PAX8-PPARγ Fusion Protein in thyroid carcinoma

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Sidney H. Ingbar Lecture, ATA 10/17/2013: PAX8-PPARγ Fusion Protein in thyroid carcinoma Ronald J. Koenig, MD, PhD Division of Metabolism, Endocrinology & Diabetes University of Michigan

Learning Objectives 1. Know what the PAX8-PPAR Fusion Protein is. 2. Know what types of thyroid cancer are associated with the PAX8-PPAR Fusion Protein. 3. Know about a clinical trial specifically designed for patients with PAX8-PPAR Fusion Protein thyroid cancer. 4. Know the normal function of PPAR. 5 Know about the Wnt pathway. (No disclosures)

Outline of today s presentation Background PPFP carcinoma patient samples Mouse model of PPFP thyroid carcinoma PPFP cell lines Summary and conclusions

Gene mutations underlie thyroid carcinoma Vu-Phan D & Koenig RJ. Mol. Cell. Endocrinol., in press (review)

PPFP is generated by a chromosomal translocation Paired Box Gene 8 (PAX8): Chr 2q13 1 2 3 4 5 6 7 8 9 10 11 12 Translocation (2;3)(q13;p25) 1 2 3 4 5 6 Peroxisome Proliferator-Activated Receptor Gamma (PPARG): Chr 3p25 DBD Fusion junction DBD LBD PAX8 PPAR PAX8-PPAR Fusion Protein (PPFP)

Functions of PAX8 and PPAR. PAX8 is a transcription factor that is essential for thyroid development and that drives the expression of many thyroid-specific genes. PPAR is the master regulator of adipogenesis, among other functions. PPAR is expressed at very low levels in normal thyroid and has no known function in that organ.

PAX8-PPAR has dominant negative activity over PPAR (transfections in U2OS osteosarcoma cells) Kroll TG, et al. Science 289:1357-60, 2000

PPAR, a putative tumor suppressor Several mouse models of cancer are exacerbated by a single allele deletion of Pparg, including a model of thyroid cancer that is unrelated to PPFP (Kato, et al. Oncogene 25:2736, 2006). Hypothesis: PPFP is oncogenic because it inhibits the tumor suppressor activity of PPAR.

Outline of today s presentation Background PPFP carcinoma patient samples Mouse model of PPFP thyroid carcinoma PPFP cell lines Summary and conclusions

Gene expression signature of PPFP follicular thyroid cancers FC(+) FC(-) NT FA OA OC PC AC MC Thomas Giordano, MD, PhD Giordano TG, et al. Clin Cancer Res. 12:1983-93, 2006

Gene expression signature of PPFP follicular thyroid cancers. FC(+) FC(-) NT FA OA OC PC AC MC AQP7 ANGPTL4 Giordano TG, et al. Clin Cancer Res. 12:1983-93, 2006.

PPFP has PPAR -like activity on the AQP7 promoter. AQP7-Luc -Cz +Cz 20 16 JEG-3 6 5 N2a 12 8 4 3 2 Fold Luc/Rluc Activity 4 0 6 5 4 FRTL-5 1 0 60 50 40 Dog thyrocytes 3 30 2 20 1 10 0 0 pcdna PAX8 PPAR PPFP pcdna PAX8 PPAR PPFP Giordano TG, et al. Clin Cancer Res. 12:1983-93, 2006.

