Open access

Identification of SIP1-modulated genes during the
epithelial-to-mesenchymal transition and interactions
with KLF factors in EMT control
Benjamin Koopmansch1, Anne Brysse2, Christine Gilles2, Jean-Michel Foidart2, Rosita Winkler1
1GIGA-Research, Molecular Oncology Unit, Université de Liège
2GIGA-Research, Laboratory of Tumor & Development Biology Unit, Université de Liège
In A431 cells, recruitment of KLF4 on
the E-cadherin promoter is diminished
more than 3 times following SIP1
forced expression.
Prm1 promoter was used as a negative
control for KLF4 binding.
The E-cadherin promoter contains 2 E-
boxes to which SIP1 is known to bind (Comijn
et al., 2001). A GC-box (putative binding site
for KLF4) is located between these two sites.
To study the interplay between SIP1 and
KLF4, we co-transfected reporter vectors
mutated for theses binding sites and a SIP1-
expression vector.
CONCLUSION
EMT (epithelial-to-mesenchymal transition) is a process characterized by the loss of epithelial properties and the gain of
mesenchymal properties. Among the transcription factors involved in EMT, SIP1 is known as a transcriptional repressor
of epithelial genes, including E-cadherin. It has been shown that SIP1 binds to DNA at 2 E-boxes separated by 40 to 50
bases. A repression model suggested that SIP1, by binding to theses 2 sites, is able to close a region of DNA and to
impair the binding of an activating factor.
KLF4, a Krüppel/Sp1-like family member, is down-regulated in some cancers (colon, oesophagus) but up-regulated in
others (breast and pancreas). To date, KLF4 is described as a positive regulator of E-cadherin and has been shown to
inhibit the EMT in MCF10A cells.
Our project aims at understanding the implication of KLF4 in the EMT in breast cancer
KLF4 is present on the E-cadherin
promoter in MCF7 cells (not
expressing SIP1), but not in SIP1-
expressing cells (MDA-MB-231 and
Hs578T).
Study of the E-cadherin promoter for the role of SIP1 and KLF4 :
RESULTS
INTRODUCTION
A431 and MCF7 cells show epithelial properties (E-cad+, Vim- and
SIP1-)
Hs578T and MDA-MB-231 show mesenchymal properties (Ecad-,
Vim+ and SIP1+).
Although KLF4 up-regulates E-cadherin and is more expressed in
«mesenchymal » cells, these cells do not express E-cadherin.
Mutation of the putative KLF4 binding site
didn’t impair the SIP1 repression.
Luciferase assay showed a potent activator
effect of the GC-box on the promoter, but
this site is not involved in the SIP1 repressive
effect.
Mutation of the E-box located at -75 is
sufficient to impair SIP1 repression of E-
cadherin promoter activity. Experiment
conducted in triplicate in MCF7 cells,
confirmed in other cell lines. This result is
different from other published results.
Although KLF4 is expressed at high levels in « mesenchymal » breast cancer
cell lines, it doesn’t bind to the E-cadherin promoter.
We postulated that SIP1 could be responsible for the absence of KLF4 on
the E-cadherin promoter. Our first results didn’t allow us to confirm this or
find the underlying mechanism.
These results prompted us to test if SIP1 is responsible for the absence of KLF4 on the E-cadherin promoter
Recruitment of KLF4 on the E-cadherin promoter in A431 and breast cancer cells: ChIP analysis