Sustained expression of LIP, a short repressive isoformof C/EBPβ, leads to epithelial-mesenchymal transition(EMT) in the mammary epithelial cells

研究例 ４細胞の癌化・転移機構の解析

11○Yuka Miura , Yohei Hirai
1Dept. of Bioscience., Grad. Sch. Sci. Technol., Kwansei Gakuin Univ.

1. Background
Upon oncogenic transformation, epithelial cells temporally display a mesenchymal phenotype (EMT), in
which a transcription factor Snail1 plays a causal role for various epithelia. Recently, another transcription
factor CCAAT/enhancer binding protein C/EBPβ has been implicated to elicit the similar response in
mammary epithelial cells. C/EBPβ mRNA has two translation initiation sites in its open reading flame,
thereby is translated into two isoforms, LAP (liver-enriched activating protein) that is composed of thetranscription and DNA binding domains, and LIP (liver-enriched inhibitory protein) that has solely DNAbinding domain. LIP is known as a competitor of LAP, and it cannot activate transcription of LAP targets.
However, no difference between the amount of LAP protein and LIP protein was observed in epithelial
and mecenchymal cells. Therefore, we focused on the temporal off-balance of expression level of LAP / LIP.
LAP

LAP

ATG

ATG

Transcriptional
activation domain

C/EBPβLIP

ATG

LIP

DNA-binding
domaincompetiton

E-cadherin↑(epithelial markers)

LAP

7. Effects of exogenous LIP on P2 cells on matrigel
LIP (off)

LIP (on)
LIP (on)DH5 + 2% matrigel
alveolimatrigel

LIP

LIP (on)

LIP (off)

W.B.

DNA-binding
domain(expression) off on long-on

E-cadherin×(epithelial markers)

LAP
LIP

Overexpression of LIP perturbed
alveolar-like morphogenesis inP2 cells cultured on Matrigel.

In contrast to on plastic, P2 cells
cultured in matrigel maintain LIPoverexpression.

LIPβ-actin

2a. Aim2b. Hypothesis

LAP

Elucidation of the possible involvement ofLAP / LIP in epithelial-mecenchymaltransition (EMT) and mecenchymalepithelial transition (MET).

integrin α1 / ZO-1 / DAPI

LIP

8. Effects of exogenous LIP on P2 cells in vivo
LIP (off)

EMTLAP

MET

epithelial phenotype

・Forced expression of LIP in epithelial cells
leads to EMT?
・Forced expression of LAP in mecenchymal
cells leads to MET?

injection P2 cells into nude mice

LIPLAP

3. Model cells and markers for EMT / METCOMMA1-D (mammary epithelial cells)
・P2 : pure epithelial population
・G6 : transformed population (fibroblastic)

METcell

CID9G6epithelial

epithelial /mecenchymal

mecenchymal○○○××◎◎◎
×keratin

P2CID9E-cadherin
vimentinLIPβ-actin

Exogenous LIP was exclusively expressed in cell nuclei.

RT-PCR(expression)

W.B.

LIP(expression) off

immunostaining

vimentin / keratin / DAPI keratin↓
LIP (long-on)

β-actinon

L-on+ - + -arginine tag

ESGGGGSPGRRRRRRRRRRRPG(day)
E-cadherin
vimentinnumber of cells

offononon

palpable cancers××××◎◎◎

weight (g)19.5 20.7 21.0 19.0 23.6 24.9 24.0

tumors (g)

000

0

0.8

0.5

2.0

spleen (g)

0.1

0.1

0.1

0.1

0.4

0.3

0.7(expression) off on on L-on
(MG132) - - + +LAP

Proteasome inhibitor(MG132) was added tothe medium.
Incubate 24h at 37℃.

In vivo, P2 cells with overexpression of LIP formed
metastatic tumors. The expression of LIP wassustained for a long period, which was accompaniedwith a dramatic alternation in the expression ofEMT-markers in the tumors.

LIP
E-cadherin
β-actinMatrigel was added to themedium (final1%) after
cells attached to dish.

LIP (long-on)(gel) - +- +E-cadherin
P-cadherin

LAP
LIP
MG132 (+)
⇒ LIP ↑, E-cad ↓ β-actin⇒ EMT

Recombinant TGFβ (2ng/mL)
was added to the medium.
Incubate 24h at 37℃.

LIPE-cadherin
P-cadherin
β-actinrTGFβ (+)⇒ EMT⇒ LAP ↓, LIP ↓LAP

1% matrigel (+)
Long-term expression of LIP⇒ LIP ↑, E-cad ↓, P-cad ↑led to clearer EMT in P2 cells.
⇒ EMT(expression)off(RTAce) + -LAP construct
ATG
ATG+ -+-gapdh

LIP(on) ⇒ no changes in mRNAlevel of TGFβC/EBPβ is a downstream ofTGFβ signaling pathway.

epithelial-like morphology
W.B.

