The Role, Relationship and Therapeutic Potential of AGL and HAS2 in - - PowerPoint PPT Presentation

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The Role, Relationship and Therapeutic Potential of AGL and HAS2 in - - PowerPoint PPT Presentation

Jan 29, 2023 472 likes •655 views

The Role, Relationship and Therapeutic Potential of AGL and HAS2 in Bladder Cancer Sunny Guin, PhD Research Scientist (Principal Investigator) Kabara Cancer Research Institute Gundersen Medical Foundation La Crosse, WI Adjunct Assistant

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Sunny Guin, PhD

Research Scientist (Principal Investigator) Kabara Cancer Research Institute Gundersen Medical Foundation La Crosse, WI Adjunct Assistant Professor Department of Biology University of Wisconsin –La Crosse La Crosse, WI

The Role, Relationship and Therapeutic Potential

  • f AGL and HAS2 in Bladder Cancer
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Constructs 50 100 150 200 250 300 350 400 T1 T2 T3 T4

AGL GPR107 OSR2 INMT ZBTB4

Read Count (103)

Guin et al, JNCI 2014

Identifying AGL as a Regulator of Bladder Cancer Growth

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  • 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

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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

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SLIDE 5

Guin et al, JNCI 2014

AGL is a Prognostic Marker in Bladder Cancer

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0.00 0.12 0.24 0.36 0.48 0.60 shCTL shAGL Limit Dextrin *

µmol per mg/cell

0.00 0.09 0.18 0.27 0.36 0.45 shCTL shAGL Glycogen

*

µmol per mg/cell

AGL GAPDH

*

0.3 0.6 0.9 1.2 1 2 3 4 5

Day

shCTL shAGL Fluorescence

Guin et al, JNCI 2014

Loss of AGL Drives Bladder Cancer Growth

300 600 900 1200

9 16 23 30 37

Tumor volume (mm3) Day

shCTL n=2:10 shAGL n=3:10

P < 0.05

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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

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SLIDE 8

UMUC3

62 38

Association of Gene Expression vs. Clinicopathologic Variables Top 100 Differentially Expressed Genes 2 4 6 8 10 12 14 16

SEMA3A HAS2 RRAGD VCAN EREG TULP3 UCHL1

Fold Change mRNA vs shCTL 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

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1 2 3 4 5 6 7 8 9 1 2 3 4

shCTL shCTL shCTL shHAS2 shAGL shCTL shAGL shHAS2

20 40 60 80 100 120 140 160

shCTL siCTL shCTL siHAS2 shAGL siCTL shAGL siHAS2

50 100 150 200 250 300

shCTL shCTL shCTL shHAS2 shCTL shAGL shAGL shHAS2

Relative HAS2 Expression (vs siCTL)

Fluorescence Intensity (103) Days

* * *

Fluorescence Intensity (103) Relative HAS2 Expression (vs siCTL) Days

  • Avg. Colony Formation
  • Avg. Colony Formation

*

0.28 0.47 0.42

shCTL shAGL1 shAGL2

siHAS2 siCTL

1 2 3 4 5 6 7 8

1 2 3 4 shCTL siCTL shCTL siHAS2 shAGL siCTL shAGL siHAS2

*

0.33 0.2

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)

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0.5 1 1.5 2 2.5 3 HAS1 HAS2 HAS3

WT AGL-KO

Relative Gene Expression (vs WT)

* * * *

Relative HAS2 Expression (vs shCTL for each)

*

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 0.2 0.4 0.6 0.8 1 1.2

HAS1 HAS3

shCTL shAGL N.D

UMUC3

Relative HAS Expression (vs shCTL)

Guin et al, CCR 2015

AGL and HAS2 Expression in Normal Bladder Tissues

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1 2 3 4 5 6 7

shCTL siCTL shCTL siHAS2 shAGL siCTL shAGL siHAS2

0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2

shCTL shCTL shCTL shHAS2 shAGL shCTL shAGL shHAS2

0.2 0.4 0.6 0.8 1 200 400 600 800 1000

Thousands

shCTL shAGL 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 200 400 600 800 1000

Thousands

shCTL shAGL

2 4 6 8 10 12 1 2 3 4

shCTL shCTL+4MU shAGL shAGL+4MU 1 2 3 4 5 6 7 shCTL shCTL 4MU shCTL 4MU+HA shAGL shAGL 4MU shAGL 4MU+HA

Thousands

Relative HA in Media per Day Relative HA in Media per Day

* *

UMUC3 T24T

HA in Media per Day (ng/ml)(103) HA in Media per Day (ng/ml)(103)

UMUC3 T24T

4MU Conc (μM) 4MU Conc (μM)

Fluorescence Intensity (103) Days Fluorescence Intensity (103)

* * * * * 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

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200 400 600 800 1000 1200 1400 1600

20 25 30 35 40 45 50

shCTL siCTL shCTL siHAS2 shAGL siCTL shAGL siHAS2

100 200 300 400 500 600 700 800 900

5 10 15 20 25 30 35 40 45

shCTL shCTL shCTL shHAS2 shAGL shCTL shAGL shHAS2

200 400 600 800 1000 1200

10 20 30

shAGL shAGL +4MU

Days Days Days

  • Avg. Tumor Volume (mm3)
  • Avg. Tumor Volume (mm3)
  • Avg. Tumor Volume (mm3)

* * *

4MU

A B C D

UMUC3 T24T UMUC3

  • Avg. Tumor Volume (mm3)

100 200 300 400 500 600 700

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

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2 4 6 8 10 12 14 16 1 2 3 4 5

shCTL siCTL shAGL siCTL shCTL siCD44 shAGL siCD44

2 4 6 8 10 12 14 1 2 3 4 5

shCTL siCTL shAGL siCTL shCTL siRHAMM shAGL siRHAMM

  • Avg. Colony/ Well

Fluorescence Intensity (103) Fluorescence Intensity (103) Days Days

* *

50 100 150 200 250

shCTL siCTL shCTL siCD44 shCTL siRHAMM shAGL siCTL shAGL siCD44 shAGL siRHAMM

* *

HA Receptors CD44 and RHAMM are Essential for Bladder Tumor Growth Driven by AGL Loss

Guin et al, BMC Cancer 2016 (under review)

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SLIDE 14
  • 5

5 10 15 20 25 30 35 siCTL siHAS2 siCD44 siRHAMM shCTL shAGL

  • 10

10 20 30 40 50 60 siCTL siHAS2 siCD44 siRHAMM

UMUC3 T24T shCTL shCTL shAGL shAGL

siCTL siHAS2 siHAS2 siCD44 siCD44 siRHAMM siRHAMM siCTL siCTL siHAS2 siHAS2 siCD44 siCD44 siRHAMM siRHAMM

% Apoptosis % Apoptosis 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

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  • 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

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SLIDE 16

Acknowledgements

Dan Theodorescu Lab Department of Surgery, University

  • f 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