1. NANOTETRAC TARGETS THE THYROID HORMONE RECEPTOR ON INTEGRIN  v  3 TO PROMOTE APOPTOSIS, DISRUPT CELL DEFENSE PATHWAYS AND BLOCK ANGIOGENESIS Paul J. Davis, MD Shaker A. Mousa, PhD Albany Medical College; Pharmaceutical Research Institute, Albany College of Pharmacy and Health Sciences, Albany, NY, USA

2. B Alberts text X

3. The thyroid hormone receptor on the extracellular domain of  v  3 binds L-thyroxine (T 4 ), 3,5,3’- triiodo-L-thyronine (T 3 ) and tetraiodothyroacetic acid (tetrac), a deaminated T 4 analogue. Tetrac blocks binding of T 4 and T 3 to the integrin and has an array of anti- cancer and anti-angiogenic properties at  v  3 that are independent of its inhibition of T 4 - and T 3 -binding to the integrin.

4. O CH 2 -CH-COOH NH 2 I - - I - - I - - I - - 3’ 5’5 3 Thyroxine (T 4 ) O HO CH 2 -CH-COOH NH 2 I - - I - - I - - 3’ 5’5 3 3,5,3’-Triiodothyronine (T 3 ) HO O CH 2 --COOH I - - I - - I - - I - - 3’ 5’5 3 Tetrac HO

5. Because unmodified tetrac is a thyromimetic within cells, we re- formulated the analogue by covalently binding it via a linker to 200 nm PLGA (poly[lactic-co- glycolic acid]) nanoparticles which limit its activities to the cell surface hormone-tetrac receptor on integrin  v  3.

6. H N H O H N O N H O N H H N O O I I O I I O O O I I O I I O H N H O O I IO I I H O PLGA nanoparticle Nanotetrac Four-to-eight tetrac moieties covalently bound via a linker to a 200 nm nanoparticle

7. At the integrin, Nanotetrac regulates via signal transducing kinases the expression of a variety of cancer-relevant and angiogen- esis-relevant genes. Nanotetrac disrupts crosstalk between  v  3 and adjacent vascular growth factor receptors (VEGFR, bFGFR, PDGFR, EGFR). Nanotetrac blocks radiation-induced activation of the integrin and controls plasma membrane ion transporters, e.g., NHE1, important to pHi and pHe.

8. ACTING AT THE INTEGRIN, NANOPARTICULATE TETRAC INDUCES A BLUEPRINT OF ANTI- CANCER GENE EXPRESSION DR of apoptosis inhibitors XIAP, MCL1, upregulation (UR) of pro- apoptotic CASP2, BCL2L14 DR of catenin genes, UR of CBY1, a nuclear inhibitor of catenin activity Downregulation (DR) of 21 of 23 differentially-regulated proto- oncogenes and 8 of 9 cyclins UR of thrombospondin (THBS1), an angiogenesis inhibitor, DR of CTSL1, progenitor endothelial cell recruiter

9. Fig. 3B Pro-apoptosis Cell Cycle, 2009

10. Fig. 2A Anti-apoptosis Cell Cycle 2009

11. Fig. 7 Cell Cycle, 2009

12. Days -10-505101520 200 400 600 800 1000 Control Tetrac 1 mg/kg (every 3rd day) Tetrac Nano 1 mg/kg (every 3rd day) Right side Days -10-505101520 100 200 300 400 500 600 700 Control Tetrac 1 mg/kg (every 3rd day) Tetrac Nano 1 mg/kg (every 3rd day) Left side Mean Tumor Volume (mm 3 ) ± SEM Effect of Tetrac and Tetrac Nanoparticles on Human Non-Small Cell Lung Carcinoma Xenograft Volume Mean Tumor Volume (mm 3 ) ± SEM Lung Cancer, 2012

13. Subcutaneous treatment effect on xenograft tumor (MPanc96-luc) after 19 days IVIS images Control (PBS)Tetrac (1 mg/kg)Nanotetrac (1 mg/kg) MPanc96-luc cells injected March 7 th, 2012 Subcutaneous daily treatment started: March 12 th, 2012 IVIS: March 30 th, 2012

