Protein Kinase C M odulates Wnt Signaling In Colon Tumoral ... · PDF file Protein Kinase C M...

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Transcript of Protein Kinase C M odulates Wnt Signaling In Colon Tumoral ... · PDF file Protein Kinase C M...

  • 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.