Základy molekulární onkologie

1. Základy molekulární onkologie Doc. MUDr. L. Boudová, Ph. D.

2. Tumorigeneze = onkogeneze = karcinogeneze = vznik a růst nádorů Nádor = abnormální populace se změněnou genetickou informací neletální poškození normální genetické informace • získané • Chemické • Fyzikální • Viry • vrozené (germline)

3. Základní hypotéza Tumour monoclonal proliferation Clonal expansion Clonality testing 1. women heterozyg. for X-linked markers 2. specific translocations 3. gene rearrangements – B, T- cells Carcinogenesis: multistep process

4. Gene expressionregulation in somatic body cells • Specific • Universal • Regulation of gene expression

5. Základní třídy regulačních genů dělení, růst, diferenciace, smrt buňky I. Protoonkogeny „podporují růst“, mutantní alely dominantní II. Nádorové supresorové geny (antionkogeny) normálně brzdí proliferaci, tzv. recesivní onkogeny III. Geny regulující apoptózu dominantní nebo recesívní vliv mutantní alely IV. Geny regulující opravu DNA mismatch repair genes, recesívní vliv mutantní al.

6. I. Protoonkogeny → onkogeny • alelické formy téhož strukturního genu • Protoonkogeny - v normálních buňkách strukturní geny → translační produkty protoonkoproteiny: • Normální role v regulaci: vedou k růstu, dělení a diferenciaci • VERSUS onkoproteiny – v nádorových buňkách vedou k neregulovanému dělení abnormálně dlouhému přežívání → vznik nádoru

7. Fyziologická proliferace • Vazba: růstový faktor - specifický receptor na buněčné membráně • Přechodná aktivace receptoru růstového faktoru, aktivuje další proteiny na vnitřní straně membrány • Přenos signálu cytosolem do jádra - second messengers • Indukce a aktivace jaderných regulačních faktorů, které iniciují transkripci DNA • Zahájení a postup buňky buněčným cyklem (dělení)

8. I. Protoonkogeny kódují: • A. Růstové faktory • B. Receptory růstových faktorů • C. Proteiny transdukující signály, nereceptorové proteinkinázy • D. Transkripční proteiny v jádře • E. Cykliny a cyklin-dependentní kinázy

9. I. A. Growth factors Normal: Stimulation of proliferation when increased = overexpression Carcinogenesis Autostimulation of tumour cells PDGF – protooncogene c-sis; Epidermal growth factor; Granulocyte macrophage stimulating factor

10. I. B. Growth factor receptors Transmembrane proteins Tyrosine kinase cytoplasmic domain Activation: transient (normal) versus persistent (tumour) Mechanisms • Hyperexpression of normal forms • Mutations • Gene rearrangements EGFR family: cerb B1, c-erbB2-Her2/neu RET

11. I. C. Signal transducing proteins biochemically heterogeneous Ras proteins = G proteins, bind GTP • ras – GTP (act.) x GDP (inact.) • ras: GTPase activity GTPase activating proteins (GAP) • Mutated ras binds GAP but persistent activation (GTP), pathologic mitogenic influence • ras mutation: the most frequent abnormality of dominant oncogenes in human tumors (20%)

12. I. C. Other signal transducing proteins Non-receptor protein kinases • Tyrosine kinase activity protooncogene abl • t(9;22)(q34;q11) - bcr-abl • chronic myelogenous leukemia; • a subset of acute lymphoblastic leukemia

13. Burkitt lymphomat(8; 14)(q24;q32)c-myc, expression dysregulationnuclear transcription factor Chronic myelogenous leukemia Reciprocal balanced t(9; 22)(q34;q11) Bcr-abl 210 kDa fusion protein Tyrosine kinase activity

14. I. D. Nuclear transcription factors • Myc, myb, jun, fos • C-myc gene of immediate early phase of growth • Regulation of cell growth and apoptosis Dysregulated expression: Burkitt lymphoma Amplification: carcinoma of the breast, colon, lung

15. Mechanisms of oncogene activation Change of the gene • structure abnormal protein • expression regulation abundant protein 1. Point mutations (ras) 2. Chromosomal lesions (transloc., inversion; hematology) 3. Gene amplification - double minutes - homogeneous staining regions

16. II. Tumour suppressor genes = brakes of cell proliferation, antioncogenes Gene Rb Gene P53, P73 BRCA, APC, NF-1, DCC, VHL, WT1

