Physio-pathological properties of colon cells are …...Physio-pathological properties of colon cells are regulated by the nucleocytoplasmic OGT Agata Steenackers CNRS/UMR 8576, Unit

Physio-pathological properties of colon cells are regulated

by the nucleocytoplasmic OGT

Agata Steenackers

CNRS/UMR 8576, Unit of Structural and Functional Glycobiology (UGSF), Lille 1 University ,

Villeneuve d’As q, France

O-GlcNAc modification

O-GlcNAcylation - dynamic and reversible PTM

nucleocytoplasmic and mitochondrial proteins

by addition of O-GlcNAc onto Ser/Thr residues

O-GlcNAcylation - controled by OGT and OGA

O-GlcNAcylation interplays with Ser/Thr

phosphorylation and regulates fundamental

cellular processes eg. cell cycle, cell signaling

and protein trafficking

OGT

OGA

O-GlcNAcylation Phosphorylation

Protein Protein Protein

Ser/Thr

GlcNAc

Ser/Thr Ser/Thr

PPase

Kinase

P

ATP H2O GlcNAc

UDP-GlcNAc UDP

ADP

H2O Pi

Cotranslational O-GlcNAcylation

60S

40S5’

OGT

G

G

G

G

G

G

Full-length protein

Nascent

polypeptide

OGT inhibition

60S

40S

OGT

3’

5’

UbUb

UbUb

Ub

UbUb

UbUb

Ub

Proteasome

Cotranslational O-GlcNAcylation – protection

from proteosomal degradation of nascent

chains proteins

G2 G1 InD 12 TPRs

G2 G1 InD 9 TPRs

G2 G1 InD 2 TPRs

MSS

ncOGT

mOGT

sOGT

TPR Catalytic Domain

OGT (O-GlcNAc transferase), key and unique

regulator of PTM

2-3% cellular glucose enter the

hexosamine biosynthetic pathway

(HBP) to produce UDP-GlcNAc

OGT uses UDP-GlcNAc, from the

nutrient dependent HBP

UDP-GlcNAc: nutritional sensor

Upregulation of HBP by

increased glucose flux directly

elevates UDP-GlcNAc levels as

well as intracellular protein O-

GlcNAcylation to modulate

their localization, stability and

activity (b-catenin & CRC)

Hexosamine biosynthetic pathway and O-GlcNAcylation

GPI

Glucose

Glutamine

Glucose

G6P F6P

Glutamate

GlcNH26P GlcNAc6P

GlcNAc1PGlycogen

synthesis

Pentose

phosphate

pathway

GFAT

Glycolysis

UDP-GlcNAc

HEXOSAMINE BIOSYNTHETIC PATHWAY

OGT

OGA

UDP

H2O

Protein

Ser/Thr

OH

Protein

Ser/Thr

OH

O-GlcNAcylation

ADP

ATP

GlcNAc SALVAGE

PATHWAYUTP

PPiUAP

AGM1

GNPNAT1

Glutamine

HK

NAGK

What is the role of OGT in cancer development ?

Glucose flux, UDP-GlcNAc pool are implicated in global O-GlcNAc levels through OGT

Role the OGT expression in cancers

What is the impact of OGT knock-down on biological properties of

colon cell lines?

shCtrl shOGT

Gu et al., 2010

Prostate cancer :

↗ OGT in cancer tissue/normal epithelium ,

associated with poor prognosis

↗ OGT and O-GlcNAc levels in cancer cell

lines/non-transformed prostate cells

siOGT growth, invasion, angiogenesis and

metastasis of prostate cancer cells (Lynch et

al., 2012)

Breast cancer :

↗O-GlcNAcylation in cancer tissues/adjacent

tissues and in metastatic lymph nodes

/breast cancer tissues.

shOGT cell migration/invasion and number

of metastatic lung nodules (Gu et al., 2010)

→ important roles in cancer initiation and

metastasis.

