups of juvenile Pacific Chinook salmon were fed five environmentally relevant concentrations of either BDE-47 (0.3-552 ng total PBDEs/g food), BDE-99 (0.3-580 ng total PBDEs/g food), or nearly equal mixtures of both congeners (0.7-690 ng total PBDEs/g food) for 39-40 days. The concentrations of circulating total thyroid hormones, thyroxine (T4) and 3,5,3'-triiodothyronine (T3), were measured using a hormone-specific time-resolved fluoroimmunoassay to determine if PBDE exposure disrupts the hypothalamic-pituitary-thyroid endocrine axis. The concentrations of both circulating T4 and T3 were altered in juvenile salmon by dietary uptake of BDE-99. Exposure to BDE-47 did not alter either T3 or T4 circulating hormone concentrations. However, exposure to a mixture of BDE-47 and BDE-99 reduced T3 in fish with lower concentrations of total whole body PBDEs than with either congener alone at equivalent PBDE whole body concentrations. Accordingly, the disruption of PBDEs on circulating thyroid hormone concentrations has the potential to impact a number of critical functions in juvenile salmon including growth, parr-smolt transformation, and immunological processes.

Src and STAT3 inhibitors synergize to promote tumor inhibition in renal cell carcinoma.

The intracytoplasmic tyrosine kinase Src serves both as a conduit and a regulator for multiple processes required for the proliferation and survival cancer cells. In some cancers, Src engages with receptor tyrosine kinases to mediate downstream signaling and in other cancers, it regulates gene expression. Src therefore represents a viable oncologic target. However, clinical responses to Src inhibitors, such as dasatinib have been disappointing to date. We identified Stat3 signaling as a potential bypass mechanism that enables renal cell carcinoma (RCC) cells to escape dasatinib treatment. Combined Src-Stat3 inhibition using dasatinib and CYT387 (a JAK/STAT inhibitor) synergistically reduced cell proliferation and increased apoptosis in RCC cells. Moreover, dasatinib and CYT387 combine to suppress YAP1, a transcriptional co-activator that promotes cell proliferation, survival and organ size. Importantly, this combination was well tolerated, and caused marked tumor inhibition in RCC xenografts. These results suggest that combination therapy with inhibitors of Stat3 signaling may be a useful therapeutic approach to increase the efficacy of Src inhibitors.

Dietary Exposure to Individual Polybrominated Diphenyl Ether Congeners BDE-47 and BDE-99 Alters Innate Immunity and Disease Susceptibility in Juvenile Chinook Salmon.

Polybrominated diphenyl ethers (PBDEs), used as commercial flame-retardants, are bioaccumulating in threatened Pacific salmon. However, little is known of PBDE effects on critical physiological functions required for optimal health and survival. BDE-47 and BDE-99 are the predominant PBDE congeners found in Chinook salmon collected from the Pacific Northwest. In the present study, both innate immunity (phagocytosis and production of superoxide anion) and pathogen challenge were used to evaluate health and survival in groups of juvenile Chinook salmon exposed orally to either BDE-47 or BDE-99 at environmentally relevant concentrations. Head kidney macrophages from Chinook salmon exposed to BDE-99, but not those exposed to BDE-47, were found to have a reduced ability in vitro to engulf foreign particles. However, both congeners increased the in vitro production of superoxide anion in head kidney macrophages. Salmon exposed to either congener had reduced survival during challenge with the pathogenic marine bacteria Listonella anguillarum. The concentration response curves generated for these end points were nonmonotonic and demonstrated a requirement for using multiple environmentally relevant PBDE concentrations for effect studies. Consequently, predicting risk from toxicity reference values traditionally generated with monotonic concentration responses may underestimate PBDE effect on critical physiological functions required for optimal health and survival in salmon.

Assimilation efficiency of PBDE congeners in Chinook salmon.

Polybrominated diphenyl ether (PBDE) flame retardants are environmental contaminants that can accumulate in biota. PBDE accumulation in an organism depends on exposure, assimilation efficiency, and elimination/metabolism. Net assimilation efficiency represents the fraction of the contaminant that is retained in the organism after exposure. In the present study, congener-specific estimates of net PBDE assimilation efficiencies were calculated from dietary exposures of juvenile Chinook salmon. The fish were exposed to one to eight PBDE congeners up to 1500 ng total PBDEs/g food. Mean assimilation efficiencies varied from 0.32 to 0.50 for BDE congeners 28, 47, 99, 100, 153, and 154. The assimilation efficiency of BDE49 was significantly greater than 100%, suggesting biotransformation from higher brominated congeners. Whole body concentrations of BDE49 significantly increased with both exposure to increasing concentrations of BDE99 and decreasing fish lipid levels, implying lipid-influenced debromination of BDE99 to BDE49. Excluding BDE49, PBDE assimilation efficiency was not significantly related to the numbers of congeners in the diets, or congener hydrophobicity, but was greater in foods with higher lipid levels. Estimates of PBDE assimilation efficiency can be used in bioaccumulation models to assess threats from PBDE exposure to Chinook salmon health and recovery efforts, as well as to their predators.

