Oncogenes & tumour suppressors Bart Vanhaesebroeck Cell Signalling Group

1. Oncogenes & tumour suppressorsBart VanhaesebroeckCell Signalling Group

2. cell signalling regulates every aspect of a cell’s life & death cancer is a consequence of deregulated cell signalling

3. growth metabolism survival differentiation migration death (apoptosis) proliferation (cell cycle progression) growth factor growth factor receptor effector region (often a tyrosine kinase) CYTOPLASM intracellular transducers create 2nd messengers NUCLEUS transcription factors DNA transcription mRNA examples: cell cycle control DNA repair anti-apoptosis proteins

4. growth factor eg. epidermal growth factor (EGF) growth factor receptor eg. EGF-receptor (EGF-R) effector region (often a tyrosine kinase) intracellular transducers create 2nd messengers eg. - Ras - protein kinases (Tyr, Ser, Thr) NUCLEUS transcription factors eg. Myc, p53 DNA transcription mRNA examples: cell cycle control : Rb, p16, CDKs DNA repair : ATM anti-apoptosis : Bcl2, Bad proteins

5. normal cell signalling is deregulated in cancer • this deregulation can occur by • mutation • gene amplification • gene translocation • gene conversion • …

6. cancer is a disease of DNA (1) chromosomes of a normal cell

7. cancer is a disease of DNA (2) chromosomes of a cancer cell

8. normal cell signalling is de-regulated in cancer • this deregulation can occur in • oncogenes • genes capable of inducing one or more characteristics of cancer cells • dominant gain-of-function: dominant in genetic terms: have an effect • even if only one of the 2 cellular copies of the gene is altered • the normal versions of the genes are called ‘proto-oncogenes’ • tumour suppressor genes • genes that inhibit tumour development = ‘brakes’ • recessive loss-of-function: recessive in genetic terms: both copies of the • gene need to be inactivated (this is the ‘classical’ theory – emerging evidence • suggests that this may not be true for all tumour suppressor genes, some (like PTEN; • see later) are ‘haplo-insufficient’, and already ‘cause trouble’ if one copy is lost).

9. growth factor eg. vascular endothelial growth factor (VEGF) growth factor receptor eg. EGF-receptor (EGF-R) effector region (often tyrosine kinase) intracellular transducers create 2nd messengers eg. - Ras - protein kinases (Tyr, Ser, Thr) NUCLEUS transcription factors eg. Myc, p53 DNA transcription mRNA examples: cell cycle control : Rb, p16, CDKs DNA repair : ATM anti-apoptosis : Bcl2, Bad proteins

10. growth factor eg. vascular endothelial growth factor (VEGF) • Avastin TM (Genentech) • blocks action of VEGF, key molecule in angiogenesis • approved by the FDA in combination with chemotherapy (intravenous 5-fluorouracil [5-FU]- • based chemotherapy) for treatment of people diagnosed with metastatic colorectal cancer • for the first time

11. examples of oncogenes • Tyrosine kinases: EGF-Receptor family members, BcrAbl • Intracellular signalling protein: Ras • transcription factor: Myc • anti-apoptotic protein: Bcl2

12. growth factor eg. vascular endothelial growth factor (VEGF) growth factor receptor eg. EGF-receptor (EGF-R) effector region (often tyrosine kinase) intracellular transducers create 2nd messengers eg. - Ras - protein kinases (Tyr, Ser, Thr) NUCLEUS transcription factors eg. Myc, p53 DNA transcription mRNA examples: cell cycle control : Rb, p16, CDKs DNA repair : ATM anti-apoptosis : Bcl2, Bad proteins

13. oncogenes • EGF-Receptor family members • overexpressed & constitutively active in breast cancer • target for (1) antibody therapy: eg. Herceptin (Genentech) = monoclonal antibody that binds the extracellular domain of the EGF-R family member HER2  inhibits the growth of cells that overexpress this EGF-R (2) tyrosine kinase inhibitor therapy: eg. IRESSA (Astra Zeneca) = small molecule that inhibits the activity of the intracellular kinase domain of the EGF-R

14. resting normal cell receptor nucleus cell membrane = hormone or growth factor (courtesy of Dr. Rob Stein)

15. gene activation cell survival & division stimulated normal cell (courtesy of Dr. Rob Stein)

