Gestational Trophoblastic Disease
Moderator: Dr. le-Ming Shih
Section 2 -
Cellular and Molecular Mechanisms in Trophoblast Invasion and Tumor Progression*
*Supported by a grant from the Canadian Institutes of Health Research
P. K. Lala
Dept. of Anatomy & Cell Biology
University of Western
London , Ontario , CANADA
The "hemochorial" type of placenta in a number of species including the human is a highly invasive
"pseudo-tumor"-like structure that invades the pregnant uterus and uteroplacental arteries to tap on the
maternal blood supply and nourish the fetus. This phenomenon poses two important biological riddles in
Our laboratory has contributed to the
understanding of both these riddles, and the present talk will focus on the second riddle. I shall
summarize studies from our laboratory to address two important issues:
- What protects the fetally-derived placenta, which can be considered as
an allograft (carrying both paternal and maternal histocompatibility genes) in the mother's uterus, from
maternal immune attack?
- What limits the invasion of the pregnant uterus by the invasive cells of the
placenta, so that the uterus remains unharmed?
- What are
the molecular mechanisms regulating placental invasiveness in order to maintain a healthy utero-placental
- What are the molecular/genetic changes associated with a loss of control of placental
invasiveness during trophoblastic tumor progression?
Cell responsible for placental invasiveness: the extravillous
trophoblast. Dr. Shih has already presented an overview of the various trophoblastic
subpopulations belonging to the normal placenta. I shall focus on the population termed as the
"intermediate trophoblast" (IT) by the pathologists, also named as the "extravillous trophoblast" (EVT)
by the placentologists. This population arises by proliferation and differentiation of cytotrophoblast
"stem cells" in certain chorionic villi (anchoring villi), wherefrom they migrate as cell columns to
invade the decidua and the uteroplacental arteries. At the base of the columns near the villous tips
wherefrom they emerge, EVT cells are highly proliferative; however, their proliferative activity is lost
progressively as they approach the decidua with a concomitant increase in cellular ploidy, and a complete
loss of proliferation within the decidua . Within the implantation site decidua they present as
(a) interstitial trophoblast dispersed within the
decidua; (b) endovascular trophoblast which invade spiral arteries in the
endometrium and myometrium, modifying them into noncontractile tubes allowing a steady flow of maternal
blood into the sinusoids. These cells acquire an endothelial phenotype (expressing VCAM-1 and ephrin B
and replace the endothelium of these arteries ;
(c) placental bed
giant cells which are non-invasive, arise by EVT cell fusion and make hPL; (d) a continuous layer
of cytotrophoblastic shell separating the decidua from the placental
Molecular mechanisms responsible for EVT invasiveness. We have succeeded
in propagating normal first trimester invasive human EVT cells from chorionic villus explant cultures
to show that they have all the phenotypic properties of the EVT cells in
situ , expressing cytokeratin-7, EGF-R, placental type alkaline phosphatase, receptor for
urokinase type plasminogen activator (uPA), HLA framework antigen, and integrin (receptors for
extracellular matrix, ECM) subunits a1, a3, a5,
av, β1 and vitronectin receptor, but not a 6 or
β4 subunits expressed by the villous trophoblast. They
express HLA-G when grown on laminin or matrigel (reconstituted ECM)
. Using in
vitro invasion assays with natural or reconstituted basement membrane substrates
found that their invasiveness depends on numerous steps: (a) binding to ECM
components laminin, collagen type IV and fibronectin, (b) degradation of the
ECM by production of matrix metalloproteases (MMP-2, MMP-9 and MT-1 MMP) and uPA (which activates MMPs),
and (c) migration through the degraded ECM. Their migration requires the
expression of Asn-linked complex type oligosaccharides  as well as binding to fibronectin via
a5β1 integrin on the EVT cell surface . The balance between the
production of MMPs and their inhibitors (tissue inhibitors of metalloproteases, TIMPs; particularly
TIMP-1), as well as uPA and its inhibitor (PAI-I),
dictates their matrix-degrading ability
While these mechanisms are identical to those of invasive and metastatic tumor cells
invasive EVT propagated in culture are neither tumorigenic nor metastatic in nude mice, and they have a
limited life span (5-15 passages)
indicating that invasiveness is an essential but not sufficient
pre-requisite for tumorigenicity or metastasis, and that EVT invasion in
situ must be controlled locally.
Molecular mechanisms regulating trophoblast growth, migration and invasion in situ. We discovered that the regulation is provided by the interaction
of the EVT with certain locally derived growth factors, growth factor binding proteins, proteoglycans and
ECM components for which EVT cells have receptors. Factors blocking EVT cell
proliferation, migration and invasiveness: Two decidua-derived factors, transforming growth factor
(TGF)-β and a TGFβ-binding proteoglycan decorin that localizes TGFβ in the decidual ECM,
inhibit EVT cell growth, migration and invasiveness independently of each other
We suggest that
decorin serves as a storage device for inactive TGFβ which is released from the TGFβ-decorin
complex and activated by EVT cell derived proteases, triggering negative control against trophoblast
over-invasion of the decidua . TGFβ negatively regulates EVT proliferation as in other
epithelial cells 
and blocks EVT invasion by (a) upregulating TIMP-1 mRNA
and protein which neutralizes MMPs ,
down-regulating uPA , (c)
upregulating PAI-I  and
(d) reducing their
migratory ability, apparently by upregulating integrins , making the cells more adhesive to the ECM.
