Nanotetrac: Targeting Thyroid Hormone Receptor for Anti-Cancer Action

1. NANOTETRAC TARGETS THE THYROID HORMONE RECEPTOR ON INTEGRIN avb3 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. X B Alberts text

3. The thyroid hormone receptor on the extracellular domain of avb3 binds L-thyroxine (T4), 3,5,3’-triiodo-L-thyronine (T3) and tetraiodothyroacetic acid (tetrac), a deaminated T4 analogue. Tetrac blocks binding of T4 and T3 to the integrin and has an array of anti-cancer and anti-angiogenic properties at avb3 that are independent of its inhibition of T4- and T3-binding to the integrin.

4. - - - - - - - - - - - I I I I I I I I I I I - - - - - - - - - - - NH2 3’ 3 HO CH2-CH-COOH O 5’ 5 Thyroxine (T4) NH2 3’ 3 CH2-CH-COOH HO O 5’ 5 3,5,3’-Triiodothyronine (T3) 3’ 3 HO CH2--COOH O 5’ 5 Tetrac

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 avb3.

6. O H H O PLGA nanoparticle I I O O O O I N N I H H I O N I O N H H O I O I I I H O N H O O N O I O H I 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 avb3 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. Cell Cycle, 2009 Fig. 3B Pro-apoptosis

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

11. Cell Cycle, 2009 Fig. 7

12. Effect of Tetrac and Tetrac Nanoparticles on Human Non-Small Cell Lung Carcinoma Xenograft Volume Left side 700 Control Tetrac 1 mg/kg (every 3rd day) 600 Tetrac Nano 1 mg/kg (every 3rd day) 500 Mean Tumor Volume (mm3) ± SEM 400 300 200 100 -10 -5 0 5 10 15 20 Days Right side 1000 Control Tetrac 1 mg/kg (every 3rd day) Tetrac Nano 1 mg/kg (every 3rd day) 800 600 Mean Tumor Volume (mm3) ± SEM 400 200 -10 -5 0 5 10 15 20 Days 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 7th, 2012 Subcutaneous daily treatment started: March 12th, 2012 IVIS: March 30th, 2012

14. 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. Inhibitory effect of avb3 MAB (LM609) on T4-stimulated angiogenesis in the CAM model T4 (total, 0.1mM) PBS T4+ LM609(10mg)

19. Table 1Inhibition of activities of pro-angiogenic factors in the CAM assay by Nanoterac (NT) (2 µg/CAM) Treatment PBS control Void PLGA nanoparticle T3 (6.5 ng/mL) T3 + NT T4 (100 nM) T4 (100 nM) + NT 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.

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

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

22. Effect of Tetrac on Cancer Cell Implants on Angiogenesis in the CAM Model Control + Tetrac (1 µM) Increased vascular proliferation Decreased vascular proliferation H1299 Bronchogenic carcinoma (1 x 106 cells/CAM) Actions of tetrac initiated nongenomically at avb3 culminate in a complex of genomic and nongenomic effects on proliferation.

23. Summary • Integrin avb3 contains a cell surface receptor for T4, T3. • Tetrac and nanoparticulate tetrac inhibit agonist hormone action at the receptor and are probes for contributions of the receptor. • In vitro, T4, T3 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.

24. Summary 2 • Acting via the cell surface receptor on avb3, 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 ERa.

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, PhD Albany Heng-Yuan Tang, MA Albany Thangirala Sudha, PhD Albany Faith B. Davis, MD Albany Murat Yalcin, DVM, PhD Turkey Sandra Incerpi, PhD Italy Osnat Ashur-Fabian, PhD Israel

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

29. PBS T4-ag T4-ag + Tetrac Angiogenesis in the CAM A PBS T4 T4 + Tetrac B C Summary of effects of T4, T4-agarose and tetrac on angiogenesis TreatmentAngiogenesis Index PBS 67  9 T4 (0.1 nM) 156  16** Tetrac (0.1 M) 76  9 T4 + tetrac 66  6 T4-agarose (total, 0.1 M) 194  28** T4-agarose + tetrac 74  7

30. HDMEC cells A B Angiopoietin-1 Angiopoietin-2 Normalized Gene Expression Normalized Gene Expression Tetrac (µM) Tetrac (µM) 0 1 3 VEGF VEGF (50 ng/ml) (50 ng/ml) Ang-2 primes vascular endothelium for action of vascular growth factors

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

32. *

33. Control (PBS) Tetrac (0.1 mg/kg) Nanotetrac (0.1 mg/kg) MPanc96-luc cells injected March 7th, 2012 Intra tumor treatment (once a week) started: March 12th, 2012 IVIS: March 30th, 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 avb3 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 T3 T4 Nucleus Blot: anti-pERK1/2 Nucleus 48 kDa - - pERK1 Blot: anti-pERK1/2 - pERK2 37 kDa - 48 kDa - - pERK1 - pERK2 Blot: anti-PCNA 37 kDa - Blot: anti-PCNA 37 kDa - - PCNA 37 kDa - - PCNA Cytosol Blot: anti-pTyrp85-PI 3-K - pTyr-p85 84 kDa - Relative I.O.D. -PI 3-K Blot: anti-p85-PI 3-K - p85-PI 3-K Cytosol 84 kDa - Blot: anti-pTyrp85-PI 3-K - pTyr-p85 84 kDa - -PI 3-K _ Relative I.O.D. T4 (M) 10-8 10-6 10-9 10-7 T3 (10-7 M) _ T3 (M) 10-8 10-6 10-10 10-9 10-7 In vitro stimulation of cell proliferation (PCNA), activation of ERKs, PI3K by thyroid hormone analogues Fig. 1

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