16. spontaneous receptor dimerisation & activation gene activation cell survival & division cancer cell (courtesy of Dr. Rob Stein)

17. growth inhibition & cell death effect of = inhibitor of receptor kinase activity (courtesy of Dr. Rob Stein)

18. deregulated signalling proteins are increasingly used for ’targeted therapies’ tumours seem to critically dependon some of these pathways : ‘Achilles heels’

19. examples of oncogenes (cont’d) Tyrosine kinases (cont’d) • BcrAbl • Philadelphia chromosome translocation = t(9;22) : fuses • * part of the bcr gene from chromosome 22 • with • * part of the abltyrosine kinase gene on chromosome 9  creates the BcrAbl fusion protein in which the Abl tyrosine kinase (1) has  kinase activity (2) localised throughout the cells (not only in the nucleus as in normal cells)  phosphorylation of substrates that  proliferation & protect from apoptosis • in chronic myelocytic leukemia (CML) • target for Gleevec (Novartis) = tyrosine kinase inhibitor  almost 100% remission in chronic phase of disease (but resistance appears to develop).

20. growth factor eg. epidermal growth factor (EGF) growth factor receptor eg. EGF-receptor (EGF-R) effector region (often tyrosine kinase) intracellular transducers create 2nd messengers eg. - Ras - protein kinases (Tyr, Ser, Thr) NUCLEUS transcription factors eg. Myc, p53 DNA transcription mRNA examples: cell cycle control : Rb, p16, CDKs DNA repair : ATM anti-apoptosis : Bcl2, Bad proteins

21. examples of oncogenes (cont’d) • Ras = intracellular signalling protein • small GTPase • controls MAP kinase protein cascade  important for proliferation & gene induction • mutated & constitutively active in many cancers • Myc = transcription factor - in Burkitt lymphoma • due to Epstein-Barr Virus (EBV): virus carried by >90% of the world's population – in severely immune-suppressed patients  EBV immune surveillance  B-cell lymphomas • How does Myc become activated?  translocation of c-myc proto-oncogene into or near one of the immunoglobulin loci  found in almost every case of Burkitt’s B-cell lymphoma in man (see lecture D. Linch & A. Khwaja)

22. examples of oncogenes (cont’d) • Bcl2 = anti-apoptotic protein = B-cell leukemia-2 (see lecture notes D. Linch & A. Khwaja) • protects against cell death • was the first ‘oncogene’ discovered which does not regulate proliferation • initially identified as a translocation breakpoint common in many B-cell lymphomas • as a result of this translocation, the bcl-2 gene comes under the control of the immunoglobulin heavy chain enhancer & is constitutively expressed in B-cells • the resulting protection from apoptosis apparently permits the survival & accumulation of aberrant B-cells that ultimately give rise to lymphoid malignancies

23. examples of tumour suppressor genes • gene regulator:Rb • transcription factor:p53 • lipid phosphatase:PTEN

24. tumour suppressor genes • genes that inhibit tumour development • classical theory: recessive (in genetic terms): both gene copies in the cell need to be • inactivated before cancer can arise • almost all genes in our cells are present in 2 redundant copies (one from mother & one from father): if one copy is lost, the other copy serves as a backup. In the case of tumour suppressor genes, this offers a measure of protection. • loss-of-heterozygosity = LOH = loss of the 2nd allele of a tumour suppressor (by gene conversion, mutation, gene deletion etc) • some people carry an inactivating mutation in a tumour suppressor gene in their sperm or eggs  offspring is more prone to lose the 2nd allele (eg. by a so-called ‘sporadic’ mutation)  predisposition to cancer. eg. familial retinoblastoma : carry mutations in Rb gene (see also lecture notes Dr. Daniel Hochhauser)

25. growth factor eg. epidermal growth factor (EGF) growth factor receptor eg. EGF-receptor (EGF-R) effector region (often tyrosine kinase) intracellular transducers create 2nd messengers eg. - Ras - protein kinases (Tyr, Ser, Thr) NUCLEUS transcription factors eg. Myc, p53 DNA transcription mRNA examples: cell cycle control : Rb, p16, CDKs DNA repair : ATM anti-apoptosis : Bcl2, Bad proteins