Outline of today s presentation Background PPFP carcinoma patient samples Mouse model of PPFP thyroid carcinoma PPFP cell lines Summary and conclusions

Thyroidal expression of PPFP in transgenic mice Ericka Diallo-Krou Melissa Dobson, PhD CAG promoter intron LoxP egfp LoxP PPFP X TPO promoter Cre Dobson ME, et al. Endocrinology. 152:4455-65, 2011

Thyroidal expression of PPFP in transgenic mice CAG promoter intron LoxP egfp LoxP PPFP X TPO promoter Cre PPFP/PPAR 40 30 20 10 0 PPFP;Cre mean±sd, n=14 Wild type PPFP;Cre Hyperplasia, No neoplasia Dobson ME, et al. Endocrinology. 152:4455-65, 2011

FTC pakt

FTC pakt

PPFP carcinomas have increased phosphoakt pakt(s473) Immunohistochemistry Normal human thyroid PPFP cancer Diallo-Krou E, et al. Endocrinology. 150:5181-90, 2009

PPFP carcinomas have increased phosphoakt pakt(s473) Immunohistochemistry Normal human thyroid PPFP cancer Combined PPFP expression with thyroidspecific Pten deletion: PPFP;PtenFF;Cre Control mice Pten deletion: PtenFF;Cre. Mouse Thyroid AKT immunoblot pakt(t308) pakt(s473) Total AKT -actin

Mouse thyroid glands Wild type PtenFF;Cre PPFP;PtenFF;Cre Dobson ME, et al. Endocrinology. 152:4455-65, 2011

PPFP;PtenFF;Cre mice develop metastatic thyroid carcinoma Tracheal Invasion H&E Myc (PPFP) TTF-1 Vascular Invasion Lung Metastases H&E Myc (PPFP) TTF-1 PAX8 (PtenFF;Cre mice have benign thyroid hyperplasia with no neoplasia). Dobson ME, et al. Endocrinology. 152:4455-65, 2011

Pioglitazone therapy of PPFP;PtenFF;Cre thyroid cancer Thyroid Area (mm 2 ) Thyroid size by ultrasound 120-110- 100-90- 80-70- 60-50- 40-30- 20-10- 0- PPFP;PtenFF;Cre Control Diet Pioglitazone 18-16- 14-12- 10-8- 6- PtenFF;Cre 4-2- Start End 0- Start End WT thyroid area 1.9±0.5 mm 2 Dobson ME, et al. Endocrinology. 152:4455-65, 2011

Pioglitazone therapy of PPFP;PtenFF;Cre thyroid cancer. Thyroid Area (mm 2 ) Thyroid size by ultrasound 120-110- 100-90- 80-70- 60-50- 40-30- 20-10- 0- PPFP;PtenFF;Cre Control Diet Pioglitazone 18-16- 14-12- 10-8- 6- PtenFF;Cre 4-2- Start End 0- Start End Pioglitazone prevents local invasion and metastases in PPFP;PtenFF;Cre mice N Local invasion Vascular invasion Distant mets Control diet 12 11 5 7 Pioglitazone 10 1 0 0 P 0.0002 0.03 0.005 (Neither invasion nor metastases were found in any PtenFF;Cre mice) WT thyroid area 1.9±0.5 mm 2 Dobson ME, et al. Endocrinology. 152:4455-65, 2011.

Adipogenic PPAR -like response to pioglitazone in PPFP;PtenFF;Cre thyroids PtenFF;Cre Control Diet Pioglitazone PPFP; PtenFF;Cre 100 Lipid droplets Dobson ME, et al. Endocrinology. 152:4455-65, 2011

Adipogenic PPAR -like response to pioglitazone in PPFP;PtenFF;Cre thyroids PtenFF;Cre Control Diet Pioglitazone PPFP;PtenFF;Cre +Pio Oil Red O Perilipin-1 PPFP; PtenFF;Cre 100 Lipid droplets Dobson ME, et al. Endocrinology. 152:4455-65, 2011

Adipogenic PPAR -like response to pioglitazone in PPFP;PtenFF;Cre thyroids PtenFF;Cre Control Diet Pioglitazone PPFP;PtenFF;Cre +Pio Oil Red O Perilipin-1 Thyroid PPFP;PtenFF;Cre Thyroid MRI Control Pioglitazone Thyroid PPFP; PtenFF;Cre 100 Lipid droplets Thyroid 40 % Fat 0 Thyroid Dobson ME, et al. Endocrinology. 152:4455-65, 2011