Toxicity of PHOS-CHEK LC-95A and 259F fire retardants to ocean- and stream-type Chinook salmon and their potential to recover before seawater entry.

Long-term fire retardants are used to prevent the spread of wildland fire, but have inadvertently entered aquatic habitats and resulted in fish kills. We examined the toxicity of two fire retardant products; PHOS-CHEK 259F and LC-95A, on Chinook salmon with two different life histories, ocean-type and stream-type, at different stages of their development. Ocean-type Chinook outmigrate to the ocean as subyearlings; while, stream-type salmon overwinter in freshwater and outmigrate as yearlings. Ocean-type and stream-type salmon were exposed to the fire retardants prior to their parr to smolt transition (presmolts) as subyearlings (stream-type and ocean-type) and yearlings (stream-type only), as well as during their transition (smolts). The salmon were exposed to eight concentrations of each retardant and a control for 96h to determine acute toxicity. Lethal concentration curves were modeled by logistic regression for each life history and life stage exposed to the two fire retardants. Among all life histories and life stages tested, PHOS-CHEK 259F was most toxic to stream-type salmon at smolt stage and PHOS-CHEK LC-95A was most toxic to ocean-type salmon at smolt stage. To determine the delayed effects of product exposures on fish health as well as for the potential of recovery, 24-hour seawater challenges were performed immediately after fire retardant exposure, as well as after a recovery period. Previous PHOS-CHEK exposure reduced survival during seawater challenge among salmon from both life histories undergoing the parr-smolt transition and was more pronounced after PHOS-CHEK LC-95A exposure. However, this delayed effect was not observed 34 or more days after either PHOS-CHEK exposure. We conclude that accidental PHOS-CHEK LC-95A or 259F drops during salmon outmigration would have adverse impacts that extend beyond the acute mortality that occurs within the immediate drop and dilution areas.

Larvae from deep-sea methane seeps disperse in surface waters.

Many species endemic to deep-sea methane seeps have broad geographical distributions, suggesting that they produce larvae with at least episodic long-distance dispersal. Cold-seep communities on both sides of the Atlantic share species or species complexes, yet larval dispersal across the Atlantic is expected to take prohibitively long at adult depths. Here, we provide direct evidence that the long-lived larvae of two cold-seep molluscs migrate hundreds of metres above the ocean floor, allowing them to take advantage of faster surface currents that may facilitate long-distance dispersal. We collected larvae of the ubiquitous seep mussel "Bathymodiolus" childressi and an associated gastropod, Bathynerita naticoidea, using remote-control plankton nets towed in the euphotic zone of the Gulf of Mexico. The timing of collections suggested that the larvae might disperse in the water column for more than a year, where they feed and grow to more than triple their original sizes. Ontogenetic vertical migration during a long larval life suggests teleplanic dispersal, a plausible explanation for the amphi-Atlantic distribution of "B." mauritanicus and the broad western Atlantic distribution of B. naticoidea. These are the first empirical data to demonstrate a biological mechanism that might explain the genetic similarities between eastern and western Atlantic seep fauna.

The impact of temperature stress and pesticide exposure on mortality and disease susceptibility of endangered Pacific salmon.

Anthropogenic stressors, including chemical contamination and temperature stress, may contribute to increased disease susceptibility in aquatic animals. Specifically, the organophosphate pesticide malathion has been detected in surface waters inhabited by threatened and endangered salmon. In the presence of increasing water temperatures, malathion may increase susceptibility to disease and ultimately threaten salmon survival. This work examines the effect of acute and sublethal exposures to malathion on ocean-type subyearling Chinook salmon held under two temperature regimes. Chinook salmon were exposed to malathion at optimal (11 °C) or elevated (19 and 20 °C) temperatures. The influence of temperature on the acute toxicity of malathion was determined by generating 96-h lethal concentration (LC) curves. A disease challenge assay was also used to assess the effects of sublethal malathion exposure. The malathion concentration that resulted in 50% mortality (LC50; 274.1 µg L(-1)) of the Chinook salmon at 19 °C was significantly less than the LC50 at 11 °C (364.2 µg L(-1)). Mortality increased 11.2% in Chinook salmon exposed to malathion at the elevated temperature and challenged with Aeromonas salmonicida compared to fish held at the optimal temperature and exposed to malathion or the carrier control. No difference in disease challenge mortality was observed among malathion-exposed and unexposed fish at the optimal temperature. The interaction of co-occurring stressors may have a greater impact on salmon than if they occur in isolation. Ecological risk assessments considering the effects of an individual stressor on threatened and endangered salmon may underestimate risk when additional stressors are present in the environment.

