Mammary Cancer and Biomarkers of Effect Jun Wang, James Mobley, - - PDF document

The Use of Proteomics in Elucidating Mechanisms of Predisposition for Mammary Cancer and Biomarkers of Effect

Jun Wang, James Mobley, Angela Betancourt, Sarah Jenkins, Susan Pinney, Frank Biro, Jose Russo and Coral Lamartiniere University of Alabama at Birmingham, Cincinnati Children’s Hospital & Fox Chase Cancer Center, USA

Cl Cl Cl Cl O O

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)

Diethylstilbestrol (D ES) C == C C2H5 C2H5 OH HO

Butyl benzyl phthalate (BBP) Genistein Resveratrol

Overarching goal: To determine the effects

• f perinatal exposure to environmental

chemicals predisposing for mammary cancer.

HO OH H

Estradiol Di-2-ethylhexyl phthalate

DEHP

BPA sport bottles Nalgene Baby Bottle

Painting

• n BPA

Office BPA H20

Lining of soda and food cans

Oral Postnatal (Prepubertal) Bisphenol A Exposure Lactating female Sprague Dawley CD rats are treated orally on days 2-20 post-partum with 25 µg

• r 250 µg BPA/kg body weight or an equivalent

volume of sesame oil. At day 50 postpartum female offspring are treated

• rally with 30 mg dimethylbenz(a)anthracene

(DMBA)/kg BW to induce mammary tumors. Palpate for mammary tumors Necropsy at 180 days post DMBA Histopathology carried out Important time line: No exposure to BPA after weaning

Dimethylbenz(a)anthracene (DMBA)

Jenkins, S, Raghuraman, N., Eltoum, I, Carpenter, M, Russo, J and Lamartiniere, CA. Environmental Health Perspectives. 117: 910-915.

Genistein Soybean Plant Lactating female Sprague Dawley CD rats were fed 25 mg genistein or 250 mg genistein/kg diet from birth until time of weaning, hence the offspring receive genistein from the mother’s

• milk. (Offspring received genistein
• nly during lactation.)

50 Day old female

• ffspring received 80 mg

DMBA/kg BW to induce mammary tumors

250 mg Genistein/ kg Diet + DMBA

Days Post DMBA 100 200

25 mg Genistein/ kg Diet + DMBA Zero Genistein in Diet + DMBA

2 4 6 8 Mammary Tumors per Rat

DMBA Induced Mammary Tumors in Rats Exposed Prepubertally to Genistein

10

Fritz, W.A., Coward, L., Wang, J. and Lamartiniere, C.A. Carcinogenesis 19: 2151- 2158.

Genistein

• The emphasis of pointing out that these experimental

animals receive BPA or genistein exposure only during the prepubertal period is to point out that these changes in susceptibility for mammary cancer are effected early in postnatal life and appear to be permanent manifestations.

• We hypothesize that these hormonally-active chemicals

elicit developmental alterations that result in the biochemical “blue print” being permanently altered and differentially expressed later in life even in the absence of the original effector.

• These alterations are termed organizational or imprinting

effects.

• Is there a precedence for this in the human population?
• Yes.
• Shu et al. investigated soyfood intake during adolescence

and subsequent risk of breast cancer among Chinese women and found that girls eating soy prior to age 15 had a lower incidence of breast cancer than those not on a soy

• diet. (Cancer Epidemiol Biomarkers Prev 2001, 10:483–

488.)

Mammary Gland Proteomics

Representative 2-D gel protein profile with the protein spots marked as differentially regulated from mammary glands of rats exposed to genistein. The identities of these spots were identified by MALDI-TOF-TOF and/or LC-MS/MS. These proteins were subsequently confirmed by western blot analysis.

pH 4 pH 7

220 kDa 15 kDa

Gelsolin PDIA3 VDBP Peroxiredoxin 2 Actin,cytoplasmic 1 Tubulin beta-5 chain Biliverdin reductase A

Bioinformatic analysis on protein expression studies suggested that BPA and genistein regulate cell proliferation and apoptosis in the mammary gland.

Ki-67 staining for cell proliferation

Apop-Tag assay for apoptosis

Cell proliferation in mammary glands of 50-day-old rats exposed prepubertally to BPA and/or genistein, and SO (Controls) from day 2 until day 20 postpartum. Ki-67 expression was measured as an indicator of cell proliferation. Values represent mean ± SE; n = 6.

a,b,c p ≤ 0.01 compared with control. d p ≤ 0.001 compared with BPA.

Jun Wang

Rate of cell apoptosis in mammary glands of 50-day-old rats exposed prepubertally to BPA and/or genistein, and SO (Controls) from day 2 until day 20

• postpartum. ApopTag staining and morphological analyses were used as

indicator of cell apoptosis. Values represent mean ± SE; n = 6. a,b,c p ≤ 0.01 compared with control. d p ≤ 0.001 compared with BPA.

