1. Elvin Estrada1, Maricarmen Colón1, Miosotis García1, Madeline S. Collazo1,
Perla Báez1, Janice Monteiro1, Abigail Ruiz1, Edward Seto2, Idhaliz Flores1
1Ponce School of Medicine and Health Sciences, Ponce, PR & 2H. Lee Moffitt Cancer Center, Tampa, FL
• Endometriosis, the presence of endometrial tissue (glands and stroma)
outside of the uterus, causes inflammation, pain and infertility.
• Despite being a common gynecologic disease and a major cause of
pain and infertility, the pathophysiology of endometriosis remains unclear.
• Genetic, inflammatory and recently epigenetic mechanisms have been
ascribed important roles in the attachment, survival, invasion and growth
of endometriotic cells at ectopic sites.
• Deacetylation of histones by histone deacetylases (HDACs), is a
mechanism for regulating expression of genes responsible for key cellular
processes including cell-cycle progression, proliferation, differentiation,
and apoptosis (Kelly et al, 2002).
• Overexpression of or dysregulated HDAC activity results in a “closed”
chromatin structure; the promoter sequences of certain genes become
inaccessible to transcription factors which results in gene silencing (de
Ruijter et al, 2003, Marks et al, 2005, Arts et al, 2003).
• Previous studies have shown that TSA, a histone deacetylase inhibitor
(HDACi), attenuates invasiveness, E-cadherin expression and
proliferation while increasing apoptosis of immortalized endometriotic
cells (Guo et al, 2007; Imesch et al, 2010).
• HDACi also decreased lesion growth and hyperalgesia in an animal
model of endometriosis and induced expression of the cell cycle regulator
p21 (Lu et al, 2010; Wu and Guo, 2008).
• Class I HDACs (HDAC1,2,3,8) have been shown to be expressed in
endometrium and alterations in their expression have been linked to
impaired endometrial differentiation (Krusche et al, 2007).
• We have shown that HDAC1/2 are overexpressed in endometriotic
cells and lesions, and that these enzymes are regulated by ovarian
steroid hormones in endometrial cells (Colón et al, under review).
• We identified growth differentiation factor 11 (GDF11), as one of the
genes most downregulated in endometriotic lesions using cDNA
microarrays.
• HDACi activates and HDAC3 represses expression of the GDF11 gene
(Zhang et al, 2004).
HDAC3 and GDF11 Expression in Endometriosis
ACKNOWLEDGEMENTS
Grant support : R01-HD050559 (I.F.); NIH-MBRS S06-GM08239 (I.F.); NIH-NIGMS
1R25GM082406 (M.C. and A.R.); U56CA126379 (TMA construction). Dr. A. Fazleabas
provided HESC and EEC cells. Dr. A. Mendoza from Southern Pathology Inc. archived
tissues.
RESULTS
1. To determine basal and steroid regulated gene expression of HDAC3
in human endometrial stromal (HESC), epithelial (EEC) and
endometriotic (Hs832cT) cell lines, and expression of HDAC3 and
gdf11 in tissues by real-time RT-PCR (qPCR).
2. To determine and correlate protein expression levels of HDAC3 and
GDF11 in endometriotic lesions obtained from patients and
endometrium of controls on a TMA by Immunohistochemistry (IHC).
INTRODUCTION
• HDAC3 was expressed in normal endometrial and endometriotic cell lines as seen
in the pathological pictures.
• The highest expression of HDAC3 was observed in the stromal endometrial cell
line (HESC).
• Ovarian steroid hormones significantly downregulated expression of HDAC3 in
human endometrial stromal cells only.
• In tissues, gene expression of HDAC3 was not significantly higher in
endometriosis vs. eutopic endometrium of women with other gynaecological
conditions (controls).
• Gene expression of gdf11 was confirmed to be significantly downregulated in
endometriotic lesions compared to controls.
• Unexpectedly gene expression levels of HDAC3 and GDF11 were positively
correlated in tissues.
• However, at the level of protein, there was a negative correlation in the expression
levels of HDAC3 and GDF11 in tissues on a TMA. Analysis is currently being
conducted to determine whether this correlation is specific to endometriosis.
• In summary, this study presents evidence for a possible role of HDACs in the
regulation of expression of GDF11 in endometriosis.
SUMMARY
METHODS
1. Gene expression of HDAC3 is upregulated in endometriotic as
compared to normal endometrium both in vivo (in tissues) and in
vitro (in cell lines).
2. Steroid hormones (E2 and P4) differentially regulate HDAC3 gene
expression in endometrial cells.
3. GDF11 is downregulated in endometriosis lesions and its
expression is negatively correlated to that of HDAC3.
AIMS
HYPOTHESES
RESULTS
RNeasy
kit (Qiagen)
HESC, EEC and Hs832cT
E2 +/- P4
24 hrs
TaqMan® Gene Expression
assays (ABI)
RNA
Endometriotic
Cells (Hs578cT)
Endometrial
Epithelial Cells
(EEC)
Endometrial
Stromal Cells (HESC)
Endometriosis Tissue Microarray
Immunohistochemistry
Archived
Human
Tissue
Samples
FFPE
Pathology
Confirma>on
TMA
construc>on
Substrate
Signal
HRP
QuickTime™anda
decompressor
areneededtoseethispicture.
Tissue Type Localization n
Proliferative
Endometrium
from Controls
(PE-Control)
Eutopic 14
Secretory
Endometrium
from Controls
(SE-Control)
Eutopic 38
Proliferative
Endometrium
from
Endometriosis
Patients
(PE-Endo)
Eutopic 22
Endometriosis
Ovary 29
Fallopian Tube 16
Peritoneum 34
Skin 4
Gastrointestinal
(GI)
7
Total 164
HDAC Gene Expression
in Endometriotic vs.
Eutopic Endometrial
Tissues
Basal and Steroid
Regulated expression
of HDAC3 in Human
Endometrial and
Endometriotic Cell
Lines