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

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 Professor Department of Biology University of Wisconsin – La Crosse La Crosse, WI

Identifying AGL as a Regulator of Bladder Cancer Growth Constructs 400 AGL GPR107 350 OSR2 Read Count (10 3 ) INMT 300 ZBTB4 250 200 150 100 50 0 T1 T2 T3 T4 Guin et al, JNCI 2014

What is AGL?  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 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

AGL is a Prognostic Marker in Bladder Cancer Guin et al, JNCI 2014

Loss of AGL Drives Bladder Cancer Growth 1200 shCTL n=2:10 1.2 shAGL n=3:10 shCTL Tumor volume (mm 3 ) 900 shAGL AGL * 0.9 Fluorescence GAPDH 600 P < 0.05 0.6 0.60 Dextrin * Limit µmol per mg/cell 300 0.48 0.3 0.36 0 0 0.24 0 9 16 23 30 37 0 1 2 3 4 5 Day 0.12 Day 0.00 shCTL shAGL 0.45 * µmol per mg/cell Glycogen 0.36 0.27 0.18 0.09 0.00 shCTL shAGL Guin et al, JNCI 2014

AGL Drives Bladder Cancer Growth Independent of its Enzymatic Activity L620P R1147G Transferase Glucosidase C Term Cheng et al, Hum Mol Genet 2009 shAGL shAGL+wtAGL shAGL+AGL L620P shAGL+AGL R1147G AGL 196 ± 12.3 138 ± 6.5* 112 ± 15.6* 121 ± 14.3* α -Tubulin Guin et al, JNCI 2014

Identifying Drivers of Bladder Cancer Growth with AGL loss 16 shCTL* shAGL1* Fold Change mRNA vs shCTL AGL 14 UMUC3 α -tubulin 12 10 Association of Gene 8 Top 100 Differentially Expression vs. Expressed Genes 6 38 4 Clinicopathologic Variables 2 0 SEMA3A HAS2 RRAGD VCAN EREG TULP3 UCHL1 62 Tumor vs Normal (T) S, G, S, G, S, G, T, S, S, G, T, S, S, G, Tumor Stage (S) Sr Sr Sr G, Sr G, Sr Tumor Grade (G) Sr Sr Patient Survival (Sr) Clinicopathologic Variable Associations Guin et al, CCR 2015

Loss of AGL Drives Bladder Cancer Growth via Hyaluronic Acid (HA) Synthase 2 (HAS2) * siHAS2 siCTL 160 Fluorescence Intensity (10 3 ) 8 Avg. Colony Formation Relative HAS2 Expression 140 shCTL siCTL 7 shCTL siHAS2 120 6 shAGL siCTL (vs siCTL) 100 5 shAGL siHAS2 * 80 0.47 4 0.42 * 60 0.28 3 * * * 40 2 1 20 0 0 shCTL shAGL1 shAGL2 0 1 2 3 4 shCTL shCTL shAGL shAGL Days siCTL siHAS2 siCTL siHAS2 UMUC3 * shHAS2 shCTL 300 9 Fluorescence Intensity (10 3 ) Relative HAS2 Expression Avg. Colony Formation 8 250 shCTL shCTL 7 * shCTL shHAS2 200 (vs siCTL) 6 shAGL shCTL shAGL shHAS2 5 150 4 0.33 100 3 0.2 * 2 * 50 1 0 0 shCTL shCTL shCTL shAGL shCTL shAGL 0 1 2 3 4 shCTL shHAS2 shAGL shHAS2 Days Guin et al, CCR 2015 T24T

AGL and HAS2 Expression in Normal Bladder Tissues hTERT TRT-HU1 WT AGL-KO 1.2 UMUC3 Relative HAS Expression (vs shCTL) 1 AGL AGL shCTL α -tubulin α -tubulin shAGL 0.8 4 Relative HAS2 Expression 3 * shCTL Relative Gene Expression (vs shCTL for each) 3.5 0.6 * shAGL 2.5 WT 3 AGL-KO 2 2.5 (vs WT) 0.4 * 2 1.5 1.5 1 0.2 * 1 0.5 * 0.5 N.D 0 0 0 HAS1 HAS3 hTERT TRT-HU1 HAS1 HAS2 HAS3 Guin et al, CCR 2015

