Sunny Guin, PhDResearch Scientist (Principal Investigator)

Kabara Cancer Research InstituteGundersen Medical Foundation

La Crosse, WI

Adjunct Assistant Professor Department of Biology

University of Wisconsin – La CrosseLa Crosse, WI

The Role, Relationship and Therapeutic Potential of AGL and HAS2 in Bladder Cancer

Constructs

0

50

100

150

200

250

300

350

400

T1 T2 T3 T4

AGL

GPR107

OSR2

INMT

ZBTB4R

ea

d C

ou

nt (1

03)

Guin et al, JNCI 2014

Identifying AGL as a Regulator of Bladder Cancer Growth

amylo-alpha-1, 6-glucosidase, 4-alpha-glucanotransferase ( glycogen

debranching enzyme)

Has 2 catalytic activities - oligo-1,4-1,4-glucanotransferase

- amylo-1,6-glucosidase

What is AGL?

Ritterson et al, Nat Rev Urol. 2015

AGL and Glycogen Storage Disease Type III

Deficiency of GDE – incomplete glycogenolysis and accumulation of abnormal

glycogen with shorter outer chains – Limit Dextrin

Glycogen Storage Disease Type III (Cori disease or Forbes disease) results from

deficiency of GDE

Liver and Muscle affected

Multiple mutations identified in AGL gene in Glycogen Storage Disease Type III

patients

GDE - glycogen debranching enzyme

Demo et al, J Hepatol. 2007

Guin et al, JNCI 2014

AGL is a Prognostic Marker in Bladder Cancer

0.00

0.12

0.24

0.36

0.48

0.60

shCTL shAGL

Limit

Dextrin *

µm

ol p

er

mg

/ce

ll

0.00

0.09

0.18

0.27

0.36

0.45

shCTL shAGL

Glycogen*

µm

ol p

er

mg

/ce

ll

AGL

GAPDH*

0

0.3

0.6

0.9

1.2

0 1 2 3 4 5

Day

shCTL

shAGL

Flu

ore

sc

en

ce

Guin et al, JNCI 2014

Loss of AGL Drives Bladder Cancer Growth

0

300

600

900

1200

0 9 16 23 30 37

Tu

mo

r v

olu

me (m

m3)

Day

shCTL n=2:10

shAGL n=3:10

P < 0.05

AGL

α-Tubulin

shAGL shAGL+wtAGL shAGL+AGL L620P shAGL+AGL R1147G

196±12.3 138±6.5* 112±15.6* 121±14.3*

Transferase Glucosidase C Term

L620P R1147G

Guin et al, JNCI 2014

Cheng et al, Hum Mol Genet 2009

AGL Drives Bladder Cancer Growth Independent of its

Enzymatic Activity

UMUC3

62

38

Association of Gene

Expression vs.

Clinicopathologic

Variables

To

p 1

00

Dif

fere

nti

ally

Ex

pre

ss

ed

Ge

ne

s

0

2

4

6

8

10

12

14

16

SEMA3A HAS2 RRAGD VCAN EREG TULP3 UCHL1

Fo

ld C

ha

ng

e m

RN

A v

s sh

CT

L

S, G,

Sr

S, G,

Sr

S, G,

Sr

T, S,

G,

Sr

S, G,

Sr

T, S,

G,

Sr

S, G,

Sr

Clinicopathologic Variable Associations

shCTL* shAGL1*

AGL

α-tubulin

Tumor vs Normal (T)

Tumor Stage (S)

Tumor Grade (G)

Patient Survival (Sr)

Guin et al, CCR 2015

Identifying Drivers of Bladder Cancer Growth with AGL loss

0

1

2

3

4

5

6

7

8

9

0 1 2 3 4

shCTL shCTL

shCTL shHAS2

shAGL shCTL

shAGL shHAS2

0

20

40

60

80

100

120

140

160

shCTLsiCTL

shCTLsiHAS2

shAGLsiCTL

shAGLsiHAS2

0

50

100

150

200

250

300

shCTLshCTL

shCTLshHAS2

shCTLshAGL

shAGLshHAS2

Re

lati

ve

HA

S2

Ex

pre

ss

ion

(vs

siC

TL

)

