Leandro S. D'Abronzo

Linking mechanistic and behavioral responses to sublethal esfenvalerate exposure in the endangered delta smelt; Hypomesus transpacificus (Fam. Osmeridae)

BackgroundThe delta smelt (Hypomesus transpacificus) is a pelagic fish species listed as endangered under both the USA Federal and Californian State Endangered Species Acts and considered an indicator of ecosystem health in its habitat range, which is limited to the Sacramento-San Joaquin estuary in California, USA. Anthropogenic contaminants are one of multiple stressors affecting this system, and among them, current-use insecticides are of major concern. Interrogative tools are required to successfully monitor effects of contaminants on the delta smelt, and to research potential causes of population decline in this species. We have created a microarray to investigate genome-wide effects of potentially causative stressors, and applied this tool to assess effects of the pyrethroid insecticide esfenvalerate on larval delta smelt. Selected genes were further investigated as molecular biomarkers using quantitative PCR analyses.ResultsExposure to esfenvalerate affected swimming behavior of larval delta smelt at concentrations as low as 0.0625 μg.L-1, and significant differences in expression were measured in genes involved in neuromuscular activity. Alterations in the expression of genes associated with immune responses, along with apoptosis, redox, osmotic stress, detoxification, and growth and development appear to have been invoked by esfenvalerate exposure. Swimming impairment correlated significantly with expression of aspartoacylase (ASPA), an enzyme involved in brain cell function and associated with numerous human diseases. Selected genes were investigated for their use as molecular biomarkers, and strong links were determined between measured downregulation in ASPA and observed behavioral responses in fish exposed to environmentally relevant pyrethroid concentrations.ConclusionsThe results of this study show that microarray technology is a useful approach in screening for, and generation of molecular biomarkers in endangered, non-model organisms, identifying specific genes that can be directly linked with sublethal toxicological endpoints; such as changes in expression levels of neuromuscular genes resulting in measurable swimming impairments. The developed microarrays were successfully applied on larval fish exposed to esfenvalerate, a known contaminant of the Sacramento-San Joaquin estuary, and has permitted the identification of specific biomarkers which could provide insight into the factors contributing to delta smelt population decline.

Transcription profiling in environmental diagnostics: health assessments in Columbia River basin steelhead (Oncorhynchus mykiss).

The health condition of out-migrating juvenile salmonids can influence migration success. Physical damage, pathogenic infection, contaminant exposure, and immune system status can affect survival probability. The present study is part of a wider investigation of out-migration success in juvenile steelhead (Oncorhynchus mykiss) and focuses on the application of molecular profiling to assess sublethal effects of environmental stressors in field-collected fish. We used a suite of genes in O. mykiss to specifically assess responses that could be directly related to steelhead health condition during out-migration. These biomarkers were used on juvenile steelhead captured in the Snake River, a tributary of the Columbia River, in Washington, USA, and were applied on gill and anterior head kidney tissue to assess immune system responses, pathogen-defense (NRAMP, Mx, CXC), general stress (HSP70), metal-binding (metallothionein-A), and xenobiotic metabolism (Cyp1a1) utilizing quantitative polymerase chain reaction (PCR) technology. Upon capture, fish were ranked according to visual external physical conditions into good, fair, poor, and bad categories; gills and kidney tissues were then dissected and preserved for gene analyses. Transcription responses were tissue-specific for gill and anterior head kidney with less significant responses in gill tissue than in kidney. Significant differences between the condition ranks were attributed to NRAMP, MX, CXC, and Cyp1a1 responses. Gene profiling correlated gene expression with pathogen presence, and results indicated that gene profiling can be a useful tool for identifying specific pathogen types responsible for disease. Principal component analysis (PCA) further correlated these responses with specific health condition categories, strongly differentiating good, poor, and bad condition ranks. We conclude that molecular profiling is an informative and useful tool that could be applied to indicate and monitor numerous population-level parameters of management interest.

