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

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)

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 Základní hypotéza

Gene expression regulation in somatic body cells Specific Universal Regulation of gene expression

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.

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

Fyziologická proliferace 1.Vazba: růstový faktor - specifický receptor na buněčné membráně 2.Přechodná aktivace receptoru růstového faktoru, aktivuje další proteiny na vnitřní straně membrány 3.Přenos signálu cytosolem do jádra - second messengers 4.Indukce a aktivace jaderných regulačních faktorů, které iniciují transkripci DNA 5.Zahájení a postup buňky buněčným cyklem (dělení)

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

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

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

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%)

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 I. C. Other signal transducing proteins

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

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

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

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

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 II. Tumour suppressor genes

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

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) 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 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 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 II. Tumour suppressor genes

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

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

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

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)

Carcinogenic agents Chemical Radiation – ionizing, UV Oncogenic microbes

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

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)

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

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

Diseases associated with Helicobacter pylori infection Chronic gastritis Peptic ulcer Gastric carcinoma Gastric MALT lymphoma DiseaseAssociation Chronic gastritisStrong causal association Peptic ulcer diseaseStrong causal association Gastric carcinomaStrong causal association Gastric MALT lymphoma* Definitive etiologic role

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%)

MALT lymphoma of the stomach