The mechanisms for negative regulatory functions of decorin on EVT cells remain to be fully identified.
Interestingly, choriocarcinoma cells are refractory to both TGFβ and decorin-mediated negative
regulation. Factors stimulating EVT cell growth: All EGF receptor ligands
(EGF, TGF-a, amphiregulin), CSF-1, VEGF and PlGF produced locally by the trophoblast and/or the
decidua stimulate EVT cell proliferation, having little effect on invasion
Migration/Invasion promoting factors: These factors are numerous. IGF-II (produced
by the EVT) stimulates their migration 
and invasiveness without influencing proliferation  by
signaling through the type II IGF receptor (IGF-RII), inhibition of adenylyl cyclase and stimulation of
MAP kinase (MAPK)
. An IGF binding protein, IGFBP-1, released by decidual cells also promotes
due to stimulation of EVT migration  because of binding of its RGD
(arginine-glycine-aspartic acid) sequence to the a5β1 integrin on the EVT
cell surface, followed by activation of focal adhesion Kinase (FAK)
and MAPK . EVT cell-derived
urokinase type plaminogen activator (uPA) exerts dual functions: promotion of invasiveness by its
catalytic domain, and promotion of migration by binding to uPA receptors on the EVT cell surface .
Indeed, derangements in the production or function of some of these migration/invasion-promoting factors
e.g. uPA/uPAR system or IGFBP-1 protein may be instrumental in the development of preeclampsia, a
trophoblastic hypoinvasive disorder . For example, reduced IGFBP-1 level in maternal serum
(apparently resulting from a low production by the decidua) during early pregnancy (15-16 weeks
gestation) may be a predictive marker for the development of this disease before the appearance of
clinical signs .
Other molecules promoting EVT cell migration are endothelin (ET)-1 made by many
cells in the placenta or the decidua 
and prostaglandin E2 (PGE2) made by the decidua. In the latter
case, PGE2 receptor class EP1 appears to play a major role .
Proliferative and invasive functions of EVT cells are not mutually
exclusive. Invasive function of EVT cells does not necessarily indicate a terminal differentiation
into a non-proliferative state. By using primary cultures of EVT cells in invasion chambers containing
matrigel and collecting those that have invaded matrigel, we discovered that even after the act of
invasion, EVT cells retained significant, but limited proliferative ability . This finding suggests
that progressive loss of proliferative ability of EVT cells in situ as they migrate to the decidua result
from a dual mechanism: endoreduplication without mitosis , and proliferation inhibition by
decidua-derived anti-proliferative factors, e.g. TGFβ and decorin .
Molecular/Genetic Mechanisms in Trophoblastic Tumor Progression
To identify molecular and genetic mechanisms leading to specific stages in trophoblastic tumor
progression, we utilized normal, premalignant and malignant human EVT (choriocarcinoma) cells as a model
system in vitro. That choriocarcinomas are malignant derivatives of EVT
cells is suggested by the fact that the EVT cell specific marker HLA-G was originally cloned from JEG-3
choriocarcinoma cells . Since premalignant EVT cell lines have not been produced from in situ
lesions, we derived them in our laboratory by genetic manipulation.
Derivation of premalignant EVT cells. We stably transfected two
different SV40 Tag-containing plasmids into a normal short-lived human first trimester EVT cell line,
HTR-8. Transfection with pSV3neoSV40 Tag having a selectable marker for neomycin resistance resulted in
the HTR-8/SVneo cell line which has identical phenotype as HTR-8 cells except for immortality (living
beyond 100 passages).
It is responsive to antiproliferative ,
antiinvasive and antimigratory  as
well as fibronectin-upregulatory signals of TGFβ, lacks in agar colony forming ability or
tumorigenicity in nude mice. Thus it is neither premalignant nor malignant, indicating that SV40 Tag
transfection per se did not induce premalignancy or malignancy. The second line RSVT2/C  was
produced by transfection with the non-selectable pRSV-Tag and selected on the basis of prolonged
longevity and derivation of an immortalized clone after a forced crisis by serum starvation in culture.
RSVT 2/C cells show higher proliferative and invasive abilities, and resistance to anti-proliferative,
anti-invasive and fibronectin-up regulatory actions of TGFβ. However, unlike choriocarcinoma
cells, they are unable to form agar colonies or tumors in nude mice. A dramatic decline in TIMP-1,
TIMP-2 and PAI-I mRNA in RSVT 2/C cells may explain their increased invasiveness . Finally,
gap-junctional intercellular communication (GJIC) as well as the expression of connexin-43 mRNA and
protein noted in parental HTR-8 cells were undetectable in RSVT 2/C cells . This
is a hallmark of carcinogenesis . These characteristics, taken together, reveal that RSVT 2/C
cells have attained a premalignant phenotype.