Linking molecular biomarkers with higher level condition indicators to identify effects of copper exposures on the endangered delta smelt (Hypomesus transpacificus)

The delta smelt (Hypomesus transpacificus) is an endangered pelagic fish species endemic to the Sacramento-San Joaquin estuary (CA, USA), and considered an indicator of ecosystem health. Copper is a contaminant of concern in Californian waterways that may affect the development and survival of this endangered species. The experimental combination of molecular biomarkers with higher level effects may allow for interpretation of responses in a functional context that can be used to predict detrimental outcomes caused by exposure. A delta smelt microarray was developed and applied to screen for candidate molecular biomarkers that may be used in monitoring programs. Functional classifications of microarray responses were used along with quantitative polymerase chain reaction determining effects upon neuromuscular, digestive, and immune responses in Cu-exposed delta smelt. Differences in sensitivity were measured between juveniles and larvae (median lethal concentration = 25.2 and 80.4 µg/L Cu(2+), respectively). Swimming velocity declined with higher exposure concentrations in a dose-dependent manner (r =  -0.911, p < 0.05), though was not statistically significant to controls. Genes encoding for aspartoacylase, hemopexin, α-actin, and calcium regulation proteins were significantly affected by exposure and were functionally interpreted with measured swimming responses. Effects on digestion were measured by upregulation of chitinase and downregulation of amylase, whereas downregulation of tumor necrosis factor indicated a probable compromised immune system. Results from this study, and many others, support the use of functionally characterized molecular biomarkers to assess effects of contaminants in field scenarios. We thus propose that to attribute environmental relevance to molecular biomarkers, research should concentrate on their application in field studies with the aim of assisting monitoring programs.

Sublethal responses to ammonia exposure in the endangered delta smelt; Hypomesus transpacificus (Fam. Osmeridae)

The delta smelt (Hypomesus transpacificus) is an endangered pelagic fish species endemic to the Sacramento-San Joaquin Estuary in Northern California, which acts as an indicator of ecosystem health in its habitat range. Interrogative tools are required to successfully monitor effects of contaminants upon the delta smelt, and to research potential causes of population decline in this species. We used microarray technology to investigate genome-wide effects in fish exposed to ammonia; one of multiple contaminants arising from wastewater treatment plants and agricultural runoff. A 4-day exposure of 57-day old juveniles resulted in a total ammonium (NH(4)(+)-N) median lethal concentration (LC50) of 13 mg/L, and a corresponding un-ionized ammonia (NH(3)) LC50 of 147 μg/L. Using the previously designed delta smelt microarray we assessed altered gene transcription in juveniles exposed to 10mg/L NH(4)(+)-N from this 4-day exposure. Over half of the responding genes were associated with membrane integrity and function, however, neurological and muscular function was also affected. Amongst the notable pathways affected by ammonium exposure, directly associated with cellular membranes, are energy metabolism through oxidative phosphorylation, cellular responses to environmental stimuli, highlighted through signal transduction and molecular interactions, cellular processes encompassing transport and catabolism, along with cell motility, development, communication and cell death. To assess these impacts further, key genes were selected as potential biomarkers and investigated using quantitative PCR analysis on fish exposed to 2.5, 5, 10, 20 and 40 mg/L NH(4)(+)-N. Quantitative PCR results indicate biphasic responses, pivoting around the estimated no-observed effect concentration (NOEC; 5.0mg/L NH(4)(+)-N) and below. Genes significantly affected by ammonia exposure include claudin-10, Keratin-15, Septin-3, Transmembrane protein 4, superfamily 4 (membrane), Tropomyosin, Myosin light chain, Calmodulin (muscular), Tubulin cofactor beta (neurological), Sirtuin-6 (development), and Rhesus associated type C glycoprotein 1 (gill- and skin-specific ammonium transporter). The quantitation of the ammonium transporter may highlight the capacity of delta smelt to contend with elevated levels of ammonia, the peak response of which may be indicative of short-term thresholds of tolerance. Our study supports the notion that exposure to ammonia results in cell membrane destabilization, potentially affecting membrane permeability, enhancing uptake and thus synergistic effects of multiple-contaminant exposure.

