Tier 1 and Done: developing in vitro cell-based assays for endocrine pathways sufficient by themselves for 21 st century risk assessment

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risk assessment Mel Andersen The Hamner Institutes for Health

1 Tier 1 and Done: developing in vitro cell-based assays for endocrine pathways sufficient by themselves for 21 st century risk assessment Mel Andersen The Hamner Institutes for Health Sciences Research Triangle Park, NC Mandersen@thehamner.org

TT21C: all routine toxicity testing will be conducted in human cells or cell lines in vitro by evaluating perturbations of cellular responses in a suite of toxicity pathway assays.

2 TT21C: all routine toxicity testing will be conducted in human cells or cell lines in vitro by evaluating perturbations of cellular responses in a suite of toxicity pathway assays. Hamner Institutes Program - Tier 1 and Done Designed-for-purpose assays for prototype signaling pathways (e.g., DNA damage, PPARα, estrogen signaling, etc.) in human cells or cells lines Map and model the pathway to understand dose-response behaviors at low levels of response based on cellular circuitry Use pathway dynamics, cellular perturbations, and reverse toxicokinetics to predict regions of safety for exposures to specific compounds

3 Some topics: The suite of tools to understand the structure and dynamics of nuclear receptor pathways Our toxicity pathway progress/plans with estrogen receptor (ER)-mediated responses in uterine cells

Provide training opportunities so toxicology

4 New extrapolation tools Understand the state-of-the art in computational cell biology Use the information to guide our own research programs with the case studies Provide training opportunities so toxicology & risk assessment communities are prepared for new approaches

5 Computational Systems Biology & Dose Response (1) Mapping toxicity pathways Bioinformatic tools ligand ER... Coactivator Coactivator 1 2 Coactivator 3 Coactivator 4 Kinase 1 Kinase 2 Kinase1 3 Kinase 4 TF1 TF2 TF3 TF4 TF5 TF6 TF7 TF8 TF9 TF10 TF11 TF13 TF12 Adapted from Bromberg et al Science (2008). (2) Modeling toxicity pathways computational methods Proliferative state Response Resting Cell Estren mer agonist

6 Hamner Project Objectives Provide validated, designed for purpose in vitro assay for estrogen pathways in human uterine epithelial cells Use in vitro assay to: Map overall ER pathway in these cells Determine role for various estrogen-receptors in IK cells using selective agonists Define dose-response models for adverse responses of specific receptor agonists and prototype compounds Develop in vitro only risk assessment for prototype chemicals

7 Why the Ishikawa uterine cell line as the model? Amphibian Metamorphosis (Frog) Androgen Receptor Binding (Rat Prostate) Aromatase (Human Recombinant) Estrogen Receptor Binding Estrogen Receptor Transcriptional Activation (Human Cell Line HeLa-9903) Fish Short-Term Reproduction Hershberger (Rat) Female Pubertal (Rat) Male Pubertal (Rat) Steroidogenesis (Human Cell Line H295R) Uterotrophic (Rat) Hamner Project - Tier 1 and Done Refers to developing an in vitro E2-pathway assay that provide results sufficient for risk assessment without to move to in life studies will the need

8 A Contemporary, though likely still incomplete Picture of the Players in E2 signaling ERα ERβ ER-36R ERRα ERRβ ERRγ

9 E2 EE ESTREN Differentiate pathway responses with ligands with some specificity for each receptors.

10 Integrating behavior across receptors/signaling pathways

11 ChIP results evaluate genomic responses After collecting genes expression data, TF analysis done from CHIP-chip or CHIP-seq.

12 The MCF-7 E2-Network

13 Membrane-ER (mer)

14 Attachment to membrane: ERα targeted to membrane through palmitoylation of cys 447 A splice variant, ER36, interacts through c-src with membrane targeting likely through c-src myristoyaltion ER66 ER46 ER36

15 Two pulse paradigm of with E2-BSA in vitro Cell impermeable E2 substitute for E2 in first pulse. Non-genomic and genomic actions synergize with each other for early responses. E2-BSA does not act on second pulse Vasudevan (2001).

