Protein Kinase C Modulates Wnt Signaling In Colon Tumoral Cell Lines - - PowerPoint PPT Presentation

Dra Martha Robles Flores Dra Martha Robles Flores Department of Biochemistry Department of Biochemistry Facultad de Medicina Facultad de Medicina Universidad Nacional Autónoma de México Universidad Nacional Autónoma de México

Protein Kinase C Modulates Wnt Signaling In Colon Tumoral Cell Lines

Wnt

Differentiation Cell cycle arrest

TG TGF-β, BM BMP

Stem cell renewal Proliferation

Tissue anatomy of the colonic epithelium

β‐catenin C Proteosomal Degradation OFF LEF/TCF TLE-1 Wnt LRP 5/6 Frizzled N P P P P P P Dvl P Nuclei LEF/TCF ON HDAC Nuclei β‐catenin acummulation Lgs/Bcl-9 Pygo

Canonical Wnt Signaling

P P

LRP5/6

Fz Wnt3a

GSK3-β/ Axin/APC β-catenin TCF/LEF Wnt gene target transcription

Fz Wnt5a

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 ?

CAMK CAMK

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

PKCβI I

Cellular proliferation, migration (HT-29) Protein levels

PKCε

Apoptosis

(CaCo-2)

Protein and mRNA levels

PKCδ

Cellular differentiat ion

(SW-480)

Protein levels

PKCβI

Cellular differentiation

(IEC-18)

Protein levels

PKCα Colon cell lines APC MI N mice model PKC I soform

NON MALIGNANT CELL LINES NON MALIGNANT CELL LINES COLON CARCINOMA CELL COLON CARCINOMA CELL LINES LINES IEC IEC-

• 18

18 (rat) (rat) 112 112-

• CoN

CoN (human) (human) RKO RKO (human) (human) SW SW-

• 480

480 (human) (human) COMPLETE COMPLETE APC APC COMPLETE COMPLETE APC APC COMPLETE COMPLETE APC APC TRUNCATED APC TRUNCATED APC Normal Wnt Normal Wnt signaling signaling Normal Wnt Normal Wnt signaling signaling Normal Wnt Normal Wnt signaling signaling 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

6±0.2 0.2 4±0.1 0.1 1

μ

2± 0. .01 01 4±0. .07 07 1

ζ

1.0 1.0 ±0.02 0.02 4±1

η

5± 0.09 0.09 2 2 ±0.03 0.03 1

ε

4±0.05 0.05 3 3 ±0.07 0.07 1

βII II

0. .1 1±0. .02 02 0. .1 1 ±0. .06 06 1 δ 0.05 0.05 ±0.04 0.04

1

βI 0.2 0.2 ±0.05 0.05 1 α RKO RKO HT HT-

• 29

29 112 CoN 112 CoN PKC PKC

DOWN DOWN-

• REGULATED

REGULATED UP UP-

• REGULATED

REGULATED

Normal Malignant

RKO GSK-3 PKC-ζ Merge IEC-18n β-cat PKC-ζ Merge

IP: GSK-3

PKC-ζ

GSK-3

HT-29 c RKOc I EC-18 n WB IEC-18n RKO

IP: β-catenin PKC ζ

β-cat

HT-29 c RKOc I EC-18 n

APC

IP: APC IP: APC

PKCδ Merge

IEC IEC-

• 18 (N)

18 (N) HT HT-

• 29 ( C )

29 ( C )

250 250 150 150 75 75 50 50

PKCα APC

IP: APC IP: APC WB: WB:

PKCδ APC APC PKCα Merge

250 250 75 75 50 50

IEC IEC-

• 18

18 (N) (N) HT HT-

• 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 SW480

1 2 3 4 5 6

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

TCF-sensitive Trancriptional activity (Fold)

0.2 0.4 0.6 0.8 1 1.2

FOP TOP

Control Wnt5a Wnt3a

RKO

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

Normalized luciferase activity

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:

20 40 60 80 100 120

PKCζ i (20 μM)

+ −

Proliferatiom (% MTT Reduction) 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

0.5 1 1.5 2 2.5 3 3.5 4

Control Vehicle Rottlerin 3uM Normalized Luciferase Activity

FOP TOP

SW SW-

• 480

480

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

0.02 0.04 0.06 0.08 0.1 0.12 Control Wnt3 Rottlerin 3uM+ Wnt3

Normalized Luciferase Activity

FOP TOP

RKO RKO

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

1 2 3 4 5 6 7 8 9 10 Normalized Luciferase Activity

Wnt3a Wnt3a Rottlerin Rottlerin

• + + + +

+ + + +

• 3μM 4

M 4μM 6 M 6μM 10 M 10μM

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.