1. Phthalates induce proliferation and invasiveness
of estrogen receptor-negative breast cancer
through the AhR/HDAC6/c-Myc signaling pathway
HSIEH ET AL.
The FASEB Journal
Vol. 26 February 2012
Iod: 0892-6638/12/0026-0778
Date : May 22,2013
Advisor: Prof. CS Chiang
Presenter: Ahmad Usama
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2. Outlines
1- Introduction
2- Materials & Methods
3- Results & Mini-conclusions
4- Discussion.
5- My Comment.
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3. 1- INTRODUCTION
Estrogen Receptor (Positive & Negative) Breast Cancer
http://www.cancer.gov/cancertopics/understandingcancer/estrogenreceptors/page18
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4. 1- INTRODUCTION
Phthalate Esters (ex: BBP & DBP)
*Phthalates induce the proliferation and invasiveness
of estrogen receptor negative human breast cancer
through the AhR / HDAC6 / c-Myc pathway .
* Phthalates absorbed through the skin or ingested.
N- Butyl Benzyl Phthalate (BBP)
Cosmetics
Nail polishes
Hair Sprays
Dibutyl Phthalate (DBP)
Plastic Toys
Food wrappers
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5. 1- INTRODUCTION
Aryl hydrocarbon Receptor (AhR)
The Genomic Pathway
Ex: Phthalates or Dioxin
released
The Aryl hydrocarbon Receptor (AhR) signaling pathway
ARNT : Aryl hydrocarbon Receptor Nuclear Translocator
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http://www.hindawi.com/journals/ijbc/2011/923250/fig1/

6. 1- INTRODUCTION
Aryl hydrocarbon Receptor (AhR)
The Non Genomic Pathway
Ex: Phthalates or Dioxin
released
cAMP
Ca +2
The Aryl hydrocarbon Receptor (AhR) signaling pathway
ARNT : Aryl hydrocarbon Receptor Nuclear Translocator
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http://www.hindawi.com/journals/ijbc/2011/923250/fig1/

7. 1- INTRODUCTION
HDAC6 (Histone Deacetylase 6)
* HDAC 6 has histone deacetylase activity and represses the transcription.
* HAT has histone acetylase activity and activates the transcription.
Activated Transcription
Repressed Transcription
7
David P. Clark. Molecular Biology: Understanding the Genetic Revolution. Elsevier Academic Press, 2008.