Jun Wang

Protein expressions of SRC-1, SRC-2 and SRC-3 from mammary gland extracts

• f 50 day old rats exposed prepubertally to BPA or/and genistein, and SO

(control). Densitometric values of Western blots were reported as a percentage

• f the controls ± SEM: a,b p ≤ 0.05 compared with control; c p ≤ 0.0001 compared

with BPA. d p ≤ 0.05 compared with genistein.

SRC: Steroid Receptor Co-regulators (positive regulators of estrogen and progesterone receptor action) are associated with cell proliferation.

Regulation of Steroid Receptor Co-regulator Proteins in Mammary Glands of 50 Day Old Rats Exposed Prepubertally to BPA and/or Genistein

 SRCs 1-3  pEGFR  VEGF-R2  Cleaved Casp 9  Cleaved Casp 3  Cleaved PARP  SRCs 1-3  pEGFR  Akts 1-3  pAkt  Wnt-1  p-Bad  Cleaved Casp 3  Cleaved PARP  P21  PTEN

 Cell Proliferation  Proliferation : Apoptosis  Apoptosis

 Proliferation : Apoptosis

BPA vs Controls Genistein vs Controls

Venn diagram depicting rate of cell proliferation and apoptosis, and differential regulation of associated proteins in mammary glands of 50 day

• ld rats exposed prepubertally to BPA or genistein compared to controls.

This data supports our hypothesis that susceptibility for chemically induced mammary cancer can be predicted by the rate of cell proliferation and cell death.

Wang, Jenkins & Lamartiniere. BMC Cancer (2014).

Prepubertal BPA or Genistein Effects in Mammary Glands of 50 Day Old Rats

15

Proteomic Biomarker Analysis in Blood of Preadolescent Girls Exposed to Bisphenol A and Genistein

Experimental Design Urine Blood Serum

Measured (CDC): (phytoestrogens, phthalates, phenols)

LC-MS/MS analysis

Data Analysis Protein Biomarkers

Proteomics

(Jim Mobley

• et. al, UAB)

(from Frank Biro, Susan Pinney, & Colleagues @ Cincinnati Children’s Hospital) (Dong-quan Chen & Angela Betancourt, UAB)

CCH to UAB

Blood Collection from Girls, and Serum Preparation

High Levels of Endocrine Active Chemicals Low Levels of Endocrine Active Chemicals

Sample 1 Sample 2 Sample 3 Sample 1 Sample 2 Sample 3

Immunodepletion with IgY-H7 Ab Reduction, alkylation, trypsin digestion

Peptides sample 1 Peptides sample 2 Peptides sample 3 Peptides sample 2 Peptides sample 1 Peptides sample 3

Label TMT 126 Label TMT 127 Label TMT 128 Label TMT 130 Label TMT 129 Label TMT 131

Combine Strong Cation Exchange Chromatography Reverse Phase Liquid Chromatography

LCMS -(MuDPIT)

Protein Identification (Database searching: SEQUEST) Protein Quantification (Data filtering: ProteoIQ) Statistical Analysis Systems Biology Analysis

Protein identification and quantification in serum from girls with high and low levels (control group) of endocrine active chemicals (Bisphenol A and genistein) in the urine. Human serum was immunodepleted of the 7 most abundant proteins and labeled with TMTs. Proteins were analyzed by on-line automated nano-LC-ESI-MS (SCX/ RP) MuDPIT (PQD-LTQXL ThermoFinnigan). Data were searched using SEQUEST, and analyzed using BioInQuires ProteoIQ software package.

Tandem mass tags Tandem mass tags

Chemical

• No. of serum

proteins identified

• No. of serum protein

differentially expressed BPA 1992 51(2.6%) Genistein 1364 34 (2.5%)

Results Summary of Proteins Identified to be Differentially Expressed in Serum of Pubertal Girls with High Urine Concentrations of BPA and Genistein

Biological Functions Bar graph representation of proteins classified by biological

• function. Analysis was carried out by PANTHER on differentially

regulated proteins identified via TMT-MS from blood of prepubertal girls with high urine concentrations of BPA and genistein.

Differentially Regulated Cancer Associated Proteins Identified by TMT-MS in Blood of Girls with High Urine Concentrations of BPA

Proteins identified using the Human Genome DB ID. Positive and negative fold change in protein expression indicate up- and down-regulation of protein expression relative to control,

• respectively. aSignificance analysis of microarray (SAM).

Protein ID

Protein Name

Group Prob- ability

• No. Unique

Peptides Fold Change (Rx/C) SAMd Q01484

ankyrin-2 plays a role in metastatic breast tumor cell invasion and migration

0.99 4 +2.21 1.69 P46013

antigen Ki-67 is a cellular marker for proliferation and used as a biomarker for cancer.