Flu

ore

sce

nce

In

ten

sity

(10

3)

Days

*

*

*

Flu

ore

sce

nce

In

ten

sity

(10

3)

Re

lati

ve

HA

S2 E

xp

ress

ion

(vs

siC

TL

)

Days

Avg

. C

olo

ny F

orm

ati

on

Avg

. C

olo

ny F

orm

ati

on

*

0.28

0.47 0.42

shCTL shAGL1 shAGL2

siHAS2 siCTL

0

1

2

3

4

5

6

7

8

0 1 2 3 4

shCTL siCTL

shCTL siHAS2

shAGL siCTL

shAGL siHAS2*

0.330.2

shCTL shAGL

shHAS2 shCTL

**

** *

Guin et al, CCR 2015

UMUC3

T24T

Loss of AGL Drives Bladder Cancer Growth via Hyaluronic Acid (HA)

Synthase 2 (HAS2)

0

0.5

1

1.5

2

2.5

3

HAS1 HAS2 HAS3

WT

AGL-KO

Re

lati

ve

Ge

ne

Ex

pre

ss

ion

(vs

WT

)

*

*

*

*

Re

lati

ve

HA

S2

Ex

pre

ss

ion

(vs

sh

CT

L f

or

ea

ch

)*

0

0.5

1

1.5

2

2.5

3

3.5

4

hTERT TRT-HU1

shCTL

shAGL

AGL

α-tubulin

hTERT TRT-HU1

AGL

α-tubulin

WT AGL-KO

0

0.2

0.4

0.6

0.8

1

1.2

HAS1 HAS3

shCTL

shAGL

N.D

UMUC3

Re

lati

ve

HA

S E

xp

ress

ion

(vs

shC

TL

)

Guin et al, CCR 2015

AGL and HAS2 Expression in Normal Bladder Tissues

0

1

2

3

4

5

6

7

shCTLsiCTL

shCTLsiHAS2

shAGLsiCTL

shAGLsiHAS2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

shCTLshCTL

shCTLshHAS2

shAGLshCTL

shAGLshHAS2

0

0.2

0.4

0.6

0.8

1

0 200 400 600 800 1000

Tho

usa

nd

s

shCTL

shAGL

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 200 400 600 800 1000

Tho

usa

nd

s

shCTL

shAGL

0

2

4

6

8

10

12

0 1 2 3 4

shCTL

shCTL+4MU

shAGL

shAGL+4MU

0

1

2

3

4

5

6

7

shCTL shCTL4MU

shCTL4MU+HA

shAGL shAGL4MU

shAGL4MU+HA

Tho

usa

nd

s

Re

lati

ve

HA

in

Me

dia

pe

r D

ay

Re

lati

ve

HA

in

Me

dia

pe

r D

ay

**

UMUC3 T24T

HA

in

Me

dia

pe

r D

ay (

ng

/ml)

(10

3)

HA

in

Me

dia

pe

r D

ay (

ng

/ml)

(10

3)

UMUC3 T24T

4MU Conc (μM) 4MU Conc (μM)

Flu

ore

sce

nce

In

ten

sity

(10

3)

Days

Flu

ore

sce

nce

In

ten

sity

(10

3)

* **

*

*

A i) ii) B i) ii)

C D

Guin et al, CCR 2015

Loss of AGL Drives Bladder Cancer Growth by HAS2 Mediated HA

Synthesis

4MU – 4-methylumbelliferone

0

200

400

600

800

1000

1200

1400

1600

20 25 30 35 40 45 50

shCTL siCTL

shCTL siHAS2

shAGL siCTL

shAGL siHAS2

0

100

200

300

400

500

600

700

800

900

0 5 10 15 20 25 30 35 40 45

shCTL shCTL

shCTL shHAS2

shAGL shCTL

shAGL shHAS2

0

200

400

600

800

1000

1200

0 10 20 30

shAGL

shAGL +4MU

Days Days

Days

Av

g.