(expression) off
vimentinβ-actinmecenchymal phenotype

keratin↑

ChIP assay

WST-1LAP (on)vimentin / keratin / DAPI

MMP3vimentin↓

cell growth↓

RT-PCRvimentin↓(expression) off
onoff+ (RTAce) + - + vimentin
gapdh

L-on+ -

0.7
0.6

LAP (off)

0.5
0.4
0.3
0.2

LAP (on)0.1

LAP binds toE-cadherin promoter*5

0.8

0on L-on

4.5: P < 0.01
*
n=6

4
3.5
3
2.5
2
1.5
1
0.5
01

2

3day

45

LAP (off)

LAP (on)Primer : E-cadherin promoter

Overexpression of LAP resulted in the cell shape change and growtharrest in G6 cells. LAP also led to the down-regulation of a mecenchymalmarker and the up-regulation of an epithelial marker. LAP elicited
cellular response opposite to LIP.⇒ LAP might induce MET.

12. Conclusion
・LIP induced EMT-like changes and abrogated morphogenic potential in mammary
epithelial cells.
LIP overexpression in the epithelial cells resulted in the formation of metastatic tumors.
・LAP induced MET-like changes in mecenchymal cells.

cell growth↑

1.8

E-cadherin↓
Arg-GFP Arg-LIP G6
1 2 3 1 2 3

L-on

LAP (long-on)

MMP2
LIP (on)primer : E-cadherin promoter

ontgfβLIP(expression) off on L-on

WST-1

macropinosomeoff

P < 0.01

zymography

1.6

Absorbance(450nm)nuclearproteoglycanoff

LAP (off)

gapdh

1.5
1.4
1.3
1.2
1.1
1
0.9
0.8
0.7
0.6
0.5

LIP protein witharginine tagcell

off

0.9

Recombinant LIP / GFP protein added tomedium was successfully delivered to nuclei. Again, LIP proteintransported to cell
Arg-LIPP2(day) 12
3
3
nuclei elicited EMTlike changes in P2
LIPcells.
β-actin
W.B.

LIP expression

immunostaining

mmp2

6. Direct introduction of LIP protein into P2 cells
LIPB3epithelial phenotype?

・Forced expression of LIP protein temporally induced EMT-like cell behaviors.
・The expression profile of the EMT-markers was consistent with that of exogenous LIP protein.

add to medium

B2MET???

snail1ChIP assay

LIP (off)

Arg-LIP

B1

vimentinmecenchymal phenotype?

LIP (on)

LIP (off)

P-cadherin

Signal intensity (relative)epithelial phenotype

off(RTAce) + P-cadherin(expression) off on L-onon

vimentinβ-actin

EMT???

A411. Effects of exogenous LAP on G6 cells

5. Effects of exogenous LIP on P2 cells
LAPA3(rTGFβ) + -Exogenous expression ofthe transgene product(LAP or LIP) became
suppressed in a week
both in P2 and G6 cells.

HA-LIPLIP

C/EBPβ / HA / DAPI

A2LAP

LAP

β-actinA1・expression (on) : 3days
・expression (L-on) : 7days(cell) P2tetLIP G6tetLAP(expression) off on L-on off on L-on

HA-LIP (on)LIP

10. Relationship between C/EBPβ and TGFβ (popular EMT inducer)

4. Generation of inducible cells (tet-off system)
HA-LIP (off)P-cad
E-cadvim
β-actin

9. Proteasome-dependent elimination of excess amount of LIP

・P2 cells express epithelial markers, keratin andE-cadherin.
・G6 cells express a mecenchymal marker, vimentin.
・CID9 cells express both epithelial andmecenchymal markers.
・EMT-transitioning cells are resemble to CID9 cells.
・P-cadherin, MMP2, MMP3 are other EMT markers.

vimentin / keratin / DAPI

LIP

LIP (on)Absorbance (450nm)

G6P2

groupA : feed tetracyclin (+) water= expression (off)groupB : feed normal water= expression (on)Signal intensity (relative)

EMT(expression) off onLAP

P2 tumor

mecenchymal phenotype

bred 2months

P2W.B.

1.4

ArgLIP

1.2
1ArgGFP

0.8
0.6
0.4
0.2
0

day1

day2

day3

・The expression profile of LIP protein was affected by cellular microenvironment.
⇒Excess amount of LIP protein was actively eliminated.
⇒Under the influence of basement membrane, the LIP-elimination was suppressed.
・Possibility : LIP also acts as a transcriptional activator in mammary.
・C/EBPβ is a downstream of TGFβ signaling pathway.