0.95 4 +3.65 1.41 P45985

mitogen-activated kinase kinase 4 (MKK4) is an important mediator of cellular responses to extracellular signals that include growth factors, hormones, cytokines and environmental stresses.

0.95 2 +3.08 1.12 Q9Y4G6

talin-2 is up-regulated in invasive breast carcinomas.

0.90 4 +2.54 1.43 Q9UL62

transient receptor potential channel 5 (TRPC5) protein is high in human breast tumors and in the circulation.

0.93 3 +2.40 1.23 Q96QB1

deleted in liver cancer 1 (DLC1) functions as a tumor suppressor in a number of common cancers, including breast.

0.83 3

• 2.95

1.24 Q9UBC3

DNA (cytosine-5)-methyltransferase 3B (DNMT3B) is essential for mammalian development and is required for genome wide de novo methylation.

0.93 2

• 2.51

1.77 Q6NUQ1

RAD50-interacting 1 plays a role in cell cycle checkpoint control after DNA damage.

0.92 2

• 2.68

1.05

Protein IDa

Protein Nameb

Group Prob- ability No. Unique Peptides Fold Change (Rx/C)c SAMd P42892

endothelin-converting enzyme (ECE-1) 1 has been implicated in the pathogenesis of a range of disease states including breast, gynecological and urological cancers, cardiovascular disease and Alzheimer’s disease.

1.00 4

• 2.12

1.72 B4DUI3

eukaryotic translation initiation factor 3 subunit J (EIF3) has been found elevated in human breast, cervical, esophageal, and lung cancers.

0.82 2

• 2.30

1.63 Q9UMY1

nucleolar 7 is reported to be a candidate tumor suppressor gene in cervical cancer

0.82 2 +2.10 1.02 Q9NQX1

PR domain zinc finger 5 (PRDM5) has growth suppressive activities and is silenced in breast,

• varian, liver, lung, colon, and other cancers.

0.97 5 +2.30 0.95

Differentially Regulated Cancer Associated Proteins Identified by TM-MS in Blood of Girls with High Urine Concentrations of Genistein

Proteins identified using the Human Genome DB ID. Positive and negative fold change in protein expression indicate up- and down-regulation of protein expression relative to control,

• respectively. aSignificance analysis of microarray (SAM).

Summary

• Our data demonstrates that prepubertal only exposure

to BPA predisposes for chemically-induced mammary cancer in rats.

• On the other hand, prepubertal exposure to genistein

via the diet suppresses chemically-induced mammary cancer in rats.

• Through the use of 2-D gels and MS, we demonstrate

the separation and quantification of proteins in mammary glands of rats exposed to two hormonally- active chemicals.

• Based on differential protein expression occurring 30

days after last exposure to genistein and BPA we hypothesize that prepubertal exposure resulted in

• rganizational effects, perhaps epigenetic alterations.
• Epigenetic studies demonstrate that prepubertal BPA

and genistein exposures differentially alter methylation patterns of promoter regions of genes involved in regulating cell proliferation, apoptosis and carcinogenesis in the mammary glands of adult rats.

Summary continuation

• Using blood of girls with high urine concentrations of

Bisphenol A and genistein, we show differentially expressed protein profiles. For this we used TMT-MS proteomic technology.

• Using western blot analysis on mammary glands of rats

we have also demonstrated cross-species and tissue validation of differentially regulated proteins from the girls’ blood.

• These data suggest that proteomics is the future of cancer

biomarker discovery because proteins (enzymes, growth factors, etc.) are the actual players in cause and prevention of cancer.

Chris Gorga Paul Clark Jun Wang Keela Dodd Angela Betancourt Rachel Kennerly Sarah Jenkins Jose Russo Isam Eltoum James Mobley Coral Lamartiniere Frank Biro This research was supported by the Breast Cancer and the Environment Research Program grants (ES/CA U01 ES012771 and U01ES019482), and a Genes, Environment and Health Initiative grant (ES/CA U01 ES016003). Contact: Coral@uab.edu

Susan Pinney

In utero exposure to Bisphenol A shifts the window of susceptibility for mammary carcinogenesis in the rat. Betancourt, A.M., Eltoum, I.A., Desmond, R.A., Russo, J, Lamartiniere, C.A. Environ Health Perspect. 118(11): doi:10.1289/ehp.1002148, 2010. Chronic oral exposure to Bisphenol A results in a non- monotonic dose response in mammary carcinogenesis and metastasis in MMTV-erbB2 mice. Jenkins S, Wang J, Eltoum I, Desmond R, and Lamartiniere CA (2011). Environ Health Perspect 119:1604–1609 . http://dx.doi.org/10.1289/ehp.1103850, 2011. Cell proliferation and apoptosis in rat mammary glands following combinational exposure to Bisphenol A and genistein. Wang J, Jenkins S and Lamartiniere CA. BMC Cancer, 14:379 doi:10.1186/1471-2407-14-379. 2014.