Transcription of Nrdp1 by the androgen receptor is regulated by nuclear filamin A in prostate cancer

Prostate cancer (PCa) progression is regulated by the androgen receptor (AR); however, patients undergoing androgen-deprivation therapy (ADT) for disseminated PCa eventually develop castration-resistant PCa (CRPC). Results of previous studies indicated that AR, a transcription factor, occupies distinct genomic loci in CRPC compared with hormone-naïve PCa; however, the cause of this distinction was unknown. The E3 ubiquitin ligase Nrdp1 is a model AR target modulated by androgens in hormone-naïve PCa but not in CRPC. Using Nrdp1, we investigated how AR switches transcription programs during CRPC progression. The proximal Nrdp1 promoter contains an androgen response element (ARE); we demonstrated AR binding to this ARE in androgen-sensitive PCa. Analysis of hormone-naive human prostatectomy specimens revealed correlation between Nrdp1 and AR expression, supporting AR regulation of NRDP1 levels in androgen-sensitive tissue. However, despite sustained AR levels, AR binding to the Nrdp1 promoter and Nrdp1 expression were suppressed in CRPC. Elucidation of the suppression mechanism demonstrated correlation of NRDP1 levels with nuclear localization of the scaffolding protein filamin A (FLNA) which, as we previously showed, is itself repressed following ADT in many CRPC tumors. Restoration of nuclear FLNA in CRPC stimulated AR binding to Nrdp1 ARE, increased its transcription, and augmented NRDP1 protein expression and responsiveness to ADT, indicating that nuclear FLNA controls AR-mediated androgen-sensitive Nrdp1 transcription. Expression of other AR-regulated genes lost in CRPC was also re-established by nuclear FLNA. Thus, our results indicate that nuclear FLNA promotes androgen-dependent AR-regulated transcription in PCa, while loss of nuclear FLNA in CRPC alters the AR-regulated transcription program.

Abstract 5750: Synergistic effects of everolimus and bicalutamide in castration-resistant prostate cancer: Results from a phase I/II clinical trial

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: We previously showed that the mTOR pathway is activated in castrate resistant prostate cancer (CRPC) while inhibition of this pathway upregulated androgen receptor (AR) signaling. Based on these data, a phase I/II clinical trial was designed to determine the efficacy and tolerability of the combination of the AR inhibitor bicalutamide and the mTOR inhibitor everolimus in CRPC patients compared with bicalutamide alone. Methods: Eligible patients had histologically confirmed disease and disease progression (PSA or radiographically) while on androgen deprivation therapy (failed the therapy); and could have less than 2 months of bicalutamide at the initiation of androgen deprivation to prevent flare. The primary endpoint is PSA response. Complete response (CR) was defined as complete disappearance of all measurable and non-measurable disease. Results: In all, 19 patients were enrolled in the study, but data from only 18 have been included because one patient passed away before he could be started on the drugs. No unexpected toxicity was observed in the 18 patients enrolled in this study. 5 patients were on placebo+bicalutamide and 13 were on everolimus+bicalutamide. Of the 13 patients on everolimus+bicalutamide, nine (69.23%) had partial response (PR, a decline in PSA by at least 30%), one (7.69%) unconfirmed PR (patients have PSA response less than two cycles), and three (23.08%) had stable disease (SD, no symptomatic deterioration, PSA increase 50%) and 4 did not, were further analyzed for levels of ErbB3. Significantly, ErbB3 levels were increased in the serum from patients who did not show any decline or partial decline in PSA, but not in the ones who responded well to the drug combination. Conclusions: The combination of everolimus+bicalutamide doubled the time to relapse in patients who had failed androgen deprivation therapy, compared to bicalutamide alone. Relapse in these patients may be related to the increase in ErbB3, hence an ErbB3 inhibitor in combination with everolimus may further benefit this group of patients. We intend to investigate in the future whether expression of ErbB3 in the serum can be used to predict whether a patient will respond to the treatment or not. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5750. doi:1538-7445.AM2012-5750

The androgen receptor is a negative regulator of eIF4E phosphorylation at S209: implications for the use of mTOR inhibitors in advanced prostate cancer