8. 1- INTRODUCTION
c-Myc gene
* c-Myc or Myc is a regulator gene that codes for a transcription factor .
* It believed to regulate the expression of 15% of all genes through binding on
Enhancer Box sequences (E-Boxes) and recruiting histone acetyltransferase
(HAT).
* Mutated version of c-Myc gene is found in many cancers which leads to
unregulated expression of many genes .
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9. Nongenomic activation of AhR and
the downstream pathway leading to activation of c-Myc expression.
Extracellular
Matrix
Cell membrane
Cytoplasm
Nucleus
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10. 2- MATERIALS & METHODS
1- Cell lines and chemicals
2- XTT studies, bromodeoxyuridine (BrdU) incorporation, and colony formation assays
3-Wound-healing (also called Cell migration) and invasion assays.
4- Flow cytometry staining.
5- Reverse transcription-PCR and qPCR assay.
6-Immunoblotting and immunoprecipitation analyses.
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11. 2- MATERIALS & METHODS
7-Total internal reflection fluorescence (TIRF) microscopy.
8-Immunofluorescence analyses.
9-Small interfering RNA (siRNA) transfection.
10- Cell fractionation.
11-Chromatin immunoprecipitation (ChIP) assay.
12-cAMP measurement.
13-Animal studies and immunohistochemistry.
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12. 3-RESULTS
I-Phthalates promote in vitro tumor growth and
invasion of cancer cells
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13. 2-RESULTS
I-Phthalates promote in vitro tumor growth and
invasion of cancer cells
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14. MINI CONCLOSION I
1- Phthalates (BBP & DBP) increase the cell proliferation , colony formation , cellular
motility (cellular migration) and cellular invasion for estrogen receptor – negative human
breast caner cell line (MDA-MB-231).
2- These increasing have a dose – dependent manner.
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15. 3-RESULTS
II-Phthalates mediate HDAC6 gene expression through
the nongenomic function of AhR
Control
treatment
BBP
treatment
1 min
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16. 3-RESULTS
II-Phthalates mediate HDAC6 gene expression through
the nongenomic function of AhR
*3’ , 4’ – DMF (Dimethoxyflavone): AhR antagonist.
*AhR siRNA: block AhR
*H89: PKA inhibitor
* CREB1: transcriptional factor for HDAC6 transcription.
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17. 3-RESULTS
II-Phthalates mediate HDAC6 gene expression through
the nongenomic function of AhR
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18. MINI CONCLOSION II
1- Aryl hydrocarbon Receptor (AhR) is higher expressed in ER-negative human breast
cancer cells than those in ER-positive human breast cancer cells.
2- The endogenous AhR is relocalized from the cytoplasm to the cellular membrane after
phthalates induction.
3- Phthalates result in acute accumulation of intracellular cAMP which is stimulated by
AhR.
4-Exposure to BBP or DBP induced dramatic recruitment of CREB1 onto the CREB1binding site after its phosphorylation at serine 133.
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19. 3-RESULTS
III- HDAC6 is required for phthalate-mediated cell growth and motility
* Ac-tubulin: Acetylated tubulin.
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20. 3-RESULTS
III- HDAC6 is required for phthalate-mediated cell growth and motility
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21. 3-RESULTS
III- HDAC6 is required for phthalate-mediated cell growth and motility
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22. 3-RESULTS
III- HDAC6 is required for phthalate-mediated cell growth and motility
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23. MINI CONCLOSION III
1-HDAC6 as a deacetylase plays a significant role in
enhancing cancer cell growth and invasion.
2-Depletion of HDAC6 blocking the phthalate-induced
cell growth , migration and invasiveness.
3- Control siRNA has weaker effect in the blocking of HDAC6 than those to
HDAC6 siRNA-1 or HDAC6 siRNA-2.
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24. 3-RESULTS
IV- Phthalates mediate c-Myc gene expression through HDAC6
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25. 3-RESULTS
IV- Phthalates mediate c-Myc gene expression through HDAC6
Confocal immunofluorescence
microscopy imaging
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26. 3-RESULTS
IV- Phthalates mediate c-Myc gene expression through HDAC6
Reciprocal immunopreciptation
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27. MINI CONCLOSION IV
* After phthalates induction, HDAC6 facilitates nuclear translocation of
the Beta-catenin to combine with LEF1/TCF4 transactivation complex, which then
promotes c-Myc expression.
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28. 3-RESULTS
V- In vivo tumor growth is mediated by phthalates
Bioluminescence imaging IVIS
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29. 3-RESULTS
V- In vivo tumor growth is mediated by phthalates
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30. MINI CONCLOSION V
*Phthalates promote tumor development of ER negative human breast cancer through a
signaling cascade to enhance tumor expression of HDAC6 and c-Myc.
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31. 4-DISCUSSION
1-phthalates induce tumor growth and invasiveness through an ER-independent non
genomic mechanism.
2-Enhanced HDAC6 expression is responsible for the increased nuclear function of
Beta-catenin to induce c-Myc expression, resulting in the promotion of proliferation
and motility in ER-negative breast cancer cells exposed to phthalates.
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32. 5- MY COMMENT
1- P.16 what is the difference between CREB1 and 133 phosphorylated serine CREB1
(CREB1-P)?
2- P.19 unexpected result of flow cytometry peak’s for DBP treatment is lower than
control for HDAC6 !!!
3- How HDAC6 increases the transcription of the oncogene c-Myc and its function is
the deactylation which leads in the normal cells to repress the transcription !!!
Ans: because HDAC6 does not work directly on c-Myc but it is just a part of a cascade to
translocate Beta-catenin from the cytoplasm into the nucleus to combine with the
transcriptional complex (ELF1/TCF4) that activates c-Myc transcription.
4- Need more in vivo confirming experiments to the in vitro results !
5- MDA-MB-231 & SK-BR-3 are human breast cancer cell lines , so in vivo experiments
need more confirmations !
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