Adipogenic PPAR -like response to pioglitazone in PPFP;PtenFF;Cre thyroids PtenFF;Cre Control Diet Pioglitazone PPFP;PtenFF;Cre +Pio Oil Red O Perilipin-1 Thyroid PPFP;PtenFF;Cre Thyroid MRI Control Pioglitazone Thyroid PPFP; PtenFF;Cre 100 Lipid droplets Thyroid 40 % Fat 0 Thyroid Adipocyte PPAR target genes induced PPFP;PtenFF;Cre +Pio/-Pio PtenFF;Cre +Pio/-Pio 17/17 3/17 Dobson ME, et al. Endocrinology. 152:4455-65, 2011

Adipogenic PPAR -like response to pioglitazone in PPFP;PtenFF;Cre thyroids PtenFF;Cre PPFP; PtenFF;Cre Control Diet Adipocyte PPAR target genes induced Pioglitazone 100 PPFP;PtenFF;Cre +Pio/-Pio Lipid droplets PtenFF;Cre +Pio/-Pio 17/17 3/17 PPFP;PtenFF;Cre +Pio Oil Red O Perilipin-1 Thyroid Thyroid Myc (PPFP) PPFP;PtenFF;Cre Thyroid MRI Control Pioglitazone TTF-1 Thyroid 40 % Fat 0 100 Thyroid Dobson ME, et al. Endocrinology. 152:4455-65, 2011

Adipogenic PPAR -like response to pioglitazone in PPFP;PtenFF;Cre thyroids PtenFF;Cre PPFP; PtenFF;Cre Control Diet Adipocyte PPAR target genes induced Pioglitazone 100 PPFP;PtenFF;Cre +Pio/-Pio Lipid droplets PtenFF;Cre +Pio/-Pio 17/17 3/17 PPFP;PtenFF;Cre +Pio Oil Red O Perilipin-1 Thyroid Thyroid Myc (PPFP) PPFP;PtenFF;Cre Thyroid MRI Control Pioglitazone TTF-1 Thyroid 40 % Fat 0 100 Thyroid Hypothesis: The therapeutic activity of pioglitazone in PPFP carcinomas is due to differentiation into adipocyte-like cells. Dobson ME, et al. Endocrinology. 152:4455-65, 2011

Adipogenic PPARγ-like response to pioglitazone in PPFP;PtenFF;Cre thyroids Adipoq Angptl4 Log 2 RNA Expression/Pgk1 Aqp7 Fabp4 Cebpa Plin1 PPFP; PtenFF;Cre PtenFF;Cre Control Diet Fabp4 Pioglitzaone 100 PtenFF;Cre CD Pio PPFP;PtenFF;Cre CD Pio PtenFF;Cre CD Pio PPFP;PtenFF;Cre CD Pio Dobson ME, et al. Endocrinology. 152:4455-65, 2011

Ratios of gene expression by genotype and Pio treatment GENE PtenFF;Cre Pio/CD PPFP;PtenFF;Cre / PtenFF;Cre (CD) PPFP;PtenFF;Cre / PtenFF;Cre (Pio) PPFP;PtenFF;Cre Pio/CD Adipoq 1.48 0.48 7.4 22.6 Agpat2 1.08 0.82 3.1 4.0 Angptl4 2.40 13.63 30.5 5.4 Aqp7 3.03 1.30 11.4 28.6 Cd36 3.93 3.54 9.7 10.7 Cebpa 1.14 3.86 7.1 2.1 Cidec 1.77 0.27 7.7 51.7 Dgat1 1.38 1.53 2.0 1.8 Fabp4 6.66 12.95 7.5 3.8 Fasn 0.72 0.87 4.0 3.3 Glut4 1.54 0.42 3.8 13.8 Gpd1 1.17 4.69 9.9 2.5 Lipe 1.37 0.86 3.5 5.6 Lpl 1.50 0.67 5.1 11.5 Pck1 2.55 1.29 7.8 15.3 Pdk4 1.32 0.96 3.6 5.0 Plin1 1.28 0.61 12.8 26.9 P<.05 Newman-Keuls test; Pio or PPFP induces gene P<.05 Newman-Keuls test; Pio or PPFP represses gene