26. Rb = retinoblastoma first identified in the rare eye tumour retinoblastoma (occurs only up to the age of 6-7) • arises from retinoblasts: cells in the embryonic retina • that will become photoreceptors • ‘sporadic’ form: afflicted children have no close relatives who • have previouslycontracted this cancer (familial form) Alfred Knutson theory(based on epidemiological studies): > sporadic form: the 2 mutations occur one after another (either during embryonic development of shortly after birth), in one of the cells of the retina  extremely rare & occurs slighly later in life (mean age: 30 months)  children mostly carry a single retinal tumour in one eye > familial form: all cells of the embryo carry 1 mutated allele of the Rb gene (including all cells of the retina).   chance of loss of 2nd allele (LOH)   frequency of retinoblastoma & occurs early (mean age: 14 months)  often multiple tumours in both eyes

27. RB RB E2F E2F G1 G1 in Rb -/- cell: loss-of-expression of Rb  brake is lost  no brakes on cell cycle progression • Rb = retinoblastoma protein • ‘pocket’ protein: binds & inhibits E2F transcription factors • ‘super’ phosphorylation of Rb (by cyclin-dependent kinases that act in cell cycle)  release of E2F from the DNA  brake is gone  allows transcription of genes important for cell cycle progression in normal cell: P P RB P phosphatases cyclinD/CDK4 P P E2F cyclin E c-Myc other S

28. STRESS (irradiation, hypoxia, anoxia, …) p53 cell death cell cycle arrest • examples of tumour suppressor genes (cont’d) • p53 • = transcription factor • in 50% of tumours: lost or (in most cases) mutated such that it can no longer bind DNA • = ‘GUARDIAN OF GENOME’: ‘senses’ DNA damage, stress • if damage is moderate: stalls cells in cell cycle until DNA is repaired • if damage is severe: induces cell death programme

29. examples of tumour suppressor genes (cont’d) p53 (cont’d): Not entirely clear how p53 works, but a very plausible pathway goes as follows: damage of cellular DNA  activation of ATM / DNA-PK (DNA-dependent protein kinase)  phosphorylation of p53  increased p53 stability  p53 accumulation & activation  induction of * cell cycle inhibitors (such as p21) * apoptosis-inducing proteins (such as Bax, Fas-receptor, ..) * IGF-BP3 (a secreted binding protein for the survival factor IGF-1) EXPRESSION OF THESE NEGATIVE REGULATORS IS LOST UPON LOSS OF p53

30. example of a dose-dependent tumour suppressor gene: PTEN

31. PI3K DISEASE: CELLS: receptor Akt ras PIP3 proliferation survival growth differentiation migration protein kinases PDK1, Akt/PKB, Btk, Itk, … PIP2 cancer inflammation diabetes adaptor proteins Gab1, Bam32, DAPP1, … + GEFs / GAPs for small GTPases of Rac, Ras, Arf families deregulation of PI3K signalling in cancer signalling by PI 3-kinases cytosol PI3K

32. : when inactivated  PI3K pathways ‘on’ PIP3 = lipid phosphatase PTEN PI3K germline PTEN mutations in some hamartoma syndromes e.g. Cowden syndrome in many sporadic cancers e.g. glioblastoma, endometrium, … PTEN +/- mice: develop cancer with 100% penetrance PIP2 under those conditions, the wild-type PTEN allele is retained, and only the dose of PTEN enzyme is altered Apparently, lowering the dose of a tumour suppressor gene can already have dire effects for cancer development, and it is thus not always necessary to lose BOTH copies of a tumour suppressor gene !!! (( Knutson theory) deregulation of PI3K signalling in cancer (cont’d) by loss of function of the PTEN tumour suppressor gene

33. summary: oncogenes and tumour suppressor genes can alter every step of cellular signalling growth factor eg. epidermal growth factor (EGF) growth factor receptor eg. EGF-receptor (EGF-R) effector region (often tyrosine kinase) intracellular transducers create 2nd messengers eg. - Ras - protein kinases (Tyr, Ser, Thr) NUCLEUS transcription factors eg. Myc, p53 DNA transcription mRNA examples: cell cycle control : Rb, p16, CDKs DNA repair : ATM anti-apoptosis : Bcl2, Bad proteins

34. THE END (thank you for your attention)