17. II. Tumour suppressor genes Cell cycle progression • Cyclins • Cyclin-dependent kinases (CDK) – phosphorylation of critical target proteins -- progression • CDK inhibitors Key regulators of the cell cycle: p16INK4a, cyclin D, CDK4, RB

18. Retinoblastoma • 60% sporadic • 40% familial – AD • Knudson two-hit hypothesis • 13q14 • LOH

19. II. Tumour suppressor genes • P53, 17p13.1 mutated in many human cancers (50%) mostly somatic mutations Molecular policeman – prevents propagation of genetically damaged cells, does not police the normal cell cycle • Surveillance • Triggering checkpoint controls (G1/S, G2/M): damaged cells • stop cell-cycle or slow down (p21) • Apoptosis (bax)

20. II. Tumour suppressor genes • p53 Li-Fraumeni syndrome • 1 p53 mutation inherited • 25x ↑ risk of developing malignancy: young, multiple, various • Sarcomas, breast cancer, leukemia, brain, adrenal tumours

21. II. Tumour suppressor genes APC – 5q21 - adenomatous polyposis coli down-regulation of growth-promoting signals APC down-regulates β- catenin (1. transcription, 2. cell adhesion) Familial adenomatous polyposis Gardner syndrome – AD Colon adenoma, osteomas, epidermal cysts, fibromatosis, dental abnormalities, Malignancies of the duodenum and thyroid

22. II. Tumour suppressor genes • VHL – 3p- • Germline: inherited RCC, renal cysts pheochromocytoma, haemangioblastoma of cerebellum, retinal angioma • Somatic mutations: sporadic conventional RCC • Influence on HIF-1; • lack of VHL: increased angiogenesis

23. III. Apoptosis regulating genes • Bcl-2, bax, bad; p53, MYC • BCL-2 – follicular lymphoma t(14;18)(q32;q21) • ↑transcription and overexpression of BCL-2 protein

24. IV. Mismatch repair genes • Spell checkers, replication errors • defective DNA repair - genome instability, microsatellite instability HNPCC = Lynch syndrome – familial, AD I. Colon (proximal) II. colon, ovary, endometrium, upper urinary tract

25. IV. Mismatch repair genes AR syndromes Xeroderma pigmentosum skin malignancies (UV – sun) defective nucleotide excision repair Ataxia – teleangiectasia ↑ IR sensitivity, developmental anomalies Single gene mutation 1% of population: heterozygous carriers Fanconi anemia Bloom syndrome ↑IR sensitivity, developmental anomalies

26. DNA repair defects • BRCA1 – 17q, BRCA2 – 13q • Nucleus, transcription regulation, DNA repair Familial BRCA1: breast, ovary, prostate, colon BRCA2: breast, also male, ovary, pancreas, melanoma • Breast carcinoma Only 3% of all breast cancers carry BRCA mutation 5-10% of breast cancers are familial (of these: 80% BRCA mutations)

27. Carcinogenic agents • Chemical • Radiation – ionizing, UV • Oncogenic microbes

28. Microbial carcinogenesis Viruses Bacteria HPV, EBV, HHV8, HBV, HTLV-1 Helicobacter pylori, Borrelia burgdorferi

29. Dysplasia of uterine cervix • Cervical intraepithelial neoplasia – CIN Morphology of dysplasia: Changes: abnormal cytology - atypia abnormal architecture • Mild – moderate – severe • HPV associated - risk – low (6, 11) high (16, 18)

30. Squamous cell carcinoma of the cervix • Most common malignancy of the female genital tract in developing countries • (x developed countries: adenocarc. of uterine body) • Association: age at first intercourse, promiscuity – HPV, low economic level, smoking • Grossly: polypoid or deeply infiltrative • Microscopy: squamous cell carcinoma

31. EBV • Infectious mononucleosis • Burkitt lymphoma • Hodgkin lymphoma • B-cell lymphomas in immunosuppressed patients (HIV, transplantation) • Nasopharyngeal carcinoma

32. Diseases associated with Helicobacter pylori infection • Chronic gastritis • Peptic ulcer • Gastric carcinoma • Gastric MALT lymphoma

33. MALT lymphoma • stomach, intestine (IPSID) chronic antigenic stimulation - Helicobacter pylori Regulation: specific activated T-cells Slow progression- 90%: stage IE, IIE (bone marrow involvement: rare, 10%)

34. MALT lymphoma of the stomach