Paul J. Davis, MD. Albany Medical College and Pharmaceutical Research Institute, Albany College of Pharmacy and Health Sciences, Albany, NY USA

Transcription

1 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

2 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 avb3.

3 HO 3 O Thyroxine (T 4 ) NH 2 CH 2 -CH-COOH 3 O NH 2 CH 2 -CH-COOH 3,5,3 -Triiodothyronine (T 3 )

4 Nongenomic Actions of Thyroid Hormone Initiated at the Cell Surface Trip230 Extracellular matrix proteins, e.g., laminin, vitronectin TRb1 ER a STAT1a p53 MAPK (ERK1/ERK2) PLC PKC STAT1a TRb1, ER a NCoR TRb1 ERa TRb1 T 4 av p300 T 3 Integrin β3 SIGNAL TRANSDUCTION p53 STAT1a, p53 rt 3 TR a1 TRa TRb1 T 4 MAPK (ERK1/ERK2) T 3 T 3 PI 3-K- Akt/PKB Na/H antiporter Amino acid transporter Cytoplasm mrna mrna T 3 T 3 Protein trafficking Serine phosphorylation of nuclear proteins Specific gene transcription Activity of ion transporters

5 Integrin avb3 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, avb3 has recently been appreciated to bind small molecules and to contain the cell surface receptor for thyroid hormone.

6 As a prototypic small molecule ligand of integrin avb3, 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 NHE1 and Na,K-ATPase.

7 Thyroid hormone and cancer cell proliferation

8 3 H-thymidine Incorporation - Fold Increase in I.O.D. - Fold Increase in Thyroid hormone and breast cancer cell proliferation MCF-7 cells: 3 H-thymidine uptake Control M E M T 4 Time (h) ICI (3 nm) 37 kda MCF-7 cells: PCNA ICI = ICI 182,780, fulvestrant T 4 T 4 -A PD

9 - Relative I.O.D. OVCAR-3 cells IP: anti-integrin αv Blot: anti-ncor - NCoR 180 kda kda - 82 kda - 37 kda - 61 kda - T 4 (10-7 M) Blot: anti-smrt Blot: anti-p300 Blot: anti-pstat1 Blot: anti-perk1/2 C sirna scrna - SMRT - p300 - pstat1 - perk1 - perk2 - Lamin B T 4 (10-7 M) C sirna scrna NCoR SMRT P300 pstat1 perk1/2 Fig. 3

10 A Blot: anti-pcna NCI-H522 cells B Blot: anti-pcna NCI-H522 cells 37 kda - - PCNA 37 kda - - PCNA 61 kda - - Lamin-B 61 kda - - Lamin-B T 4 (M) T 3 (M) C D Thymidine Incorporation CPM x NCI-H522 cells NCI-H510A cells ntegrin β ntegrin α - GAPDH 0 T 4 (M) T 3 (M) RT-PCR H510A, small cell lung carcinoma cells H522, NSCLC cells

11 -Relative I.O.D. A 62 kda - 62 kda - 37 kda - 37 kda - NCI-H510A cells IP: anti-er-α Blot: anti-phosphoer-a (118) Blot: anti-er-α Blot: anti-perk1/2 Blot: anti-pcna - per-a - ER-a - perk1 - perk2 - PCNA 61 kda - - Lamin-B a per perk1/2 PCNA 0 ICI (nm) T 3 (10-7 M) T 4 (10-7 M) B CPM X Thymidine Incorporation ICI = ICI 182,780, 0 ICI (nm) T 3 (10-7 M) T 4 (10-7 M)

12 Relative I.O.D. Relative I.O.D. U87MG (GBM) cells 48 kda - 37 kda - 37 kda - 84 kda - T 3 Nucleus Blot: anti-perk1/2 Blot: anti-pcna Cytosol Blot: anti-ptyrp85-pi 3-K Blot: anti-p85-pi 3-K - perk1 - perk2 - PCNA - ptyr-p85 -PI 3-K 48 kda - 37 kda - 37 kda - Nucleus Blot: anti-perk1/ T 4 Blot: anti-pcna perk1/2 PCNA - perk1 - perk2 - PCNA 84 kda perk1/2 PCNA ptyr-p85-pi 3-K p85-pi 3-K - p85-pi 3-K 84 kda - Cytosol Blot: anti-ptyrp85-pi 3-K - ptyr-p85 -PI 3-K 2 T 4 (M) T 3 (10-7 M) T 3 (M) In vitro stimulation of cell proliferation (PCNA), activation of ERKs, PI3K by thyroid hormone analogues Fig. 1

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

14 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).