The antiandrogen bicalutamide is widely used in the treatment of advanced prostate cancer (PCa) in many countries, but its effect on castration-resistant PCa (CRPC) is limited. We previously showed that resistance to bicalutamide results from activation of mechanistic target of rapamycin (mTOR). Interestingly, clinical trials testing combinations of the mTOR inhibitor RAD001 with bicalutamide were effective in bicalutamide-naïve CRPC patients, but not in bicalutamide-pretreated ones. Here we investigate causes for their difference in response. Evaluation of CRPC cell lines identified resistant vs sensitive in vitro models, and revealed that increased eIF4E(S209) phosphorylation is associated with resistance to the combination. We confirmed using a human-derived tumor xenograft mouse model that bicalutamide pre-treatment is associated with an increase in eIF4E(S209) phosphorylation. Thus, AR suppressed eukaryotic initiation factor 4E (eIF4E) phosphorylation, while the use of antiandrogens relieved this suppression, thereby triggering its increase. Additional investigation in human prostatectomy samples showed that increased eIF4E phosphorylation strongly correlated with the cell proliferation marker Ki67. Small interfering RNA-mediated knockdown (k/d) of eIF4E-sensitized CRPC cells to RAD001+bicalutamide, whereas eIF4E overexpression induced resistance. Inhibition of eIF4E phosphorylation by treatment with CGP57380 (an inhibitor of mitogen-activated protein kinase-interacting serine–threonine kinases MAP kinase-interacting kinase 1 (Mnk1/2), the eIF4E upstream kinase) or inhibitors of extracellular signal-regulated kinase 1/2 (ERK1/2), the upstream kinase-regulating Mnk1/2, also sensitized CRPC cells to RAD001+bicalutamide. Examination of downstream targets of eIF4E-mediated translation, including survivin, demonstrated that eIF4E(S209) phosphorylation increased cap-independent translation, whereas its inhibition restored cap-dependent translation, which could be inhibited by mTOR inhibitors. Thus, our results demonstrate that while combinations of AR and mTOR inhibitors were effective in suppressing tumor growth by inhibiting both AR-induced transcription and mTOR-induced cap-dependent translation, pre-treatment with AR antagonists including bicalutamide increased eIF4E phosphorylation that induced resistance to combinations of AR and mTOR inhibitors by inducing cap-independent translation. We conclude that this resistance can be overcome by inhibiting eIF4E phosphorylation with Mnk1/2 or ERK1/2 inhibitors.

eIF4E Phosphorylation in Prostate Cancer

Prostate cancer (PCa) progression involves a shift from endocrine to paracrine and eventually autocrine control resulting from alterations in molecular mechanisms in the cells. Deregulation of RNA translation is crucial for tumor cells to grow and proliferate; therefore, overactivation of the translation machinery is often observed in cancer. The two most important signal transduction pathways regulating PCa progression are PI3K/Akt/mTOR and Ras/MAPK. These two pathways converge on the eukaryotic translation initiation factor 4E (eIF4E) which binds to the protein scaffold eIF4G upon mechanistic target of rapamycin (mTOR) activation and is phosphorylated by the mitogen-activated protein kinase (MAPK) interacting protein kinases (Mnk1/2). This review describes the role of eIF4E in mRNA translation initiation mediated by its binding to the methylated 5′ terminal structure (m7G-cap) of many mRNAs, and the ability of many tumor cells to bypass this mechanism. Hormonal therapy and chemotherapy are two of the most prevalent therapies used in patients with advanced PCa, and studies have implicated a role for eIF4E phosphorylation in promoting resistance to both these therapies. It appears that eIF4E phosphorylation enhances the rate of translation of oncogene mRNAs to increase tumorigenicity.

Abstract 5051: Androgen receptor-mediated regulation of p14ARF transcription in prostate tumor cells

The p14ARF tumor suppressor is often deleted or silenced in malignancies. Prostate tumors are an exception, where p14 expression is elevated. To understand this phenomenon, we assessed the expression of p53 pathway members, which are most effected by p14ARF. The expression of androgen receptor (AR), a pivotal prostate cancer regulator, which is also affected by p14arf and MDM2 was analyzed as well. The studies used archival prostate tumor tissues obtained from prostatectomies performed at the Veterans Affairs-Northern California Health Care System in Mather California between 1996 and 2002 to better define the relationship between these interrelated networks. A prostate tumor tissue array consisting of 78 tumors of differing stages and grades was constructed to evaluate correlations between multiple parameters. Immunohistochemical studies assessed expression of the proliferation marker Ki67, p53, MDM2, MDM4, p14ARF, and the AR in the nuclear and cytoplasmic compartments of tumor and adjacent cells. p53, MDM4, p14ARF and AR were detected in nuclear and cytoplasmic compartments of tumor and non-tumor cells, but were predominantly nuclear. MDM2 expression was primarily cytoplasmic in tumor cells. Multivariate analysis of the immunohistochemical markers identified a strong correlation between expression of p14ARF and AR. Studies utilizing the prostate CWR22 xenograft and LNCaP cell line models revealed that castration or androgen deprivation resulted in reduced p14arf levels and that this effect correlated with a precipitous decline in E2F1-3a levels. In a reciprocal analysis, RB ablation enhanced p14ARF transcription, arguing that the E2F/RB pathway mediates AR-dependent p14ARF expression. However, we also identified an AR binding site located ∼40 KB upstream of the p14ARF gene. Chromatin immunoprecipitation (ChIP) studies showed that in prostate cells this site was bound by AR. ChIP studies also revealed E2F1 and E2F3 were present at the p14ARF promoter. Together, the studies argue p14ARF is a direct transcriptional target of AR and that AR and E2F collaborate to promote p14ARF expression. . Citation Format: Maria Mudryj, Salma Siddiqui, Stephen J. Libertini, Alan P. Lombard, Benjamin Mooso, Leandro D9Abronzo, Frank Melgoza, Alexander Borowsky, Christiana Drake, LiHong Qi, Paramita M. Ghosh. Androgen receptor-mediated regulation of p14ARF transcription in prostate tumor cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5051. doi:10.1158/1538-7445.AM2015-5051

