GSK-3 -New Therapeutic Target in Renal Cell Carcinoma

8/13/2019 GSK-3 -New Therapeutic Target in Renal Cell Carcinoma

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http://kidneycancersurvivalrateudif.wordpress.com

http://www.unckidneycenter.org/kidneyhealthlibrary/glomerulardisease.html


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ObjectivesAim:Show that genetic depletion/ pharmacological

inhibtion of GSK-3 results in decreased renal cancercell proliferation and survivalShow aberrant GSK-3 b nuclear over expression inRCC cell lines and most human renal carcinomasShow a synergistic anti-cancer effect of GSK-3inhibitor and Docetaxel in RCC.


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IntroductionRenal cell carcinoma (RCC) is highly resistant tochemotherapy and progressive

High apoptotic thresholdActivation of Nuclear Factor- kb (NF- kb )Increased expression of Bcl-2 and XIAP (anti-apoptoticmolecules)


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IntroductionGlycogen synthase kinase-3

Pluripotent serine/threonine kinase

Important for epithelial cell homeostasis oncogeneactivation increase proliferation2 types: a and bInhibition of GSK-3 apoptosis due to decreased

expression of NF- kb target genes Bcl-2, XIAP (chroniclymphocytic leukaemia and pancreatic cancer cells)


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Method

Immuno-histochemistry

Nuclear-cytosolicfractionation

Expressionpattern ofGSK-3 b

Smallmoleculeinhibitor

RNAinterference

Westernblotting Q- RT-

PCR

MTS BrDUassay

Effect of GSK-3 inactivation


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GSK-3B is expressed and active in human renal cancer cells

Western blot:Higher levels of GSK-3 b expression in RCC cell

lines compared with normal renal cellIncreased phosphorylation of GS moreinactive GS

Higher levels of phosphorylation of glycogensynthase( GS)

Lesser conversion of glucose to glycogen


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Figure 1A

Protein lysates from the indicated RCC cell lines were used andnormal kidney is a control

separated by SDS-PAGE,transferred to PVDF membrane

probed with antibodies against GSK-3 b, phospho-glycogensynthase(pGS) and total glycogen synthase (GS)

pGS( inactive GS,phosphorylated by GSK-3) was found tohave higher intensity bands in RCC cell lines compared to

normal kidneyA ratio of pGS to total GS would show how much protein areactive/inactive


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Figure 1AKidney ACHN KRC/Y Caki1 Caki2 A704 A498

GSK-3 b

pGS

Total GS

b-Actin

Loading control

RCC celllines


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Figure 1B

Kidney ACHN Caki1 Caki2 KRC/Y A498 KH39 KU19-20

C Nu C Nu C Nu C Nu C Nu C Nu C Nu C Nu

GSK-3

NF kb p65

H3

SOD

Using western blot withnuclear/cytosolic fractionation


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Figure 1C:Expression of GSK-3

Patient 1 Patient 2Patient 3 Patient 4

N T N T

N T N T

C Nu C Nu C Nu C Nu

GSK-3

pGS

SOD

H3

b -Actin

GSK-3

tumornormal

Figure 1D:Nuclear/cytosolic fractionation was used

nucleus

cytoplasm

If GSK-3 b activated enters nucleusIf GSK-3 b inactivated found in cytoplasm


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Immunohisotchemical analysis

GSK-3 b isaccumulated in thenucleus of renal cancercells

pGS expression isseen

Weak cytoplasmicexpression of gsk-3bin a fraction ofglomerular andtubular epithelialcells in normalkidney


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Total GSK-3 nuclear pGS positive

Histological type

Oncocytoma 2 0 0 Clear cell 64 60 a 62 b

Other 10 8 7

pT stage (malignant tumours only)

1 53 50 52

2 7 6 7

3 14 12 10

Grade (malignant tumours only)

1 27 26 27

2 43 39 39

3 4 3 3

Total 74 RCCs and 2oncocytomas

68 69

Table 2. Results of immunohistochemical study for GSK-3 and pGS

Abbreviations: pGS=phospho-glycogen synthase; RCC=renal cellcarcinoma.
http://www.nature.com/bjc/journal/v101/n12/fig_tab/6605437t2.htmlhttp://www.nature.com/bjc/journal/v101/n12/fig_tab/6605437t2.htmlhttp://www.nature.com/bjc/journal/v101/n12/fig_tab/6605437t2.htmlhttp://www.nature.com/bjc/journal/v101/n12/fig_tab/6605437t2.html


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Pharmacological inhibition and genetic depletion of GSK-3decrease proliferation and survival of renal cancer cells

