Dra Martha Robles FloresDra Martha Robles Flores Department of BiochemistryDepartment of Biochemistry Facultad de MedicinaFacultad de Medicina Universidad Nacional Autónoma de MéxicoUniversidad Nacional Autónoma de México

Protein Kinase C Modulates Wnt Signaling In Colon Tumoral Cell Lines

Wnt

Differentiation Cell cycle arrest

TGF-β, BMP

Stem cell renewal Proliferation

Tissue anatomy of the colonic epithelium

β‐catenin C

Proteosomal Degradation

OFFLEF/TCF

TLE-1

Wnt

LRP 5/6 Frizzled

N

PP P

P P P

Dvl

PPP

Nuclei

LEF/TCF

ON

HDAC

Nuclei

β‐catenin acummulation

Lgs/Bcl-9 Pygo

Canonical Wnt Signaling

P P

LRP5/6 FzWnt3a

GSK3-β / Axin/APC

β-catenin

TCF/LEF

Wnt gene target transcription

FzWnt5a

Dvl

G Protein

Ca++ mobilization

PKC?

Canonical Wnt Signaling (Wnt/ β -catenin)

Non-canonical Wnt Signaling (Wnt/Ca++)

Planar cell polarity Negative modulation of TCF actions

Dvl PLC

IP3 DAG

?

CAMKCAMK

RoR

Heider et al, 2004;

Perletti, 1998

Yu, 2003, Murray, et

al, 1999

Black, 2001, Cerda et

al, 2006.

Goldstein, 1995, Assert, 1999.

Frey 1997, Hizli, 2006,

Black 2000.

References

Cancer cell lines proliferation and

migration

Protein and mRNA levels

PKCβ II

Cellular proliferation,

migration (HT-29) Protein levelsPKCε

Apoptosis (CaCo-2)

Protein and mRNA levels

PKCδ

Cellular differentiation

(SW-480) Protein levelsPKCβ I

Cellular differentiation

(IEC-18) Protein levelsPKCα

Colon cell linesAPC MIN mice modelPKC Isoform

NON MALIGNANT CELL LINESNON MALIGNANT CELL LINES COLON CARCINOMA CELL COLON CARCINOMA CELL LINESLINES

IECIEC--1818 (rat)(rat)

112112--CoNCoN (human)(human)

RKORKO (human)(human)

SWSW--480480 (human)(human)

COMPLETE COMPLETE APCAPC

COMPLETE COMPLETE APCAPC

COMPLETE COMPLETE APCAPC

TRUNCATED APCTRUNCATED APC

Normal Wnt Normal Wnt signalingsignaling

Normal Wnt Normal Wnt signalingsignaling

Normal Wnt Normal Wnt signalingsignaling

Altered Wnt Altered Wnt signaling signaling (constitutively (constitutively active)active)

APC

PKCδ

Truncated APC

Comparative expression profile of PKC isoforms in colon cultured cell linesde colon

66±±0.20.244±±0.10.111μμ

22±± 00..010144±±00..070711ζζ

1.0 1.0 ±±0.020.0244±±1100ηη

55±± 0.090.092 2 ±±0.030.0311εε

44±±0.050.053 3 ±±0.070.0711ββIIII

00..11±±00..020200..1 1 ±±00..060611δδ

000.05 0.05 ±±0.040.0411ββ II

000.2 0.2 ±±0.050.0511αα

RKORKOHTHT--2929112 CoN112 CoNPKCPKC

DOWNDOWN--REGULATEDREGULATED UPUP--REGULATEDREGULATED

Normal Malignant

RKO

GSK-3 PKC-ζ Merge

IEC-18n

β-cat PKC-ζ Merge

IP: GSK-3

PKC-ζ

GSK-3

HT-29c RKOc IEC-18n

WB

IEC-18n

RKO

IP: β-catenin

PKC ζ

β-cat

HT-29c RKOc IEC-18n

APC

IP: APCIP: APC

PKCδ Merge

IECIEC--18 (N) 18 (N)

HTHT--29 ( C ) 29 ( C )

250250

150150

7575

5050

PKCα

APC

IP: APCIP: APC WB:WB:

PKCδ

APC

APC

PKCα Merge

250250

7575

5050

IECIEC--18 18 (N) (N)

HTHT--29 ( C ) 29 ( C )

GOALS

¿What is the biological meaning of PKCα/δ interaction with APC?

