24/10/2015 Pituitary signalling : from Zebrafish to Clinical - PowerPoint PPT Presentation

24/10/2015 Pituitary signalling : from Zebrafish to Clinical Therapy Hypothalamic releasing and inhibiting hormones Superior hypophysial artery Portal vein S.Melmed Inferior October 2015 hypophysial UCSF CME artery Hypophysial vein Li-Ng JCEM 2008 Melmed NEJM 2006 Pituitary Tumorigenesis Pituitary compression Parasellar compression Hormone hypersecretion Oncogene activation Mutations Hypogonadism Visual field disturbance GH Acromegaly Mutations Tumor suppressor inactivation Stromal and Unknown changes Thyroid failure Headache ACTH Cushing Cell cycle dysregulation epigenetic events Adrenal failure Cranial nerve palsy PRL Prolactinoma Parasellar invasion FSH/LH Nonsecreting TSH TSHoma Mixed Co-morbidities Shrink Surgery Aggressive Replace Normal pituitary Microadenoma Macroadenoma Mortality or Medical Rx deficient ablate Radiation hormone Humoral factors Hyperplasia Proliferative restraint Senescence mass Melmed JCI 2003 Melmed Nat Rev Endocrinol 2011 1

24/10/2015 Pituitary Tumor Signalling Pathways Receptor subtype expression Number of (%) tumors SSTR1 SSTR2 SSTR3 SSTR4 SSTR5 44 GH 61 88 4 78 111 Ben-Shlomo, Trends Endocr Metab , 2010 Proliferative Mitogenic hormones and growth factors constraints GHRHR, CHRHR, GNRHR p15, p16 CDK4 Cyclin D p18, p19 CDK6 P GH( µ g/L ) CDK P P SSTR Before After inhibitors p21, p27 Cyclin E 80 CDK2 50 µ g Octrotide or placebo p57 P P P 70 Senescence M M M 60 P P P P p53 Rb 50 P P Control day 40 G 1 E2Fs CDK2 30 Chromosomal Hormone S Cyclin A G 0 instability 20 Octreotide G 2 DNA damage CDK1 M 10 Cyclin B 0 8am 10 12 2pm 4 6 8 10 12pm 2 4 6am Cell Cell cycle proliferation disruptors Melmed, JCI , 2009 Lamberts, JCEM , 1985 Melmed Nat Rev Endocrin 2011 Cell Origin and Receptor Profile Acidophil Stem Cell Adenoma Θ sstr2 Θ D2 M 1 2 3 4 5 6 Gigantism Tumor receptor expression • 15-year-old female, height 179 cm Acidophil • Headaches, diplopia, sleepy Stem cell • Shoe size 11, tight rings 1.0 mg 0.75 • Primary amenorrhea, no 0.5 Cabergoline galactorrhea 30 mg Surgery SRL • Jaw prognathism, incisor gap 100 GH 50 10 Acidophil mg/L Stem cell Clonal expansion IGF-1 1200 400 GH: Baseline 64 8000 PRL Lactotroph Somatotroph Mammosomatotroph 2-h OGTT 71 ng/mL 4000 IGF-1: 845 (<660) 1000 500 PRL: 8700 (<23) 0 PRL GH GH PRL 15.5 16 16.5 17 17.5 18 23 α subunit: 3.7 (<1.2) GH PRL α subunit Age (years) Maheshwari JCEM 2000 2

24/10/2015 STAT3 Activates GH Promoter STAT3 Inhibitor Suppresses GH Potential STAT binding site Rat Gh promoter -2500 -2000 -1500 -1000 -500 +1 -2040/-1806 -1118/-663 -119/+165 Primer 1 Primer 2 Primer 3 STAT3 inhibitor ( µ M) ChIP 0 50 75 100 125 GH PRL STAT3 0.8 3.5 Input * * ng/ml/10 3 GH3 cells ng/ml/10 3 GH3 cells 0.6 3 2.5 IgG STAT3 P-STAT3 2 * -fold 0.4 * * 1.5 1 GH 0.2 0 Primer 1 Primer 2 Primer 3 0.5 S31-201 0 0 PRL 40 4 control 50 75 100 125 ( µ M) control 50 75 100 125 ( µ M) * ZsGreen * STAT3 inhibitor STAT3 inhibitor 0 STAT3 50 30 3 100 β -actin * 150 µ M luc luc activity activity 20 2 * 10 1 0 0 Zhou JCI 2015 Zhou JCI 2015 pGL4.10 -4192/+167 -1752/+167 -4192/+167 -1752/+167 STAT3 Inhibitor Suppresses hGH STAT3 Inhibitor Suppresses GH-tumor Secretion in vitro Proliferation and GH in vivo Tumors RT-PCR GH mRNA 1.2 WST-1 N=17 N=4 Control 1.2 1.0 1.5 P<0.0001 2.0 S3I-201 0.8 0.8 1.5 * -fold * 0.6 GH mRNA 1.0 GH mRNA * 1.0 0.4 0.4 0.5 Volume Weight Stat3 0.2 0.5 ** ** 0 140 900 * 0 1.2 S3I-201 ( µ M) treated 0 0 50 100 150 0 50 100 150 700 * control 100 * BrdU 1.0 Western ** 120 mm 3 * 500 * Western mg -fold * * 60 Staining (%) 0.6 N=15 N=6 300 80 * P-STAT3 * 2.0 2.0 P<0.0001 100 20 0.2 STAT3 40 1.5 1.5 0 2 4 6 8 10 12 14 Control Rx Control Rx Days GH GH 0 1.0 1.0 GH 0 50 100 S3I-201 ( µ M) 0.5 0.5 0 50 100 150 50 100 150 0 S3I-201 (mM) 0 0 50 100 150 0 50 100 150 Serum GH Serum IGF-1 GH RIA 1.2 6.0 3.0 N=13 N=8 1.0 ** 0.8 4.0 2.0 2.0 1.5 P<0.0001 -fold -fold 1.0 0.6 *** ** *** 0.8 1.0 0.4 2.0 1.0 GH GH 0.6 *p<0.05; **p<0.01; ***p<0.001 0.2 0.4 0.5 0 0 0 0.2 0 50 100 150 Control Rx Control Rx 0 0 S3I-201 (mM) 0 50 100 150 0 50 100 150 STAT3 inhibitor (mM) STAT3 inhibitor (mM) Zhou JCI 2015 Zhou JCI 2015 3

