Tumorigenesis results from genetic alterations that occur in a stepwise manner giving rise to cells with increasingly
cancer-like characteristics. We used in vitro propagated first trimester human extravillous trophoblast (EVT) cells to identify genetic changes responsible for the transition of the EVT from a normal to premalignant stage. The model used consisted of a normal invasive EVT (HTR8) cell line and its premalignant derivative (RSVT2/C) generated by transfection with the SV40 Tag and selected using a forced crisis regimen. RSVT2/C display increased proliferative, migratory and invasive behavior, unresponsiveness to anti-proliferative and anti-invasive signals of
TGFbeta and a deficiency in gap junctional intercellular communication. These cells, however, were unable to form colonies on soft
agar or
tumors in nude mice and are thus defined as premalignant. Differential display revealed 18 gene sequences, 7 with unknown and 11 with known identity, showing altered expression between the normal HTR8 and premalignant RSVT2/C cell lines. The known sequences include the potential
tumor suppressors
insulin-like growth factor binding protein (IGFBP)-5 and
fibronectin (FN) and potential protooncogenes such as
chromokinesin (KIF4), alternative splicing factor (SF2),
dynein,
DNA polymerase epsilon (DNApol epsilon) and
NF-kappaB activating
kinase (NAK). The role of the remaining 4 genes upregulated in the premalignant EVT is presently unknown and these are
FK506 binding protein (
FKBP) 25,
histone protein (HP1Hs)-gamma,
nucleoporin (Nup) 155 and an 82 kDa acidic human
protein. The functional role of
IGFBP-5 was examined in the control of proliferation, migration and invasiveness of RSVT2/C cells measured in vitro.
IGFBP-5 alone had no effect on these properties of RSVT2/C cells. Furthermore, unlike normal EVT cells, RSVT2/C cells exhibited refractoriness to the migration stimulating signals of
IGF-II, which was explained by the loss or downregulation of the
IGF type 2 receptor (IGF-R2). RSVT2/C cells, however, expressed the
IGF type 1 receptor (IGF-R1) and responded to
IGF-I by increased proliferation. This response was blocked with increasing concentrations of
IGFBP-5. These results suggest that the loss of
IGFBP-5 and possibly IGF-R2, both of which can sequester
IGF-I from IGF-R1, permits unhindered proliferation of the premalignant EVT in an
IGF-I rich environment of the fetal-maternal interface. The functions of the other differentially expressed genes, some of which are essential for cell cycle progression or cell survival require further investigation.