Tu

mo

r V

olu

me

(m

m3)

Av

g.

Tu

mo

r V

olu

me

(m

m3)

Av

g.

Tu

mo

r V

olu

me

(m

m3)

*

*

*

4MU

A B

C D

UMUC3 T24T

UMUC3

Av

g.

Tu

mo

r V

olu

me

(m

m3)

0

100

200

300

400

500

600

700

0 10 20 30 40

shAGL

shAGL+4MU

4MU

Days

T24T

*

Guin et al, CCR 2015

4-Methylumbelliferone Reduce Bladder Xenograft Growth Driven

by AGL Loss

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5

shCTL siCTL

shAGL siCTL

shCTL siCD44

shAGL siCD44

0

2

4

6

8

10

12

14

0 1 2 3 4 5

shCTL siCTL

shAGL siCTL

shCTL siRHAMM

shAGL siRHAMM

Avg

. C

olo

ny/

We

ll

Flu

ore

sce

nce

In

ten

sity

(10

3)

Flu

ore

sce

nce

In

ten

sity

(10

3)

Days Days

*

*

0

50

100

150

200

250

shCTLsiCTL

shCTLsiCD44

shCTLsiRHAMM

shAGLsiCTL

shAGLsiCD44

shAGLsiRHAMM

**

HA Receptors CD44 and RHAMM are Essential for Bladder Tumor

Growth Driven by AGL Loss

Guin et al, BMC Cancer 2016 (under review)

-5

0

5

10

15

20

25

30

35

siCTL siHAS2 siCD44 siRHAMM

shCTL

shAGL

-10

0

10

20

30

40

50

60

siCTL siHAS2 siCD44 siRHAMM

UM

UC

3T

24

Ts

hC

TL

sh

CT

Ls

hA

GL

sh

AG

L

siCTL

siHAS2

siHAS2

siCD44

siCD44

siRHAMM

siRHAMM

siCTL

siCTL

siHAS2

siHAS2

siCD44

siCD44

siRHAMM

siRHAMM%

Ap

op

tos

is%

Ap

op

tos

is

A C

B D

siCTL

siCTL 20µm

20µm

20µm

20µm

20µm

20µm

20µm

20µm

20µm 20µm 20µm 20µm

20µm 20µm 20µm 20µm

**

*

Guin et al, BMC Cancer 2016 (under review)

Loss of CD44 or RHAMM Induce Apoptosis in Low AGL Expressing

Bladder Cancer Cells

AGL KO transgenic mice studies

Therapeutic inhibition of the HAS2-HA-CD44/RHAMM axis in AGL low bladder

cancer model

Summary

AGL is a new regulator of bladder cancer growth

AGL is a prognostic marker in bladder cancer

Loss of AGL drives bladder cancer growth via Hyaluronic acid (HA) Synthase 2 (HAS2)- HA-CD44/RHAMM axis

Ongoing Studies

Acknowledgements

Dan Theodorescu Lab

Department of Surgery, University

of Colorado, Denver, CO, USA

Dan Theodorescu, MD, PhD

All Lab members

Sigma-Aldrich Research Biotech,

Saint Louis, MO, USA

Andrea Spencer, PhD

Scott Knight, PhD Heather Holemon, PhD

Department of Pathology, Cleveland

Clinic, Cleveland, OH, USA

Sounak Gupta, PhD

Donna Hansel, MD

Center for Regulatory and Environmental Analytical

Metabolomics (CREAM), Department of Chemistry,

University of Louisville, Louisville, KY, USA

Pawel Lorkiewicz, PhD Andrew N. Lane, PhD

Teresa W.-M. Fan, PhD

Funding

Cancer League of Colorado, Inc

BCAN Young Investigator Award NIH Funding (RO1) to Dr. Theodorescu

Gundersen Medical Foundation

Kabara Cancer Research Institute

La Crosse, WI, USA

Darby Oldenburg, PhD

Steve Cash, BS