ANTI-APOPTOTIC AND PROANGIOGENIC PROPERTIES OF ENDOGENOUS THYROID HORMONE

ANTI-APOPTOTIC AND PROANGIOGENIC PROPERTIES OF ENDOGENOUS THYROID HORMONE AND ITS ACTION ON P-GLYCOPROTEIN MAY BLUNT RESPONSE TO CONVENTIONAL CHEMOTHERAPY Paul J. Davis, MD Albany Medical College and Pharmaceutical Research Institute, Albany College of Pharmacy and Health Sciences, Albany, NY USA

MECHANISMS OF THYROID HORMONE ACTION • Thyroid hormone (L-thyroxine, T 4; 3, 5, 3’-triiodo-L-thyronine, T 3) acts via genomic and nongenomic mechanisms. The genomic pathway depends primarily upon formation of intranuclear complexes of T 3 with the nuclear thyroid hormone receptor proteins (TRs). • The nongenomic pathways include actions initiated at a cell surface receptor for T 4 and T 3 on the extracellular domain of integrin avb 3.

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

Nongenomic Actions of Thyroid Hormone Initiated at the Cell Surface T 4 T 3 Extracellular matrix proteins, e. g. , laminin, vitronectin Integrin V PLC PKC ne a br em a m as T 4 T 3 m TR∆ 1 TRb 1, ER STAT 1 TR TRb 1 p 53 PHORYL E PHOS PR OT NCo. R TRb 1 Amino acid transporter (ERK 1/ERK 2) STAT 1 , p 53 PI 3 -KAkt/PKB EIN TR TRb 1 ER Na+/H+ antiporter MAPK ATION SERIN AF FI CK ING TRb 1 ER STAT 1 Trip 230 r. T 3 SIGNAL TRANSDUCTION MAPK (ERK 1/ERK 2) Pl p 53 β 3 p 300 Nu cle us Gene ption Transcri Cytoplasm m. RNA Protein m. RNA is Synthes T 3 Protein trafficking Serine phosphorylation of nuclear proteins Specific gene transcription Activity of ion transporters T 3

Integrin avb 3 is primarily expressed by dividing cells and thus concentrated and activated in cancer cells and rapidly-dividing endothelial cells that serve cancers. Usually viewed as a sensing mechanism for extracellular matrix (ECM) proteins and critical to cellmatrix and cell-cell interactions that underlie tissue organization, avb 3 has recently been appreciated to bind small molecules and to contain the cell surface receptor for thyroid hormone.

As a prototypic small molecule ligand of integrin avb 3, thyroid hormone importantly alters transcription of differentiallyregulable genes important to cancer cell function and angiogenesis— and a) regulates integrin vascular growth factor receptor function, b) alters the state of the actin cytoskeleton, c) changes intracellular protein trafficking and d) controls the function of plasma membrane NHE 1 and Na, KATPase.

Thyroid hormone and cancer cell proliferation

Thyroid hormone and breast cancer cell proliferation MCF-7 cells: 3 H-thymidine uptake ** 3 H-thymidine - Fold Increase in Incorporation Control ** Time (h) 1 2 4 6 24 ICI (3 n. M) _ _ _ - Fold Increase in I. O. D. 37 k. Da- T 4 -A PD _ _ _ _ _ + + + ICI = ICI 182, 780, fulvestrant MCF-7 cells: PCNA T 4 24

OVCAR-3 cells IP: anti-integrin αv Blot: anti-NCo. R - NCo. R Blot: anti-SMRT - SMRT 180 k. Da - Blot: anti-p 300 * ** * Blot: anti-p. STAT 1 - p. STAT 1 82 k. Da - Blot: anti-p. ERK 1/2 - p. ERK 1 - p. ERK 2 37 k. Da - 16 * 14 NCo. R SMRT P 300 * p. STAT 1 ** ** * * p. ERK 1/2 * 12 * 8 6 | T 4 (10 -7 M) * * 10 - Lamin B 61 k. Da - - Relative I. O. D. - p 300 180 k. Da - | C _ + si. RNA _ + | sc. RNA _ | + 4| 2 T 4 (10 -7 M) 0 Fig. 3 | C _ + | si. RNA _ + sc. RNA _ + |

A B Blot: anti-PCNA NCI-H 522 cells 37 k. Da - - PCNA - Lamin-B 61 k. Da - _ T 4 (M) 61 k. Da - T 3 (M) 10 -9 10 -8 10 -7 10 -6 C - Lamin-B _ 10 -9 10 -8 10 -7 10 -6 D Thymidine Incorporation 30 NCI-H 522 cells NCI-H 510 A cells - Integrin β * * 20 * * 15 * * * 10 * * - GAPDH OV 5 - Integrin α CA NC R- 3 INC H 52 I-5 2 10 A CPM x 104 25 0 T 4 (M) T 3 (M) _ _ 10 -8 _ 10 -7 10 -6 _ _ _ 10 -9 _ _ 10 -8 10 -7 RT-PCR H 510 A, small cell lung carcinoma cells H 522, NSCLC cells