14. Treatment (per implant) Tumor weight (gm.) Control (Vehicle)0.0423 Void NP0.0417 Tetrac (3 µg)0.0422 Tetrac (10 µg)0.0125 Nanotetrac (3 µg)0.0120 Nanotetrac (10 µg)0.0089 Effect of Nanotetrac on human glioblastoma U87MG xenografts in nude mice (16-day dose/implant study)

15. Nanotetrac has been effective against xenografts of human glioma/glioblastoma, breast, prostate, pancreatic, kidney, (NSCLC and non-small cell) lung, colon and thyroid cells.

16. The anti-angiogenic properties of Nanotetrac involve vascular growth factor gene expression (VEGFA, bFGF), growth factor receptor gene expression (EGFR), interaction of growth factors (VEGF, bFGF, PDGF, EGF) with their specific cell surface receptors, release of vascular growth factors (bFGF), expression of the cancer cell THBS1 (TSP1) gene and endothelial cell motility. This is a broadly-based set of anti- angiogenic actions.

17. Effect of tetrac (10 mg/kg, i.p. daily) or Nanotetrac (1.0 mg/kg, i.p. daily) on pancreatic tumor angiogenesis

18. PBS T 4 (total, 0.1  M) T 4 + LM609(10  g) Inhibitory effect of  v  3 MAB (LM609) on T4- stimulated angiogenesis in the CAM model

19. Table 1 Inhibition of activities of pro-angiogenic factors in the CAM assay by Nanoterac (NT) (2 µg/CAM) Treatment PBS control Void PLGA nanoparticle T 3 (6.5 ng/mL) T 3 + NT T 4 (100 nM) LPS + NT Bradykini (5 µg/mL) Bradykini + NT Angiotensin II (5 µg/mL) Angiotensin II + NT VEGF (2 µg/mL) VEGF + NT bFGF (1 µg/mL) bFGF + NT bFGF + VEGF + TNF-α bFGF + VEGF + TNF-α + NT Materials and Methods CAM assay was performed in duplicate X3 by our peviously published method [23]. P values by ANOVA compared single and multiple agent-containing samples with control or samples with and without Nanotetrac (NT). All comparisons were significant at least at P < 0.01. PBS phosphate-buffered saline, PLGA poly[lactic-co-glycolic acid], LPS lipopolysaccharide, VEGF vascular endothelial growth factor, bFGF basic fibroblast growth factor, TNF-α tumor necrosis factor-α. T 4 (100 nM) + NT

20. Fig. 5 Anti-angiogenesis Cell Cycle, 2009

21. THBS1 mRNA expression ControlTetrac NP TetracControlTetrac NPTetrac VEGFA THBS1, thrombospondin 1 VEGFA, vascular endothelial growth factor A Human medullary thyroid carcinoma cells

22. Effect of Tetrac on Cancer Cell Implants on Angiogenesis in the CAM Model + Tetrac (1 µM)Control Increased vascular proliferation Decreased vascular proliferation H1299 Bronchogenic carcinoma (1 x 10 6 cells/CAM) Actions of tetrac initiated nongenomically at  v  3 culminate in a complex of genomic and nongenomic effects on proliferation.

23. Integrin  v  3 contains a cell surface receptor for T 4, T 3. Tetrac and nanoparticulate tetrac inhibit agonist hormone action at the receptor and are probes for contributions of the receptor. In vitro, T 4, T 3 act at the integrin receptor via ERK1/2 to support tumor cell proliferation (breast, glioma, head-and-neck, thyroid, ovary, pancreas, kidney, lung, prostate) and angiogenesis; tetrac and Nanotetrac block these actions. Summary

24. Summary 2 Acting via the cell surface receptor on  v  3, Nanotetrac coherently modulates expression of multiple genes relevant to tumor cell survival. Receptor-initiated actions of thyroid hormone analogues also include modulation of crosstalk between the integrin and nearby vascular growth factor receptors and crosstalk with nuclear hormone receptor proteins, such as ER .

