ANTI-APOPTOTIC AND PRO- ANGIOGENIC 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 T3 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  v  3.

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

Integrin STAT1  TR  1, ER PLC PKC TR  1 ER  T4T4 TR  1 p300 NCoR mRNA Trip230 MAPK (ERK1/ERK2) Plasma membrane Cytoplasm Nucleus Gene Transcription T3T3 TR  STAT1 , p53 T4T4 TR ∆  1 rT 3 TR  1 PI 3-K- Akt/PKB VV β3β3 T3T3 mRNA Extracellular matrix proteins, e.g., laminin, vitronectin p53 SIGNAL TRANSDUCTION Protein Synthesis SERINE PHOSPHORYLATION TR  1 ER STAT1    p53 PROTEIN TRAFFICKING T3T3 T3T3 T3T3 MAPK (ERK1/ERK2) Na+/H+ antiporter Amino acid transporter Protein trafficking Serine phosphorylation of nuclear proteins Specific gene transcription Activity of ion transporters Nongenomic Actions of Thyroid Hormone Initiated at the Cell Surface

Integrin  v  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 cell- matrix and cell-cell interactions that underlie tissue organization,  v  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  v  3, thyroid hormone importantly alters transcription of differentially- regulable 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 NHE1 and Na,K-ATPase.

Thyroid hormone and cancer cell proliferation

Time (h) _ ____ + ICI (3 nM) MCF-7 cells: 3 H-thymidine uptake - Fold Increase in 3 H-thymidine Incorporation Control * * * * 37 kDa- MCF-7 cells: PCNA PD ++ __ + T4T4 ++ _ __ _ T 4 -A + + _ _ _ _ _ - Fold Increase in I.O.D. Thyroid hormone and breast cancer cell proliferation ICI = ICI 182,780, fulvestrant

T 4 (10 -7 M) _ + _ + _ + - NCoR IP: anti-integrin αv Blot: anti-NCoR Blot: anti-pSTAT1 - pSTAT1 Blot: anti-SMRT Blot: anti-p300 Blot: anti-pERK1/2 CsiRNAscRNA |||| - SMRT - pERK1 - pERK2 - p300 - Lamin B T 4 (10 -7 M) _ + _ + _ + CsiRNAscRNA |||| Fig kDa - 37 kDa - 82 kDa kDa - - Relative I.O.D. * * * * * * * * * * * * * * * * OVCAR-3 cells

A NCI-H522 cells Blot: anti-PCNA - PCNA 37 kDa - T 3 (M) _ B Thymidine Incorporation C T 4 (M) T 3 (M) __ ____ CPM x 10 4 __ Blot: anti-PCNA - PCNA - Lamin-B 37 kDa - 61 kDa - T 4 (M) _ NCI-H522 cells - Lamin-B 61 kDa - * * * * * * * * * * * * * - GAPDH - Integrin α - Integrin β OVCAR- 3 NCI-H522 NCI-510A D H510A, small cell lung carcinoma cells H522, NSCLC cells RT-PCR

NCI-H510A cells ICI (nM) T 3 (10 -7 M) _ _ T 4 (10 -7 M) _ __ ____ _ ++ ___ Blot: anti-PCNA - PCNA 37 kDa - - pERK1 - pERK2 Blot: anti-pERK1/2 Blot: anti-phosphoER-  (118) 62 kDa - IP: anti-ER- α Blot: anti-ER- α - pER-  62 kDa - Thymidine Incorporation - ER-  ICI (nM) T 3 (10 -7 M) _ _ T 4 (10 -7 M) _ __ ___ _ + __ B A CPM X 10 4  -Relative I.O.D. 61 kDa - - Lamin-B * * * + + * * + * * * * * * * * * ICI = ICI 182,780,

U87MG (GBM) cells Blot: anti-pERK1/2 Nucleus Blot: anti-PCNA Blot: anti-pTyrp85-PI 3-K Cytosol T 4 (M) _ 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) _ Blot: anti-pTyrp85-PI 3-K Cytosol 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

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

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 Low-grade thyromimetic within cells TH antagonist at integrin  v  3 TH receptor

Resveratrol αvβ3αvβ3 pERK1/2 activation PD98059 Cytosol Nucleus pSer15-p53 NS-398 Apoptosis αvβ3αvβ3 Thyroid Hormone pERK1/2 activation PD98059 X Cell Proliferation [COX-2]-pERK1/2 complexing Plasma membrane Tetrac Thyroid hormone inhibits induction by RV of Ser-15 phosphorylation of p53

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.

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

Data represent mean ± SD, n=8; *p<0.01, indicating significant stimulation of angiogenesis. PBS, phosphate-buffered saline. A B b-FGF (1.0 µg/ml) PBS (Control) DITPA (0.01 µM)VEGF (2.0 µg/ml) T 4 (0.1 µM)T 4 -agarose (0.1 µM) Mean Vessel Branch Treatment Points ± SD PBS (Control) 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*

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

By a variety of mechanisms, thyroid hormone, specifically T 4, has been shown to influence the activity and abundance of P-glycoprotein (P-gp; MDR1; ABCB1), 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.

MDR1 EGFR transcription Nucleus Plasma Membrane Cytoplasm Na +, K + - ATPase Na + /H + antiporter P-glycoproteinEGFR Chemotherapeutic agent efflux EGF Integrin αvαv β3β3 αvαv β3β3 αvαv β3β3 K+K+ Na + H+H+ Results of T 4 action Increased EGF input to P-gp [Na + ] i Ca 2+ pH i pH e + Calmodulin T4T4 Bold blue arrows indicate stimulatory action of tetrac/Nanotetrac on Na +, K + - ATPase, Na + /H + antiporter and EGF receptor T4T4

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  v  3 (= tetrac, Nanotetrac). The affinity of the thyroid hormone receptor on  v  3 is higher for T 4 than T 3; at physiological concen- trations, free T 4 supports tumor cell proliferation and cancer cell sur- vival pathway gene transcription. Nonmalignant, hypervascular skin disorders, such rosacea, may be thyroid hormone-supported.

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

VEGF Tetrac _ A B 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 h. Int Angiol, 2006

Green: phosphoER-  Red: Nucleoprotein Control NCI-H522 cells Tetrac + T 4 L-T 4 A B NCI-H510A NCI-H522 - ER-  - GAPDH ER-  gene expression Relative I.O.D. C IP: anti-integrin- α v T₄T₄ IgG - ER- α  Promoter _ + - Input Control * * + +

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

EC Migration (RFU) x 10 3 Effects of L-T 4 or T 4 Nanoparticles (NP) on Endothelial Cell Migration toward Vitronectin (VN) Chamber Upper Lower T4T4 T 4 -NP VN Boyden apparatus