Colon cancer :

↗ O-GlcNAcylation of colon cancer tissues

/adjacent tissues.

siOGT colony formation of HT29 cells

(Mi et al., 2011)

Lynch et al., 2012

Overexpression of OGT

3D Culture

Olivier Van-Stichelen et al., 2014

OGT expression and O-GlcNAcylation level in colon cell lines

0

1

2

3

4

5

6

7

8

9

OGT/TBP OGA/TBP NAGK/TBP GFAT/TBPOGT OGA NAGK GFAT

0

1

2

3

4

5

6

7

8

9 HCT 116

HT 29

CCD 841CoN

mR

NA

re

lati

ve

ex

pre

ssio

n

1.qPCR analysis to the mRNA levels of O-GlcNAc cycling enzymes 2. Western Blot of O-GlcNAc cycling enzymes

No correlation between

mRNA profile expression

and protein pattern

NAGK GFAT OGT OGA

HCT116 colorectal carcinoma, HT29 colorectal adenocarcinoma, CCD841 fetal

G6P Is

Glutamine Glucosamine

Glucose

G6P F6P

Glutamate

GlcNH26P GlcNAc6P

GlcNAc1P

GFAT

UDP-GlcNAc

HEXOSAMINE BIOSYNTHETIC

PATHWAY

OGT

OGA

UDP

H2O

Protein S/T

ADP

ATP

SALVAGE

PATHWAY

UTP

PPi

UDP-GlcNAc

pyrophosphorylase

GlcNH26P

AcTGK

Protein S/T-GlcNAc

Protein S/T

GlcNAc

NAGK

130-

95-

170-

130-

95-

72-

55-

95-

55-

CC

D8

41

HT

HC

T

WB: α-O-GlcNAc

WB: α-OGA

WB: α-OGT

WB: α-GFAT

WB: α-tubulin

B

GFAT

OGT

OGA

3. O-GlcNAcylation rate depends on glucose

concentration and glutamine

CCD841CoN HT29 HCT116

α-tubulin

α-O-GlcNAc

55-

95-130-170-

72-

55-

OGT silencing decreased cell adhesion

00

10

20

30

40

50

60

15' 30' 1h 2h 4h5’ 3 ’ 1h 2h 4h

0,60

0,50

0,40

0,30

0,20

0,10

0,00

Time

Ad

he

sio

nra

te

(Ab

sorb

an

ce a

t 4

90

nm

) siCrl

siOGT

Ad

he

sio

nra

te

(Ab

sorb

an

ce a

t 4

90

nm

)

00

10

20

30

40

50

600,60

0,50

0,40

0,30

0,20

0,10

0,005’ 3 ’ 1h 2h 4h

Time

0

0,2

0,4

0,6

0,8

1

1,2

15' 30' 1h 2h 4h

0,60

0,40

0,20

0,00 5’ 3 ’ 1h 2h 4h

Ad

he

sio

n r

ate

(Ab

sorb

an

ce a

t 4

90

nm

)

0,80

1,00

1,20

HT29 HCT116

CCD841

siO

GT

siO

GT

si C

trl

siO

GT

70 -

100 -

130 -

55 -

100 -

130 -

40 -

si C

trl

si C

trl

WB: a-O-GlcNAc

WB: a-OGT

HCT 116HT 29CCD841CoN

OGT silencing reduced OGT expression as well O-

GlcNAcylation in the three colon cell lines

n.s. band

OGT silencing decreases cell proliferation and colony formation

siCrl siOGT

Time (hours)

00

25

50

75

00

25

50

75

00

0,00

0,25

0,50

0,75

1,00

1,25

1,50

1,75

2,00

0 24 48 72 96

Ce

llg

row

th(A

49

0 n

m)

***

***

***

***HCT116

00

25

50

75

00

25

50

75

00

0 24 48 72 96

0,00

0,25

0,50

0,75

1,00

1,25

1,50

1,75

2,00

0 24 48 72 96

Time (hours)

Ce

llg

row

th(A

49

0 n

m)

**

**

*

HT29

00

25

50

75

00

0,00

0,25

0,50

0,75

1,00

0 24 48 72 96

Ce

llg

row

th(A

49

0 n

m)

Time (hours)