Toxicity of forest fire retardant chemicals to stream-type chinook salmon undergoing parr-smolt transformation.

Long-term fire retardants are used to prevent the spread of wildland fires. These products are normally applied by aircraft and are intended specifically for terrestrial application, but fire retardants have entered aquatic habitats by misapplication and/or accidental spills and have resulted in fish mortalities. The authors examined the toxicity of two fire retardant products, PHOS-CHEK 259F and LC-95A, to salmon undergoing parr-smolt transformation. Yearling stream-type chinook salmon at the smolt stage were exposed to eight concentrations of each retardant in freshwater and a no-PHOS-CHEK control for 96 h to determine acute toxicity. Concentrations of the products that caused 50% mortality were 140.5 and 339.8 mg/L for 259F and LC-95A, respectively, and could occur during accidental drops into aquatic habitats. Damage to gill tissues seen in histopathological sections was attributed to fire retardant exposure. Un-ionized ammonia levels, from 259F, were sufficient to cause acute mortality; but additional factors, indicated by increased phagosome prevalence in the gills, might have contributed to mortality during LC-95A exposure. Seawater and disease challenges were performed to determine sublethal effects of product exposures on fish health. Although PHOS-CHEK exposure did not adversely affect chinook salmon's susceptibility to Listonella anguillarum, exposure did significantly reduce seawater survival. Reduced salmon survival resulting from prior fire retardant exposure during their transition from freshwater rearing environments to seawater may decrease the abundance of salmon populations.

Dispersal of deep-sea larvae from the intra-American seas: simulations of trajectories using ocean models.

Using data on ocean circulation with a Lagrangian larval transport model, we modeled the potential dispersal distances for seven species of bathyal invertebrates whose durations of larval life have been estimated from laboratory rearing, MOCNESS plankton sampling, spawning times, and recruitment. Species associated with methane seeps in the Gulf of Mexico and/or Barbados included the bivalve "Bathymodiolus" childressi, the gastropod Bathynerita naticoidea, the siboglinid polychaete tube worm Lamellibrachia luymesi, and the asteroid Sclerasterias tanneri. Non-seep species included the echinoids Cidaris blakei and Stylocidaris lineata from sedimented slopes in the Bahamas and the wood-dwelling sipunculan Phascolosoma turnerae, found in Barbados, the Bahamas, and the Gulf of Mexico. Durations of the planktonic larval stages ranged from 3 weeks in lecithotrophic tubeworms to more than 2 years in planktotrophic starfish. Planktotrophic sipunculan larvae from the northern Gulf of Mexico were capable of reaching the mid-Atlantic off Newfoundland, a distance of more than 3000 km, during a 7- to 14-month drifting period, but the proportion retained in the Gulf of Mexico varied significantly among years. Larvae drifting in the upper water column often had longer median dispersal distances than larvae drifting for the same amount of time below the permanent thermocline, although the shapes of the distance-frequency curves varied with depth only in the species with the longest larval trajectories. Even species drifting for >2 years did not cross the ocean in the North Atlantic Drift.

Trends in organic pollutants and lipids in juvenile Snake River spring Chinook salmon with different outmigrating histories through the Lower Snake and Middle Columbia Rivers.

A three-year field study was conducted from 2006 to 2008 to monitor the spatial and temporal trends of organic pollutants in migrating juvenile Snake River spring Chinook salmon (Oncorhynchus tshawytscha) sampled from the Lower Snake and Middle Columbia River Basins. Specifically, hatchery-reared juvenile salmon were monitored as they navigated the Federal Columbia River Power System (FCRPS) by either transport barge (Barged) or remained in the river (In-River) from Lower Granite Dam to a terminal collection dam, either John Day Dam or Bonneville Dam. Levels of polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), and organochlorine (OC) pesticides were detected in the bodies of both In-River and Barged salmon during the 2006, 2007 and 2008 outmigrating season. At the terminal dam, In-River fish had greater concentrations of persistent organic pollutants POPs than Barged salmon. Of the POPs detected, dichlorodiphenyltrichloroethanes (DDTs) were found at the greatest concentrations in the salmon bodies. These elevated lipid-normalized concentrations in the In-River fish were due to lipid depletion in all years as well as increased exposure to POPs in some years as indicated by an increase in wet weight contaminant concentrations. Salmon were also exposed to polycyclic aromatic hydrocarbons (PAHs) as indicated by the phenanthrene (PHN) signal for biliary fluorescent aromatic compounds (FACs) at the hatcheries or prior to Lower Granite Dam. There were detectable levels of biliary FACs as fish migrated downstream or were barged to the terminal dam. Therefore, the potential exists for these organic pollutants and lipid levels to cause adverse effects and should be included as one of the variables to consider when examining the effects of the FCRPS on threatened and endangered juvenile salmon.