24/10/2015 Lapatinib Decreases HER2CA Tumor Size Targeted Prolactinoma Therapy and Prolactin Levels PRL 350 HER2-GH 3 Vehicle 300 � � � � � � � � � � � � � � � � � � � � � � � � � PRL (ng/mL) 250 Gefitinib 200 * Lapatinib 150 ** 100 50 Vlotides Cancer Res 2009 0 –5 2 7 Tumor volume Time (days) 1000 140 Vehicle PRL mRNA Tumor volume (mm 3 ) HRG EGF 800 120 Gefitinib * >1500 100 1000 600 PRL mRNA (%) Lapatinib 500 80 (ng/mL) JM JM JM JM Y701 Y701 PRL ** Y701 Y701 400 100 60 TK TK TK 50 * Y845 Y845 Y920 Y920 Y891 Y845 Y845 Y920 40 Y891 Y920 Y891 Y891 200 Lactation Y1148 Y1148 * Y1148 Y1173 Y992 Y992 Y1173 Y1148 Y1173 Y992 Y992 Y1173 Impotence Normal potency Y1045 Y1045 Y1068 Y1068 Y1086 Y1045 Y1045 Y1086 Y1068 Y1068 20 Y1086 Y1086 0 –8 –4 0 4 8 12 14 0 1 4 6 10 Bromocriptine Time (months) vehicle gefitinib lapatinib EGFR HER2 HER3 Vehicle Gefitinib Lapatinib Time (days) * P <0.05, ** P <0.01 vs vehicle Fukuoka Mol Endocrinol 2011 Lapatinib Suppresses Both Prolactin mRNA Lapatinib Decreases Tumor Size and Prolactin Levels in Estrogen-induced Prolactinoma and Secretion in Human Prolactinomas Pituitary weight 17 β -estradiol H&E PRL EGFR HER2 250 200 Weight (mg) * 150 100 50 Fischer 344 rat 0 Vehicle Lapatinib PRL mRNA PRL secretion Vehicle Lapatinib 120 120 6000 Serum PRL 100 100 PRL secretion (%) PRL mRNA (%) 5000 80 80 Serum GH 4000 100 60 * 60 PRL (ng/mL) * ** 80 GH (ng/mL) ** 40 40 3000 ** 60 20 ** 20 2000 40 0 0 1000 0 0.1 1 10 10 0 0.1 1 10 10 20 (µM) (µM) 0 0 0 Vehicle Lapatinib Vehicle Lapatinib Lapatinib Gefitinib Lapatinib Gefitinib * P <0.05, ** P <0.01 vs vehicle * P <0.05 H&E, hematoxylin and eosin Fukuoka Mol Endocrinol 2011 Fukuoka Mol Endocrinol 2011 4

24/10/2015 Aggressive Prolactinoma: Cushing Disease Lapatanib 1250 mg/day 11988 mm 3 350 300 250 PRL (ng/mL) 200 Harvey Cushing, MD 150 998 mm 3 100 937 mm 3 50 0 1 2 3 4 5 6 7 Time (months) Minnie G.– 1910 Rx 6 months Pre-Rx Cushing Bull Johns Hopkins Hosp. 1932 Cooper Endocr 2013 Stewart Best Pract Res Clin Endocrinol Metab . 2009 Challenges in Diagnosis and Treatment Cushing Disease of Cushing Disease Imaging • Average tumor size ~6 mm • ~40% tumors not visible • 10% of normal population micro- Median survival 4.6 years if adenoma inadequately controlled Life expectancy no different than in 1930 Hormone hypersecretion Central mass effects Therapy � � ACTH → � cortisol � Pituitary hormone deficiencies • Surgery: 60–70% initial remission Physical and biochemical diagnosis • Obesity � Visual field disturbance 20–60% recurrence • Hypertension • Signs , symptoms and � Headache • Pituitary radiation: Hypopituitarism • Diabetes hypercortisolemia overlap with other � Parasellar invasion • Adrenalectomy or cortisol synthesis • Osteoporosis illnesses Surgery inhibitor: Side effects and morbidity • Hirsutism • Cyclic or intermittent hypercortisolemia Medical Rx • Muscle weakness Radiation • Moon face • Acne Melmed JCI 2003 5