***

***

**

CCD841

HT 29 HCT 116 CCD 841CoN

siCtrl

siOGT

0

0

0

0

0

0

0

HCT 116 Crl HCT 116 siOGT HT 29 Crl HT 29 siOGTHCT 116

Crl

HCT 116

siOGT

HT 29

siOGT

HT 29

Crl

**600

500

400

300

200

100

0N

um

be

r o

f co

lon

ies

45% decrease

18% decrease

22% decrease

CCD 841CoN

T=0

T=24

siCtrl

T=0

T=24

siOGT

148 µm

315 µm

321µm

HT 29

230 µm

303 µm

60 µm

297 µm

HCT 116

245 µm

315 µm

188 µm

314 µm

OGT silencing expression inhibited cell migration

Wound healing assays

0

50

100

150

200

250

300

HCT 116

Crl

HCT 116

siOGT

HT 29 Crl HT 29

siOGT

CCD 841

Crl

CCD 841

siOGT

*** ***

*

Wid

th (

µm

) a

t 2

4h

Cell migration and cytoskeletal proteins in colon cell lines

siO

GT

si C

trl

WB: β-tubulin

WB: actin

WB: GAPDH

siO

GT

si C

trl

HCT 116 HT 29 CCD841CoN

siO

GT

si C

trl

70 -

55 -

40 -

40 -

35 -

55 -

70 -

55 -

40 -

70 kDa

100 kDa

130 kDa

55 kDa

170 kDa

40 kDa

35 kDa

25 kDa

siO

GT

si C

trl

HCT 116 HT 29 CCD841CoN

siO

GT

si C

trl

siO

GT

si C

trl

OGT silencing reduced migration of CCD841CoN compared

to cancer cell lines using the Transwell system

0

10

20

30

40

50

60

70

80

90

HCT 116 Crl HCT 116 HT 29 Crl HT 29 siOGT CCD 841 Crl CCD 841HCT116

Crl

HCT116

siOGT

HT29

Crl

HT29

siOGT

CCD841

Crl

CCD841

siOGT

Nu

mb

er

of

mig

rate

dce

lls ***

*

Visualizations the proteins by Bleu Coomassie Cytoskeletal proteins profile

Cytoskeleton network in colon cell lines A

ctin

Tu

bu

lin

V

incu

lin

siCrl siOGT siCrl siOGT siCrl siOGT

CCD 841 HT 29 HCT 116

OGT silencing greatly affected the cytoskeletal networks and cell morphology, particularly in CCD841CoN cells. The cell shape appeared stocky

and stunted whereas the microfilament network, responsible for cell migration, was less extended.

Conclusion

OGT silencing decreased O-GlcNAcylation level, proliferation, adhesion

and migration of HT29, HCT116 and CCD841 cell lines

We showed that OGT expression is not only necessary for the biological

properties of cancer cells but also for normal cells

OGT silencing

↘ cell proliferation ↘ cell adhesion

↘ cell migration

Perspectives

In vivo model : fat mice (high carbohydrate diet) and C57Bl6 mice (regular diet) injected with AOM

(azoxymethane).

Glc or GlcNH2

force-feeding

b-catenin ↗

O-GlcNAcylation ↗

AOM

injection

Analysis of OGT andb-catenin

expression, level of O-GlcNAcylation

Correlation between nutrition and

colorectal cancer development

Glucose

Glucose

Proteasomal degradation

UDP-GlcNAc

Cell proliferation

β--catenin

OGT

OGA

G

β--catenin

β--catenin

G

TCF

ARN pol II

CRC development

Target gene transcription

pp

pp

Ub Ub

APC Axin GSK3β

CK1α

(Olivier Van-Stichelen et al., 2012)

WB: 0-GlcNAc

0 5.5 25

Glcucose (mM)

0 5.5 25 Glc (mM)

Glu ose ↑ - increased intracellular

protein O-GlcNAcylation

WB: β-catenin

Previously : O-GlcNAcylation and β-catenin stability

Acknowledgments

Unit of Structural and Functional Glycobiology, CNRS UMR 8576, Villeneuve d’Asc

Prof. Tony Lefebvre, Ikram El Yazidi-Belkoura, Vanessa Dehennaut, Anne-Sophie Vercoutter-Edouart,