25. Summary 3 Nanotetrac also blocks tumor cell repair of radiation-induced double- strand DNA breaks Nanotetrac prolongs intracellular residence time of doxorubicin, etoposide and cisplatin, apparently via NHE1 and consequent alterations in pHi and pHe that affect P-glycoprotein and organic cation transporter function.

26. COLLABORATORS Shaker A. Mousa, PhD Albany Hung-Yun Lin, PhDAlbany Heng-Yuan Tang, MAAlbany Thangirala Sudha, PhDAlbany Faith B. Davis, MDAlbany Murat Yalcin, DVM, PhDTurkey Sandra Incerpi, PhDItaly Osnat Ashur-Fabian, PhDIsrael

28. Hypothyroid Median Survival: 10.1 mos Non-hypothyroid Median Survival: 3.1 mos Hercbergs AA et al, Anticancer Res, 2003

29. PBS A T4T4 T 4 + Tetrac B Summary of effects of T 4, T 4 -agarose and tetrac on angiogenesis Treatment Angiogenesis Index PBS67  9 T 4 ( 0.1 nM ) 156  16** Tetrac ( 0.1  M )76  9 T 4 + tetrac66  6 T 4 -agarose (total, 0.1  M) 194  28** T 4 -agarose + tetrac74  7 C PBS T 4 -agT 4 -ag + Tetrac Angiogenesis in the CAM

30. Normalized Gene Expression Angiopoietin-1Angiopoietin-2 Normalized Gene Expression 013 Tetrac (µM) VEGF (50 ng/ml) Tetrac (µM) VEGF (50 ng/ml) A B Ang-2 primes vascular endothelium for action of vascular growth factors HDMEC cells

31. CV-1, monkey kidney fibroblast 293T, human kidney epithelial cell

32. Treatment Tumor weight (gm) PBS (Control) 0.123 * Nanotetrac (1mg/kg)0.066 Nanotetrac (1mg/kg) (microfluidizer) 0.073 *

33. Control (PBS)Tetrac (0.1 mg/kg) Nanotetrac (0.1 mg/kg) MPanc96-luc cells injected March 7 th, 2012 Intra tumor treatment (once a week) started: March 12 th, 2012 IVIS: March 30 th, 2012

34. Effect of tetrac (10 mg/kg, i.p. daily) or tetrac PLGA nano (1.0 mg/kg, i.p. daily) on human pancreatic cancer xenografts

35. Integrins are 24 heterodimeric structural proteins of the plasma membrane that are critical to cell- cell and cell-ECM matrix protein interactions. Integrin  v  3 binds protein molecules—vitronectin, fibronectin, osteopontin—and recently has been appreciated to bind small molecules. The latter include thyroid hormone and its analogues, resveratrol and dihydrotestosterone.

36. U87MG (GBM) cells Blot: anti-pERK1/2 Nucleus Blot: anti-PCNA Blot: anti-pTyrp85-PI 3-K Cytosol T 4 (M) _ 10 -9 10 -8 10 -7 10 -6 - pERK1 - pERK2 - PCNA - pTyr-p85 48 kDa - 37 kDa - 84 kDa - T 3 (10 -7 M) Relative I.O.D. -PI 3-K T 3 (M) _ 10 -9 10 -8 10 -7 10 -6 Blot: anti-pTyrp85-PI 3-K Cytosol 10 -10 Blot: anti-p85-PI 3-K - p85-PI 3-K Blot: anti-pERK1/2 Nucleus Blot: anti-PCNA - pERK1 - pERK2 - PCNA 84 kDa - 48 kDa - 37 kDa - Relative I.O.D. - pTyr-p85 -PI 3-K T3T3 T4T4 Fig. 1 In vitro stimulation of cell proliferation (PCNA), activation of ERKs, PI3K by thyroid hormone analogues

37. Index of double-stranded DNA breaks Tetrac-induced Radiosensitization