A NCI-H 510 A cells IP: anti-ER-α Blot: anti-phospho. ER-a (118) - p. ER-a 62 k. Da - Blot: anti-ER-α - ER-a 62 k. Da - Blot: anti-p. ERK 1/2 37 k. Da - - p. ERK 1 - p. ERK 2 Blot: anti-PCNA - Lamin-B 61 k. Da - 6 * * -Relative I. O. D. 5 4 -a p. ERK 1/2 PCNA * * 3 ** * * 2 ++ ++ + 1 * * 0 ICI (n. M) T 3 (10 -7 M) T 4 (10 -7 M) B _ 20 _ _ _ _ + + 20 _ + _ 2 20 + + + _ _ _ ICI = ICI 182, 780, Thymidine Incorporation 20 * * CPM X 104 10 + + 5 T 3 (10 -7 M) (10 -7 * * 15 ICI (n. M) T 4 2 M) _ _ _ 0 20 _ _ + 20 _ + _ 20 + + _ _

U 87 MG (GBM) cells T 3 T 4 Nucleus Blot: anti-p. ERK 1/2 48 k. Da - - p. ERK 1 - p. ERK 2 37 k. Da - Blot: anti-PCNA 37 k. Da - Nucleus Blot: anti-p. ERK 1/2 48 k. Da - - p. ERK 1 - p. ERK 2 37 k. Da - Blot: anti-PCNA - PCNA 37 k. Da - - PCNA - p. Tyr-p 85 -PI 3 -K 84 k. Da - Blot: anti-p 85 -PI 3 -K Relative I. O. D. Cytosol Blot: anti-p. Tyrp 85 -PI 3 -K - p 85 -PI 3 -K Relative I. O. D. 84 k. Da - Cytosol Blot: anti-p. Tyrp 85 -PI 3 -K - p. Tyr-p 85 -PI 3 -K 84 k. Da - T 4 (M) _ 10 -9 10 -8 10 -7 10 -6 T 3 (10 -7 M) T 3 (M) _ 10 -10 10 -9 10 -8 10 -7 10 -6 In vitro stimulation of cell proliferation (PCNA), activation of ERKs, PI 3 K by thyroid hormone analogues Fig. 1

Hypothyroid Median Survival: 10. 1 mos Non-hypothyroid Median Survival: 3. 1 mos Hercbergs AA et al, Anticancer Res, 2003

Spontaneous or medically-induced hypothyroidism has been shown to favorably affect the clinical courses of GBM (Cleveland Clinic), breast cancer (MD Anderson Cancer Center), renal cell carcinoma (TKI therapy at multiple centers) an head -and neck cancers (Cleveland Clinic).

Thyroid hormone has anti-apoptotic activity in cancer cells

RV = resveratrol

I - I 3’ HO 3 CH 2 -CH-COOH O 5’ NH 2 5 I - Thyroxine (T 4) I - I 3’ 3 CH 2 -CH-COOH O HO 5’ NH 2 5 I - 3, 5, 3’-Triiodothyronine (T 3) I - HO I 3’ 3 CH 2 --COOH O 5’ I - 5 I - Tetrac Low-grade thyromimetic within cells TH antagonist at integrin avb 3 TH receptor

Thyroid Hormone Resveratrol Tetrac α vβ 3 PD 98059 p. ERK 1/2 activation Plasma me mbrane PD 98059 Cell Proliferation p. ERK 1/2 activation X Cytosol p. Ser 15 -p 53 NS-398 [COX-2]-p. ERK 1/2 complexing Apoptosis Nucleus Thyroid hormone inhibits induction by RV of Ser-15 phosphorylation of p 53

Thyroid hormone is pro-angiogenic by a variety of mechanisms. This is relevant to support by the hormone of cancer-related vascularization and to vascularity of nonmalignant conditions, such as skin diseases.

Angiogenesis in the CAM A B C PBS T 4 + Tetrac PBS T 4 -ag + Tetrac Summary of effects of T 4, T 4 -agarose and tetrac on angiogenesis Treatment Angiogenesis Index PBS 67 9 T 4 (0. 1 n. M) 156 16** Tetrac (0. 1 M) 76 9 T 4 + tetrac 66 6 T 4 -agarose (total, 0. 1 M) 194 28** T 4 -agarose + tetrac 74 7

A PBS (Control) T 4 (0. 1 µM) T 4 -agarose (0. 1 µM) DITPA (0. 01 µM) VEGF (2. 0 µg/ml) b-FGF (1. 0 µg/ml) B Treatment PBS (Control) Mean Vessel Branch Points ± SD 87 ± 9 T 4 (0. 1 μM) 148 ± 7* T 4 -agarose (0. 1 μM) 167 ± 8* DITPA (0. 01 μM) 134 ± 11* DITPA (0. 1 μM) 170 ± 9* VEGF (2. 0 μg/ml) 168 ± 10* b-FGF (1. 0 μg/ml) 174 ± 8* Data represent mean ± SD, n=8; *p<0. 01, indicating significant stimulation of angiogenesis. PBS, phosphate-buffered saline.