NCT01655719: Pioglitazone therapy of PPFP thyroid carcinomas Multi-site clinical trial. Local or metastatic disease not amenable to therapy with surgery or RAI. We screen archival tumor blocks from patients with FVPTC or FTC for the PAX8- PPAR translocation. Pioglitazone has very low toxicity; please consider referring patients to our trial!

Outline of today s presentation Background PPFP carcinoma patient samples Mouse model of PPFP thyroid carcinoma PPFP cell lines Summary and conclusions

PPFP increases cell invasiveness and anchorage-independent growth. PCCL3 thyroid cells stably expressing PPFP or DBD mutant AAA Dang Vu-Phan, PhD Colony formation in soft agar Colonies/5000 cells EV PPFP AAA Invading cells/5000 Invasion through Matrigel EV PPFP AAA Vu-Phan D, et al. Endocr Relat Cancer. 20:725-39, 2013.

The Connectivity Map (CMAP) CMAP is a collection of genome-wide transcriptional expression data from cultured human cells treated with bioactive small molecules, combined with pattern-matching algorithms that together enable the discovery of functional connections between drugs, genes and diseases.

CMAP connects PPFP with Wnt The PPFP gene signature was analyzed in CMAP. 70 bioactive small molecules elicited gene expression changes that were enriched for PPFP profile genes. 16 of those 70 have been reported to be Wnt pathway modulators.

Wnt signaling overview Niehrs C. Nature Reviews Molecular Cell Biology (2012) 13:767.

Wnt/Tcf pathway Anastas JN and Moon RT. Nat Rev Cancer. 13:11-26, 2013

PPFP induces a subpopulation of Wnt/TCF-activated cells Vu-Phan D, et al. Endocr Relat Cancer. 20:725-39, 2013

PPFP induces a subpopulation of Wnt/TCF-activated cells And pioglitazone further increases the GFP+ subpopulation Vu-Phan D, et al. Endocr Relat Cancer. 20:725-39, 2013

TCF_GFP+ and TCF_GFP cells are hierarchically organized TCF_GFP cells beget TCF_GFP cells TCF_GFP+ cells beget TCF_GFP+ and cells TCF_GFP (left) and TCF_GFP+ (right) cells were sorted by FACS, expanded in culture, and reanalyzed for GFP status. Vu-Phan D, et al. Endocr Relat Cancer. 20:725-39, 2013

TCF_GFP+ cells are enriched in colony formation and invasiveness Vu-Phan D, et al. Endocr Relat Cancer. 20:725-39, 2013

Wnt/TCF-responsive genes are induced in a mouse model of PPFP thyroid carcinoma Vu-Phan D, et al. Endocr Relat Cancer. 20:725-39, 2013

Summary and conclusions.1 PPFP is found in FTCs and FVPTCs. PPFP can antagonize PPAR or can be PPAR -like, depending on context. In a mouse model of PPFP thyroid cancer, the PPAR agonist pioglitazone is highly therapeutic and is pro-adipogenic. We have begun a clinical trial of pioglitazone in patients with PPFP thyroid carcinomas.

Summary and conclusions.2 PPFP expression enhances the ability of PCCL3 thyroid cells to invade through Matrigel and to have anchorage independent growth. These actions require an intact PPAR DBD within PPFP.

Summary and conclusions.3 PPFP induces a subpopulation of Wnt/TCF activated cells. The PPFP-induced TCF+ population is enriched in colony forming and invading cells. The PPFP cells are hierarchically organized by TCF status. Based upon this, we hypothesize that the TCF+ fraction harbors PPFP thyroid cancer stem cells.

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