Studies have been done to suggest that GSK-3 is importantfactor of NF-kb- mediated cancer cell survival and

proliferation in pancreatic and chronic lymphocytic leukaemia(CLL)To determine if GSK-3 is essential for RCC cell survival and

proliferation, 3 small molecule inhibitors were used:AR-A014418 (ATP-competitive)SB-216763 (ATP-competitive)TDZD8 ( non ATP competitive)


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All 3 inhibitors can decrease viability of ACHN renal cancercellAnti-cancer effect of AR-A014418 was tested on 6 other renal

cancer cell lines: KH39,KU19-20,Caki1,Caki2,KRC/Y andA498Ar-A014418 is a potent and specific GSK-3 inhibitorInhibition of GSK-3 decreased renal cancer cell viability in a

dose and time dependent manner


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Using BrDU incorporation assay:Found that pharmacological inhibition of GSK-3 suppresses

proliferation of renal cancer cells

Using Hoest staining

Found a dose-dependent induction of apoptosis in AR-A014418-treatedcancer cellsResults suggest that GSK-3 is a positive regulator of renal cancer cell

proliferation and survival

To determine if the inhibitory effect was specific to GSK-3b,GSK-3a and 3b was depleted in ACHN using siRNA

Result: depletion of GSK-3 b significant decrease in renal cancer cellsurivival, accompanying apoptotic morphological changes observed byHoest stainingGSK-3 a does not affect cancer cells


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Most sensitive

cell line


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Scrambled siRNACheckuniquenessof drugs

Noapoptosis


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Figure 4A ACHN Caki124 h 48 h

0 25 50 25 50 0 25 50 25 50

24 h 48 hAR-A014418 M

pGS

Total GS

PARP

Cleaved PARP

XIAP

Bcl-2

-Actin


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Figure 4B,C


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Figure 4D,E,FInput Ig p65

Bcl-2

XIAP

DMSO AR-A DMSO AR-A DMSO AR-A

GSK-3

H3

SOD

WCL C Nu

PARPCleave

PARPXIAP

Bcl-2

-Actin


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AR-A014418 and Docetaxel synergistically suppresssurvival of renal cancer cells

Chemotherapeutic effect for RCC is very limited askidney cancer is intrinsically chemoresistantIncreased expression of Bcl-2 and XIAP is importantreasonHo: whether inhibition of GSK-3 could be useful incombination with conventional chemotherapeutic agent intreatmentDocetaxel is well establised drug with limited cytotoxiceffect in clinical RCCResult: inhibition of GSK-3 sensitised ACHN and Caki1cells to Docetaxel decrease cell survivability


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Figure 5


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In a nutshell. GSK- 3b has important function in pathogenesis of humancancerGSK-3 is a positive regulator of RCC cell survival,

proliferation and chemoresistanceGSK-3 b aberrant nuclear accumulation in most renal cellInactive GSK-3 b is able to translocate to nucleus from

cytoplasm but rapidly degraded by proteosomal pathway within nucleus of cancer cell


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In a nutshell Factors leading to progression of RCC:

Increased activity of NF- kb Increased expression of anti apoptotic molecules (Bcl-2 andXIAP)GSK-3 inhibitors inactivation of NF- k b sensitisedcancer cells to chemotherapy conventional chemotherapydrugs can be more effectiveThus, work has identified GSK-3 b as a novel potentialtherapeutic target in RCC


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Reference:Bilim.V.,Ougolkov.A.,Yuuki.K., Naito.S., Kawazoe.H.,, Muto.A.,Oya.M., Biladeau.D., Motoyama.T. and Tomita.Y. (2009). Glycogensynthase kinase-3: a new therapeutic target in renal cell carcinoma.

British Journal of Cancer (101) 2005-2014

Fang.X.,Yu.S.X.,Lu.Y.,Bast,R.C.Jr.,James R. Woodgett, andMills.G.B. Phosphorylation and inactivation of glycogen synthasekinase 3 by protein kinase A.(2000) PNAS.(97) 11960-11965Mishra.R.(2010).Glycogen synthase kinase 3 beta: can it be target fororal cancer. molecular cancer. (9)

Narayan.G, Prasad. S.B.(2012). A NEW INSIGHT OF GSK3 b REGULATION:IMPLICATIONS IN CANCER THERAPY. Journalof Scientific Research . (56) 25-34


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Thank you

Presented by:

Farhath JabienBBSD1 1012A

UOB: 09074657

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GSK-3 -New Therapeutic Target in Renal Cell Carcinoma