Annick Pierce, Sophie Groux-Degroote, Marlene Mortuaire , Steffi Baldini, Maite Leturcq, Jeanne

Vermuse , Moyira Aquino-Gil , Nao Yamakawa

INSERM U908, Lille 1 University, Villeneuve d’Asc , F ance

Xuefen Le Bourhis, Sylvain Julien, Leo Aubert

CCMIC platform (BICeL, Campus Lille 1)

Christian Slomianny and Elodie Richard

Thank you for your attention !

Glucose

Glucose

Proteasomal degradation

UDP-GlcNAc

Cell proliferation

β--catenin

OGT

OGA

G

β--catenin

β--catenin

G

TCF

ARN pol II

CRC development

Target gene transcription

pp

pp

Ub Ub

APC Axin GSK3β

CK1α

Previously :

WB: 0-GlcNAc

0 5.5 25

Glcucose (mM)

0 5.5 25 Glc (mM)

Glu ose ↑ - increased intracellular

protein O-GlcNAcylation

WB: β-catenin

G6P Is

Glucose

Glutamine Glucosamine

Glucose

G6P F6P

Glutamate

GlcNH26P GlcNAc6P

GlcNAc1P Glycogen

synthesis

Pentoses

phosphates

pathway

GFAT

Glycolysis

UDP-GlcNAc

HEXOSAMINE BIOSYNTHETIC

PATHWAY

OGT

OGA

UDP

H2O

Protein

Protein

Ser/Thr

OH

Ser/Thr

OH

O-GlcNAcylation

ADP

ATP

SALVAGE

PATHWAY

UTP

PPi

UDP-GlcNAc

pyrophosphorylase

Phospho-GlcNAc mutase

GlcNH26P

AcT

Glutamine Glucosamine

GK

CCD 841CoN

siCrl siOGT

O-Gl NA ylation and β-catenin stability

Wnt/β-catenin pathway is modified in 90%

of cases of colorectal cancers by genetic

alteration of β-catenin or one member of the

destruction complex: APC, axin, GSK3β or

CK1α

Glucose

Glucose

Proteasomal degradation

UDP-GlcNAc

Cell proliferation

β--catenin

OGT

OGA

G

β--catenin

β--catenin

G

TCF

ARN pol II

CRC development

Target gene transcription

pp

pp

Ub Ub

APC Axin GSK3β

CK1α

Consequence

β-catenin sta ility→ uncontrolled cell proliferation

Previously

Cell culture: high glucose ↗ β-catenin expression

(A) and stimulation of cell proliferation (B)

A B

- 4 O-GlcNAc sites at the N-term of β-catenin:

S23/T40/T41/T112

- 2 of those in the D-box: T40/T41

- T41 is key residue for β-catenin degradation

D-Box C-ter

Wild type (WT)S23T40

T41T112

O-GlcNAc Tetramutant (4M)A23

A40

A41

A112

(Olivier Van-Stichelen et al., 2014) (Olivier Van-Stichelen et al., 2012)

OGT

OGA

O-GlcNAcylation Phosphorylation

Protein Protein Protein

Ser/Thr

GlcNAc

Ser/Thr Ser/Thr

PPase

Kinase

P

AT H2O GlcNAc

UDP-GlcNAc UDP

Pyrophosphate

H2O Pi

OGT

OGA

Protein

Protein

Ser/Thr

OH

Ser/Thr

OH O-GlcNAcylation

3’ 60S

40S 5’

OGT

G

G

G

G

G

G

Full-length protein

Nascent

polypeptide

60S

40S

OGT

3’

5’

Ub Ub

Ub Ub

Ub

Ub Ub

Ub Ub

Ub

Proteasome

G2 G1 InD 12 TPRs

G2 G1 InD 9 TPRs

G2 G1 InD 2 TPRs

MSS

ncOGT

mOGT

sOGT

TPR Catalytic Domain

OGT (O-GlcNAc transferase), key and unique

regulator of PTM