Abstract 1725: Increased phosphorylation of eIF4E induces resistance to treatment with mTOR inhibitors alone or together with AR antagonists in advanced prostate cancer

The standard of care for patients with recurrent prostate cancer (PCa) is the use of androgen receptor (AR) antagonists, but the treatment ultimately fails, resulting in the development of castration resistant PCa (CRPC). Patients with CRPC are frequently continue to express an active AR, despite castration resistance, and AR inhibitors remain effective in these patients for several months. We previously showed that upregulation of mammalian target of rapamycin (mTOR) activity upon use of AR antagonists contributed to acquired resistance to this therapy, and that a combination of an mTOR inhibitor and an AR antagonist overcame resistance to AR antagonists alone (Wang et al, Oncogene, 2008;27(56):7106-17). Based on our data, a Phase II clinical trial was conducted to determine the efficacy of the combination of the mTOR inhibitor RAD001 and the AR antagonist bicalutamide in bicalutamide-naive CRPC patients (ClinicalTrials.gov: NCT00814788). This study, which was recently concluded, showed a response rate of 75% with this combination with the historical control of 25%. The overall goal of this project was to define pathways that results in resistance to combinations of mTOR and AR inhibitors in patients with CRPC. Comparison of various mTOR inhibitors: the mTORC1 inhibitor RAD001, a mTORC1/C2 dual inhibitor INK128 and a mTORC1/C2/PI3K triple inhibitor BEZ-235 either alone or in combination with AR antagonists bicalutamide and enzalutamide in various prostate derived cell lines including C4-2, PC-346C, 22Rv1 and CWR-R1, identified cells that were resistant (CWR-R1, PC-346C) vs those that were sensitive (22Rv1, C4-2) to these inhibitors. Investigation of the base-line molecular profile of these cells demonstrated that those that expressed high levels of phosphorylated form of eIF4E (S209) were resistant to mTOR inhibitors. Downregulation of eIF4E phosphorylation by siRNA resulted in sensitivity of CRPC cells to the combination of the mTOR inhibitors with AR antagonists. Investigation of the mechanism by which eIF4E phosphorylation levels increased in certain CRPC cells but not in others revealed that expression and transcriptional activity of the AR negatively correlated with the levels of eIF4E phosphorylation. In cells with high basal levels of phospho-eIF4E, bicalutamide further increased eIF4E phosphorylation, whereas those with low eIF4E levels were not further affected. The ability of AR inhibition to suppress eIF4E phosphorylation was mediated by MAP kinase interacting kinase (Mnk), and the ability of some cells to phosphorylate eIF4E, but not others, correlated with the levels of Mnk phosphorylation. Based on these studies, we predict that patients with high basal PSA who express low levels of Mnk phosphorylation are the ones who are likely to respond to the combination of an mTOR inhibitor and an AR antagonist. Citation Format: Leandro S. D’Abronzo, Michael Crapuchettes, Ryan E. Beggs, Swagata Bose, Salma Siddiqui, Yu Wang, Blythe Durbin-Johnson, Chong-Xian Pan, Paramita Ghosh. Increased phosphorylation of eIF4E induces resistance to treatment with mTOR inhibitors alone or together with AR antagonists in advanced prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 236.