Using differential display (DD) of reverse-transcribed mRNA, we identified a total of 18 different
gene sequences with differential expression between the normal HTR-8 and the premalignant RSVT2/C cells
Of these sequences, 7 (up or down-regulated) genes are of unknown
identity, while the remainder include potential tumor suppressors such as insulin-like growth factor
binding protein (IGFBP)-5 and fibronectin (hFN) as well as genes with oncogenic potential such as
Chromokinesin (KIF4), alternative splicing factor (SF) 2, dynein, human DNA polymerase ε subunit p12
and NF-κB-activating kinase (NAK). The function of the remaining 4 genes FK506 binding protein
(FKBP5), histone protein (HP1Hs)-γ, nucleoporin (Nup) 155 and an 82kDa acidic human protein, which
are upregulated in the premalignant EVT, remains unknown. We found that loss of
IGFBP-5 in conjunction with the loss of type 2 IGF receptor (1GF-IIR) in the premalignant RSVT2/C cells has emancipated them from the negative
regulatory control of IGFBP-5 on IGF-1 dependent proliferation of these cells.
Loss of IGF-IIR, a recognized tumor-suppressor gene was unique to the
premalignant EVT, unrelated to Tag transfection alone, since this was not seen in HTR-8/SVneo cells .
TGFβ is the key factor controlling EVT cell growth, migration and invasiveness in situ ,
whereas premalignant EVT 
as well as malignant EVT i.e. choriocarcinoma cells  are resistant to
negative regulation by TGFβ. As a first step in identifying TGFβ signaling defect(s) responsible
for TGFβ resistance in premalignant and malignant EVT, we compared the expression of TGFβ signaling
molecules in the normal (HTR-8), premalignant (RSVT2/C) and malignant (JAR and JEG-3 choriocarcinoma) EVT
cells . RT-PCR analysis revealed that all these cell lines expressed the mRNA for TGFβ1, β2 and β3,
TGFβ receptors I, II and III and post-receptor signaling genes Smad 2, 3, 4, 6 and 7 with the
exception that TGFβ 2 and Smad3 were undetectable in choriocarcinomas. Smad 3 loss in the latter was
verified at the protein level in immunoblots. TGFβ -induced Smad3 phosphorylation and translocation to
the nucleus was demonstrable in the normal and premalignant, but not in malignant EVT cells . Thus,
Smad 3 disruption, which has not been reported in any other cancer,
constitutes at least one of the TGFβ signaling defect(s) in choriocarcinomas that may account for
TGFβ resistance. Indeed, we found that introduction of dominant negative Smad3 into normal EVT
(HTR8/SVneo) cells made them partially resistant to growth inhibitory effects of "nodal", another member
of the TGFβ superfamily .
To test whether TGFβ response can be restored by restitution of Smad3 in JAR cells we made stable
Smad3-transfected JAR cell clones . Since anti-invasive effects of TGFβ in the normal EVT are
combined results of upregulation of PAI-1 ,
a down regulation of uPA
and a down regulation
of TIMP-1 , we tested the above TGFβ responses in one of the Smad 3 restituted clones JAR Smad 3c
This clone exhibited further upregulation of Smad 3 in the presence of TGFβ 1. The basal
levels of PAI-1 mRNA and protein as well as uPA proteins were found to be very low in JAR and JAR-Smad3c
cells, as compared to normal EVT cells. However, TGFβ1 significantly upregulated PAI-1 and
downregulated uPA in JAR-Smad3/c but not in wild type JAR cells . Similarly, the expression of
TIMP-1 mRNA was found to be low in JAR as well as JAR-Smad3c cells as compared to normal EVT cells .
Exposure to TGFβ 1 upregulated TIMP-1 mRNA and secreted protein in Smad3 restituted JAR as well as in
normal EVT cells but not in wild type JAR cells. However, in vitro functional assays revealed that in
spite of significant restoration of uPA/PAI-1 as well as TIMP-1 responses to TGFβ , Smad3 restitution was
insufficient in full restoration of antiinvasive function of TGFβ . Thus additional factors
(possibly low Smad4 expression and/or other unidentified defects downstream of Smad or in
Smad-independent signaling) may also contribute to TGFβ unresponsiveness in JAR cells. These
possibilities are currently being explored.
Extravillous trophoblast cells responsible for the invasive function of the placenta utilize similar
molecular mechanisms for their invasiveness as cancer cells, however, unlike cancer cells, their
proliferative, migratory and invasive functions are stringently controlled in situ by certain
decidua-derived factors, in particular, TGFβ. Progression of normal EVT cells to a premalignant
state is associated with changes in the expression of many genes some of which can explain their
premalignant behavior. Loss of anti-invasive effects of TGFβ on the premalignant EVT is explained
by down-regulation of TIMP-1, TIMP-2, and PAI-1 genes. Loss of TGFβ sensitivity in the malignant
EVT (choriocarcinoma) is partially explained by the loss of expression of a transcription factor Smad3.
Whether genetic changes noted in our premalignant EVT cell lines occur in situ remain to be investigated
. Availability of cell lines derived from in situ lesions representing various steps in
trophoblastic tumorigenesis  should help future studies in this field.
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