¿What is the biological meaning of β-catenin interaction with PKCζ?

¿Can PKC modulate canonical Wnt signaling?

IEC-18 (Normal)

RKO SW4800

1

2

3

4

5

6 TOPFlash (functional TCF sites) FOPFlash (mutated TCF sites)

TC F-

se ns

iti ve

T ra

nc ri

pt io

na l

ac tiv

ity (F

ol d)

0

0.2

0.4

0.6

0.8

1

1.2

FOP TOP

Control Wnt5a Wnt3a

RKO

0

5

10

15

20

25

30

35

FOPFLASH TOPFLASH

Wnt 3a

PKCζ i (20μM)

-

-

-

+

- + +

- - +

SW480 RKO

PKCζ selective inhibitor blocks β-catenin-mediated transcriptional activity in both RKO and SW480 colon carcinoma cell lines

N or

m al

iz ed

lu ci

fe ra

se a

ct iv

ity

Psuper PKC RNAi (μg) 0 1 2

1

0.8

0.6

0.4

0.2

PKCζ

0 1 2

Actin

pSuper.PKCz.RNAi (μg)

PKCζ knockdown blocks in a dose-dependent manner the β-catenin-mediated transcriptional activity

WB:

0

20

40

60

80

100

120

PKCζ i (20 μM) +−

Pr ol

ife ra

tio m

(%

M TT

R ed

uc tio

n)

SW480 cells WB:

c-myc

c-myc

GAPDH

PKCζi (20 μM)

+−

PCR

Effect of PKCζ knockdown on cell proliferation and Wnt target gene expression (c-myc)

actin

0

0.5

1

1.5

2

2.5

3

3.5

4

Control Vehicle Rottlerin 3uM

N or

m al

iz ed

L uc

ife ra

se A

ct iv

ity

FOP

TOP

SWSW--480480

Effect of PKCδ inhibition on β-catenin-mediated transcriptional activity

0

0.02

0.04

0.06

0.08

0.1

0.12

Control Wnt3 Rottlerin 3uM+ Wnt3

N or

m al

iz ed

L uc

ife ra

se A

ct iv

ity

FOP TOP

RKORKO

Effect of  PKCδ inhibition on β‐catenin‐mediated  transcriptional activity

0 1 2 3 4 5 6 7 8 9

10

N or

m al

iz ed

L uc

ife ra

se A

ct iv

ity

Wnt3aWnt3a RottlerinRottlerin

-- + + + ++ + + +

-- 33μμM 4M 4μμM 6M 6μμM 10M 10μμMM

SUMMARY

• PKC isoforms associate in vivo with key Wnt canonical proteins: PKCζ, upregulated in malignant cells, interacts with GSK3β and with β-catenin mainly in cancerous cells. PKCδ, downregulated in malignant cells, interacts with APC in both normal and malignant cells.

• Pharmacological inhibition of PKCζ, or its decreased expression blocked in a dose-dependent manner canonical Wnt activation in cancer cell lines, suggesting that PKCζ modulates in a positive way canonical Wnt activation.

• Pharmacological inhibition of PKCδ or its decreased expression, improved in a dose-dependent way canonical Wnt activation in RKO cancer cells, suggesting that PKCδ modulates in a negative way canonical Wnt activation.

Altogether, our results indicate that PKC isoforms play an essential role in the regulation of canonical

Wnt pathway.