15 Thyroid hormone has anti-apoptotic activity in cancer cells

16 RV = resveratrol

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18 HO 3 O Thyroxine (T 4 ) NH 2 CH 2 -CH-COOH 3 O NH 2 CH 2 -CH-COOH 3,5,3 -Triiodothyronine (T 3 ) HO 3 O Tetrac CH 2 --COOH Low-grade thyromimetic within cells TH antagonist at integrin avb3 TH receptor

19 Thyroid Hormone Resveratrol Tetrac PD98059 PD98059 perk1/2 activation perk1/2 activation Cytosol X NS-398 pser15-p53 Cell Proliferation [COX-2]-pERK1/2 complexing Nucleus Apoptosis Thyroid hormone inhibits induction by RV of Ser-15 phosphorylation of p53

20 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.

21 A Angiogenesis in the CAM B PBS T 4 T 4 Tetrac C PBS T 4 -ag 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 nm) Tetrac (0.1 M) 76 9 T 4 tetrac 66 6 T 4 -agarose (total, 0.1 M) T 4 -agarose tetrac 74 7

22 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 Mean Vessel Branch 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 Data represent mean ± SD, n=8; p<0.01, indicating significant stimulation of angiogenesis. PBS, phosphate-buffered saline.

23 Inhibitory Effect of avb3 MAB (LM609) on T4- stimulated Angiogenesis in the CAM Model PBS T 4 (total, 0.1 M) CAM Treatment # of branch pts ± SEM % Inhibition ± SEM PBS 73 ± 8 T4(0.1uM) 170 ± 16 0 T4LM609(10ug) 109 ± 9 64 ± 9 T 4 LM609(10 g)

24 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.

25 Na, K - ATPase Na /H antiporter P-glycoprotein EGFR T 4 ph e Chemotherapeutic agent efflux T 4 EGF β3 K Na β3 Na H β3 Plasma Membrane Cytoplasm Results of T 4 action [Na ] i Ca 2 Calmodulin ph i Increased EGF input to P-gp MDR1 transcription EGFR transcription Bold blue arrows indicate stimulatory action of tetrac/nanotetrac on Na, K - ATPase, Na /H antiporter and EGF receptor

26 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.

27 SUMMARY 2 It is desirable to have a specific pharmacologic antagonist of thyroid hormone actions at integrin avb3 (= tetrac, Nanotetrac). The affinity of the thyroid hormone receptor on avb3 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.

28 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

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30 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 h. B VEGF Tetrac Int Angiol, 2006

31 Relative Bound DNA Relative I.O.D. A ER-a gene expression - ER-a - GAPDH ER-a gene expression ER-α 0 NCI-H510A NCI-H522 B NCI-H522 cells Green: phosphoer-a Red: Nucleoprotein C IP: anti-integrin-αv - ER-α Promoter nput Control Control T₄ IgG L-T NCI-H-522 cells Tetrac Tetrac T Con ICI T₄ ICI T₄

32 -Fold Increase in nucleosome content -Fold Increase in nucleosome content A Nucleosome ELISA BHP cells FTC 236 cells RV (μm) T 4 (10-7 M) B 15 Nucleosome ELISA BHP 2-7 cells 10 BHP cells 5 0 Tetrac (10-7 M) RV (10 μm) T 4 (10-7 M)

33 Chamber Effects of L-T 4 or T 4 Nanoparticles (NP) on Endothelial Cell Migration toward Vitronectin (VN) 25 EC Migration (RFU) x Upper Lower T 4 T 4 -NP VN VN VN Boyden apparatus