Inhibitory Effect of vb 3 MAB (LM 609) on T 4 stimulated Angiogenesis in the CAM Model PBS T 4+ LM 609(10 mg) T 4 (total, 0. 1 m. M)

By a variety of mechanisms, thyroid hormone, specifically T 4, has been shown to influence the activity and abundance of P-glycoprotein (P-gp; MDR 1; ABCB 1), a plasma membrane efflux pump that serves to shorten intracellular retention time of traditional chemotherapeutic agents that are ligands for the protein (doxorubicin, etoposide, paclitaxel, etc. ). Thus, thyroid hormone may support chemoresistance.

Na+, K+ ATPase Na+/H+ antiporter T 4 p. He K+ Chemotherapeutic agent efflux EGFR T 4 Na+ αv β 3 Integrin αv β 3 n Integri + [Na+]i Ca 2+ + Calmodulin leus Nuc MDR 1 transcription EGFR transcription EGF Plasma Membrane Cytoplasm H+ Na Results of T 4 action P-glycoprotein p. Hi Increased EGF input to P-gp Bold blue arrows indicate stimulatory action of tetrac/Nanotetrac on Na+, K+ - ATPase, Na+/H+ antiporter and EGF receptor

SUMMARY There are multiple implications of all of these functions of thyroid hormone recently recognized to occur on cancer cells and on angiogenesis. • Normal thyroid function may support tumor cell proliferation and limit effectiveness of chemotherapy. • Induced or spontaneous hypothyroidism may impede cancer cell function.

SUMMARY 2 • It is desirable to have a specific pharmacologic antagonist of thyroid hormone actions at integrin avb 3 (= tetrac, Nanotetrac). • The affinity of the thyroid hormone receptor on avb 3 is higher for T 4 than T 3; at physiological concentrations, free T 4 supports tumor cell proliferation and cancer cell survival pathway gene transcription. • Nonmalignant, hypervascular skin disorders, such rosacea, may be thyroid hormone-supported.

COLLABORATORS Shaker A. Mousa, Ph. D Hung-Yun Lin, Ph. D Heng-Yuan Tang, MA Thangirala Sudha, Ph. D Faith B. Davis, MD Murat Yalcin, DVM, Ph. D Sandra Incerpi, Ph. D Osnat Ashur-Fabian, Ph. D Albany Albany Turkey Italy Israel

A Effect of tetrac on tube formation by human dermal microvascular endothelial cells (HDMEC). In this 3 -dimensional human microvascular endothelial cell sprouting assay, cells were mixed with gelatin-coated Cytodex-3 beads and the mixture suspended in endothelial basal medium (EBM) with 15% normal human serum, mixed and cultured overnight in a CO 2 incubator. The “EC-beads” were then placed in a fibrinogen solution and thrombin added. After polymerization of the fibrin, EBM and 20% human serum were added and samples incubated for 24 -48 h. B VEGF Tetrac + _ Int Angiol, 2006 + + 1. 0 2. 5

A ER-a gene expression - ER-a - GAPDH 5 -H I NC B A 10 22 5 I-H C Relative I. O. D. ER-a gene expression 6 ER-α 4 2 0 NCI-H 510 A NCI-H 522 N NCI-H 522 cells C IP: anti-integrin-αv Green: phospho. ER-a Red: Nucleoprotein - ER-α Promoter - Input Control T₄ Control _ + Ig. G NCI-H-522 cells Tetrac + T 4 Relative Bound DNA L-T 4 2. 4 * * 1. 9 + + 1. 4 0. 9 0. 4 Con ICI T₄ ICI + T₄

A in nucleosome content -Fold Increase Nucleosome ELISA _ _ T 4 (10 -7 M) RV ( M) _ + 1 _ 10 1 10 + _ + B -Fold Increase in nucleosome content Nucleosome ELISA Tetrac (10 -7 M) _ RV (10 _ M) T 4 (10 -7 M) _ + _ _ + + _ + _ + + _ _ + + +

Effects of L-T 4 or T 4 Nanoparticles (NP) on Endothelial Cell Migration toward Vitronectin (VN) 20 15 10 5 Chamber EC Migration (RFU) x 103 25 Upper Lower 0 0 Boyden apparatus 0 VN T 4 -NP VN