16 Or using a transgenic mouse: transgenic mouse with ERα that lacks DNA -The NERKI (non-classical ER knock-in) - AA/- mouse non-classical allele on a null background Called AA/+ and AA/- mice

17 Responses due to membrane E2 receptors in the uterus Membrane receptors alone do not lead to uterotrophic assay weight increase (top) Neither do they upregulate progesterone receptors (PR) O'Brien, J. E., T. J. Peterson, et al. (2006). "Estrogen-induced proliferation of uterine epithelial cells is independent of estrogen receptor alpha binding to classical estrogen response elements." J. Biol. Chem. 281(36):

Responses due to membrane E2 receptors in the uterus Non-genomic pathways mediate uterine cell proliferation Uterine weight gain (water imbibition) mediated by genomic responses O'Brien, J.

18 Responses due to membrane E2 receptors in the uterus Non-genomic pathways mediate uterine cell proliferation Uterine weight gain (water imbibition) mediated by genomic responses O'Brien, J. E., T. J. Peterson, et al. (2006). "Estrogen-induced proliferation of uterine epithelial cells is independent of estrogen receptor alpha binding to classical estrogen response elements." J. Biol. Chem. 281(36):

19 A new player on the block,

What are the different functions of the two

Is GPER modulatory rather than activational?

ER... Coactivator Coactivator 1 2 Coactivator 3

20 What are the different functions of the two membrane receptors? Is GPER modulatory rather than activational? New evidence suggests that GPER controls expression of ER36. IK Cell Proliferation with agonists TF1 TF2 TF3 TF4 ER... Coactivator Coactivator 1 2 Coactivator 3 Kinase 1 Kinase 4 ligand Kinase 2 Coactivator 4 Kinase 3 TF5 TF6 TF7 TF8 TF9 TF10 Adapted from Bromberg et al Science (2008). TF13 TF12 TF11

21 Early results with ethinyl estradiol (EE) in IK cells ER expression Proliferation ALP induction ER target gene induction Progesterone Receptor

22 Specific Aims: 1. Collect dense data stream in the IK cells, dose and time for: Proliferation, transcriptomics, Ca 2+ influx, kinase panels, phosphoproteomics, chip-seq 17β estradiol (E2), ethynyl estradiol (EE) G1 (specific GPER agonist) PPT (specific ESR1/ERα agonist) DPN (specific ESR2/ERβ agonist) Estren (4-estren-3α,17β-diol/mER agonist)

23 Specific Aims: 3. Apply bioinformatic tools to infer pathway circuitry controlling gene expression and phenotypic responses & generate a CSBP model to describe the dose-response. 4. Conduct a detailed rat uterotrophic assay with EE to map/model in vivo E2-pathway and compare results to in vitro data. 5. Apply validated in vitro E2-assays to several estrogenic compounds (genistein, methoxychlor, etc.) and conduct in vitro-only risk assessments.

24 Creating a CSBP Model need a hypothesis so. With increasing E2 exposures, GPER activates ER36 synthesis Increasing ER36 interactions produces c-src mediated ultrasensitive MAPK cascades, trigerring proliferation Proliferative state Enhanced ERα activation through c- src enhances E2 specific gene transcription Response Resting Cell Estradiol Concentration

25 Thoughts on progress with case studies: It is necessary to understand pathway circuitry in order to use information for safety assessment purposes. What role does differential affinity for the three key receptors ERα, mer and GPER play in responses to environmental compounds? How much detail will be required as we move to subsequent pathway case studies? Is each pathway unique or will we quickly discover recurring motifs that modulate dose response across pathways, e.g., negative feedback, and bistability and ultrasensitivity controlling activational processes?

Hamner TT21C Sponsors Programmatic Support and Estrogen Pathway: ACC-LRI (2008-2009) NIEHS Superfund Basic Research Program Dow Chemical Company Exxon Mobil Foundation Dow Corning CropLife America

26 Hamner TT21C Sponsors Programmatic Support and Estrogen Pathway: ACC-LRI ( ) NIEHS Superfund Basic Research Program Dow Chemical Company Exxon Mobil Foundation Dow Corning CropLife America Agilent Technologies Illumina, Inc. DNA-damage and oxidative stress pathways: Hamner TT21C Team Rebecca Clewell Caroline LeSommer Bin Sun Boris Reidel Susan Ross Pergentino Balbuena Sandeep Singhal Patrick McMullen Sudin Bhattacharya Joe Trask Linda Pluta Qiang Zhang Manda Edwards Ed LeCluyse Rusty Thomas Unilever

Progress and Future Directions in Integrated Systems Toxicology. Mary McBride Agilent Technologies

Progress and Future Directions in Integrated Systems Toxicology Mary McBride Agilent Technologies 1 Toxicity testing tools of the late 20 th century Patchwork approach to testing dates back to the 1930