Loss of AGL Drives Bladder Cancer Growth by HAS2 Mediated HA Synthesis A i) ii) B i) ii) HA in Media per Day (ng/ml)(10 3 ) HA in Media per Day (ng/ml)(10 3 ) * Relative HA in Media per Day Relative HA in Media per Day 2 7 * 1 1.6 UMUC3 UMUC3 T24T T24T Thousands Thousands 1.8 6 1.4 1.6 0.8 1.2 5 1.4 shCTL shCTL 1.2 1 4 0.6 shAGL shAGL 1 0.8 3 0.8 0.4 0.6 2 0.6 0.4 0.4 0.2 1 0.2 0.2 0 0 0 0 shCTL shCTL shAGL shAGL shCTL shCTL shAGL shAGL 0 200 400 600 800 1000 0 200 400 600 800 1000 siCTL siHAS2 siCTL siHAS2 shCTL shHAS2 shCTL shHAS2 4MU Conc ( μ M) 4MU Conc ( μ M) C D 12 Fluorescence Intensity (10 3 ) * Fluorescence Intensity (10 3 ) 7 Thousands * 10 shCTL 6 * shCTL+4MU * 8 5 * shAGL 4 6 shAGL+4MU 3 4 2 2 1 0 0 shCTL shCTL shCTL shAGL shAGL shAGL 0 1 2 3 4 Days 4MU 4MU+HA 4MU 4MU+HA Guin et al, CCR 2015 4MU – 4-methylumbelliferone

4-Methylumbelliferone Reduce Bladder Xenograft Growth Driven by AGL Loss A B Avg. Tumor Volume (mm 3 ) UMUC3 Avg. Tumor Volume (mm 3 ) T24T 1600 900 800 1400 shCTL siCTL shCTL shCTL 700 1200 shCTL siHAS2 shCTL shHAS2 600 shAGL siCTL 1000 shAGL shCTL * 500 shAGL siHAS2 shAGL shHAS2 800 400 600 * 300 400 200 200 100 0 0 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 Days Days C D T24T 1200 UMUC3 700 Avg. Tumor Volume (mm 3 ) Avg. Tumor Volume (mm 3 ) 600 1000 shAGL shAGL shAGL+4MU 500 shAGL +4MU 800 400 600 * * 300 400 200 4MU 4MU 200 100 0 0 0 10 20 30 0 10 20 30 40 Days Days Guin et al, CCR 2015

HA Receptors CD44 and RHAMM are Essential for Bladder Tumor Growth Driven by AGL Loss 16 Fluorescence Intensity (10 3 ) Fluorescence Intensity (10 3 ) 14 14 shCTL siCTL shCTL siCTL 12 * shAGL siCTL 12 * shAGL siCTL 10 shCTL siCD44 10 shCTL siRHAMM shAGL siCD44 8 shAGL siRHAMM 8 6 6 4 4 2 2 0 0 0 1 2 3 4 5 0 1 2 3 4 5 Days Days * 250 * Avg. Colony/ Well 200 150 100 50 0 shCTL shCTL shCTL shAGL shAGL shAGL siCTL siCD44 siRHAMM siCTL siCD44 siRHAMM Guin et al, BMC Cancer 2016 (under review)

Loss of CD44 or RHAMM Induce Apoptosis in Low AGL Expressing Bladder Cancer Cells A C 60 * shCTL * 50 Apoptosis 40 UMUC3 siCD44 siRHAMM siCTL siHAS2 20µm 20µm 20µm 20µm 30 shAGL 20 % 10 siRHAMM siCTL siCTL siCD44 siHAS2 20µm 20µm 0 20µm 20µm siCTL siHAS2 siCD44 siRHAMM -10 B D shCTL 35 shAGL * 30 shCTL 25 Apoptosis 20 siCTL siRHAMM siHAS2 siCD44 20µm 20µm 20µm 20µm T24T 15 shAGL 10 % 5 siRHAMM siCTL 20µm 20µm siCD44 20µm 20µm 0 siHAS2 siCTL siHAS2 siCD44 siRHAMM -5 Guin et al, BMC Cancer 2016 (under review)

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  AGL KO transgenic mice studies  Therapeutic inhibition of the HAS2-HA-CD44/RHAMM axis in AGL low bladder cancer model

Acknowledgements Dan Theodorescu Lab Center for Regulatory and Environmental Analytical Department of Surgery, University Metabolomics (CREAM), Department of Chemistry, of Colorado, Denver, CO, USA University of Louisville, Louisville, KY, USA  Dan Theodorescu, MD, PhD  Pawel Lorkiewicz, PhD  Andrew N. Lane, PhD  All Lab members  Teresa W.-M. Fan, PhD Gundersen Medical Foundation Sigma-Aldrich Research Biotech, Kabara Cancer Research Institute Saint Louis, MO, USA La Crosse, WI, USA  Andrea Spencer, PhD  Darby Oldenburg, PhD  Scott Knight, PhD  Steve Cash, BS  Heather Holemon, PhD Department of Pathology, Cleveland Funding Clinic, Cleveland, OH, USA  Cancer League of Colorado, Inc  Sounak Gupta, PhD  BCAN Young Investigator Award  Donna Hansel, MD  NIH Funding (RO1) to Dr. Theodorescu