Philip L. Ho, MD Urologic Oncology Fellow The University of Texas - PowerPoint PPT Presentation

Stat3 Transgenic Mice as a Model for Human Basal Subtype of Invasive Bladder Cancer Philip L. Ho, MD Urologic Oncology Fellow The University of Texas at MD Anderson Cancer Center Houston, TX

Disclosures • None

Outline • Cancer Stem Cells • Keratin 14, Basal subtype • Stat3 – Cancer – Transgenic mouse tumors – Mechanism and Targets

Introduction • Minimal improvement in bladder urothelial carcinoma patient outcomes have been made • Standard of care remains surgery and chemotherapy – No targeted therapies • Need to further understand biology in order to develop targeted therapies

Cancer Stem Cell Model Intratumoral Heterogeneity CSC 2. Self-renewal CSC 1. Tumor-initating potential 3. Ability to Cancer Stem Cell CSC differentiate - Recapitulate Progenitor Cells heterogeneity of original tumor Differentiated Cell

Bladder Cancer Stem Cell Markers Ho, PL. Kurtova, A., Chan, KS. Nature Reviews Urology 2012

Keratins (K) • Structural proteins  intermediate filaments • Expression – Cell-type – Differentiation – Function • Cancer – Diagnosis – Prognosis Karantza, V. Oncogene 2011

Bladder Cancer – Basal Subtype and K14 McConkey MDACC TCGA Basal Kim UNC The Cancer Genome Atlas Research Network. Nature 2014 Choi et al. Cancer Cell 2014 Damrauer et al. PNAS 2014

K14 Gene Expression Correlates with Survival Overall Survival – Multivariable analysis HR (low-high) p K14 1.51 (1.11 – 2.03) 0.0077 Stage 1.32 (1.20 – 1.45) <0.001 Grade 1.32 (1.00 – 1.74) 0.047 Gender 1.02 (0.82 – 1.27) 0.86 Age 1.45 (1.23 – 1.71) <0.001 Volkmer et al. PNAS 2012

K14 Protein Expression Correlates with Survival Multivariable analysis Multivariable analysis HR (low-high) p HR (low-high) p K14 2.11 (1.18 – 3.80) 0.0038 K14 2.42 (1.19 – 4.93) 0.032 Stage 23.32 (2.72 – 199.83) 0.0038 Stage 0.89 (0.66 – 1.20) 0.16 Grade 1.29 (0.27 – 6.10) 0.87 Grade 0.89 (0.61 – 1.32) 0.55 Gender 0.87 (0.63 – 1.19) 0.41 Gender 1.20 (0.47 – 3.05) 0.71 Age 1.03 (1.01 – 1.06) 0.042 Age 1.03 (1.00 – 1.06) 0.069 Volkmer et al. PNAS 2012

K14 Reporter Construct Tm+/K14+ cells Transduced with hK14.tdTomato (Tm) virus FACS Tm-/K14- cells DAPI Tm K14

K14 Reporter Construct Cells Tm- 5000 7/7 500 5/7 50 1/7 10 0/7 A B Tm- sphere number Fold Change sphere number C Tm+ Cells Tm+ 5000 7/7 500 7/7 50 6/7 10 2/7

Bladder Cancer – Basal Subtype and K14 … Stat3 TCGA subgroup McConkey group

Stat3 – basal subtype Choi, W, Porten , S, Kim, S, Willis, D,… McConkey, DJ. Cancer Cell 2014

STAT Proteins • Signal Transducer and activator of transcription (STAT) Proteins • Family of proteins that mediate • Growth factor signaling • Apoptosis • Angiogenesis • Immune responses • Tumor development Bromberg. Stat proteins and oncogenesis. Journal of Clinical Investigation. May 2002

STAT3 activation • Target genes include – Antiapoptotic proteins – Proliferation-associated proteins – Proangiogenic factors • Regulator of EMT transcriptions factors – Twist, Snail Bromberg. Journal of Clinical Investigation. May 2002 Chan et al. Oncogene 2008. Pedranzini et al. Journal of Clinical Investigation. 2004. Wendt et al. JAK STAT. 2014

STAT3 and Cancer • Constitutively active in many human malignancies • Naturally occurring mutations in Stat3 have yet to be identified • Dysregulation may play role in constitutive activation of Stat3 Bromberg. Journal of Clinical Investigation. May 2002 Myers Jr. et al. Science 323 (2009): 723-24.

STAT3 and Bladder Cancer Nuclear, active Stat3 was present in 100% of high-grade, invasive human UC tumors but only in 37.5% of low ‐ stage tumors. Stat3 was also found to co ‐ localize with CK14 Ho, PL …Chan, KS. Cancer Research 2012

2 Pathways of Bladder Cancer Model? Dinney, CP, McConkey, DJ et al. Cancer Cell 2004 Ho, PL., Kurtova, A., Chan, KS. Nature Reviews Urology 2012

K5.Stat3C Transgenic Mice Stat3C Bovine keratin 5 promoter  K5.Stat3C transgenic mice with constitutively active Stat3 driven by the basal keratin 5 promoter were exposed to nitrosamine (BBN) through their drinking water.

K5.Stat3C Transgenic Mice Stat3-transgenic Wild-type Ho, PL & Chan, KS. Cancer Research 2012

K5.Stat3C Transgenic Mice Ho, PL & Chan, KS. Cancer Research 2012

K5.Stat3C Tumors – Sphere-forming Assay Ho, PL & Chan, KS. Cancer Research 2012

Stat3  ?

Stat3  ? SOX2 Immunofluorescence staining of SOX2 in Stat3-driven lesions

Activation of Stat3 Yu et al. Nature Reviews Immunology. January 2007

siRNA inhibition of Src and Jak A â JAK2 inhibitor - Ruxolitinib Src inhibitor - Dasatinib â

Conclusion • K14 – marker for cancer stem cells • Stat3 – drives basal subtype of invasive bladder cancer • Stat3 transgenic mouse – Invasive bladder cancer model – Preclinical studies to investigate inhibitory drugs

Acknowledgements Baylor College of Medicine MD Anderson Cancer Center - Keith S. Chan PhD - Ashish M. Kamat MD - Seth P. Lerner MD - David J. McConkey PhD - Colin P. N. Dinney MD Chan Lab - Antonnia Kurtova - Jing Xiao PhD - Erica J. Lay - Yanting Qi PhD - Ross E. Krasnow MD