Molecular and Cell Biology of Tumors Lucia Knopfová, PhD. Prof. RNDr. Jana Šmardová, CSc. Institute of Experimental Biology Faculty of Science MU Brno Bi9910en 6. Genetic instability Molecular and Cell Biology of Tumors (1) Sustaining proliferative signaling (2) Evading growth suppressors (3) Resisting cell death (4) Enabling replicative immortality (5) Inducing angiogenesis (6) Activating invasion and metastasis (7) Genome instability and mutation (8) Tumor-promoting inflammation (9) Deregulating cellular energetics (10) Avoiding immune destruction Hallmarks of cancer Hanahan D. and Weinberg R.A., Cell 144 (2011) 646-674 (1) Sustaining proliferative signaling (2) Evading growth suppressors (3) Resisting cell death (4) Enabling replicative immortality (5) Inducing angiogenesis (6) Activating invasion and metastasis (7) Genome instability and mutation (8) Tumor-promoting inflammation (9) Deregulating cellular energetics (10) Avoiding immune destruction Hallmarks of cancer Hanahan D. and Weinberg R.A., Cell 144 (2011) 646-674 Carl O. Nordling Ø  1953: it was first speculated that neoplastic transformation does not occur in a single step Ø  An architect Carl O. Nordling studied deaths frekvencies for cancer patients of different age (from 25 to 74 years) and found the death rate increased proportionally with the sixth power of the age. Ø  he deduced that a cancer cell was the end-result of at least six successive mutations Nordling C.O. A new theory on cancer–inducing mechanism. Br.J.Cancer 7: 93-112, 1953. Genetic instability of tumors •  Tumors develop via gradual accumulation of genetic (and epigenetic) changes of specific genes driving cell division, cell death and other important cellular functions. •  Calculations based on the known mutation rate in somatic cells (10-6 per gene per cell generation / 10-9 per nucleotide per cell generation) showed that such accumution of mutations would not be possible during lifetime... •  What is the mechanism of this accumulation? Multistep cancerogenesis accompanied by sequence of clonal expansions Weinberg RA. The Biology of Cancer. Garland Science 2007 Genetic instability tumors 1.  Accumulation of all necessary mutations is achieved by normal mutation rates in combination with waves of clonal expansion, that may be caused by positive selection of „precancerous“ cells. 2.  Accumulation is enabled by genetic instability (i.e. „mutator hypothesis“). Instability means increased inherent rate of genetic change. Ø  Most tumors are genetically instable Level of genetic instability •  Absence of genetic instability would not ensure sufficient genetic variability to overcome additional selection barriers during multilevel cancerogenesis. •  Excessive instability would cause extensive DNA damage and subsequently induction of apoptosis. •  Similar findings achieved during research of bacterial fitness (reproduction capacity): there must be balance between positive and negative effects of genetic variability (secured by mutations) – sufficient variability to survive in changing and selective environment but not too high to threaten viability. •  Principle „just-right instability“ •  Extent of genetic instability increases during cancerogenesis Increasing tolerance to the increasing levels of ß-catenin and to chromosomal instability! Fodde R et al, Nat Rev Cancer 1 (2001) 55-66 Genetic model of CRC cancerogenesis Level of genetic instability endogenous DNA damage Exogenous DNA damage smoking, alcohol, radiation… Mutationrate Repair systems Level of genetic instability DNArepaircapacity Mutator hypothesis mutator hypothesis Accumulation of mutations is enabled by enhanced genetic instability, that results from (germinal or somatic) defect in DNA repair systems and cell cycle checkpoints Significance of DNA repair and cell cycle checkpoints is confirmed by the fact, that congenital defects in these systems predispose to cancer development (hereditary cancer syndromes). Types of genetic changes in tumors 1.  Minor changes in DNA sequence 2.  Changes in chromosome numbers 3. Chromosomal translocations 4. Gene amplification Lengauer C et al, Nature 396 (1998) 643-649 missense mutations, small deletions and insertions (e.g. missense mutations of Kras occur in 80 % of pancreatic tumors, missense mutations of TP53 are present in almost 50% of all tumors..) loss or gain of whole chromosomes (e.g. Loss of chromosome 10 in glioblastomas associated with inactivation of tumor suppressor PTEN; gain of chromosome 7 in papillary renal cancer associated with duplication of oncogene c-Met) fusion of (parts of) different chromosomes or parts of the same chromosome that are normally not connected (may lead to fusions between different genes) (e.g. Philadelphia chromosome in leukemias) amplification of N-myc in 30 % of neuroblastomas •  Genetic instability has multiple levels. 1. DNA sequence instability •  This type of instability is rather rare in tumors, if present it has serious impact. Errors occur during DNA replication (error-prone vs error-free DNA polymerases – proof-reading) and because of imperfect DNA repair systems. •  Defects in DNA polymerases are not common in tumors, but 2 main systems of DNA damage repair may be disrupted: 1. Nucleotide-excision repair - NER – responsible for NERassociated instability - NIN 2. Mismatch repair - MMR – responsible for microsatellite instability (MIN) DNA sequence instability Ø  Nucleotide excision repair - NER – associated with „NERassociated instability“ - NIN Ø  Mismatch repair - MMR – associated with microsatellite instability (MIN) Xeroderma pigmentosum Hereditary non-polyposis colorectal carcinoma - HNPCC = Lynch syndrome Nucleotide excision repair – NER Xeroderma pigmentosum x Mismatch repair - MMR Lynch syndrome •  Mutations of MMR and NER genes are recessive (at the cellular level!), i.e. one “functional” allele is sufficient to preserve normal repair, only after inactivation of second allele there is accumulation of mutations. •  Carriers of one germ-line mutation in MMR genes are predisposed for cancer – Lynch syndrome is dominant! •  × heterozygots in NER genes do not have increased risk of cancer!! (it may be explained by the fact that the second mutation is not per se enough to increase the rate of mutations, there must be an exogenous mutagenic factor, e.g. UV!) DNA sequence instability Ø  Repair of DNA double strand breaks by homologous recombination DNA sequence instability Ø  Repair of DNA double strand breaks by homologous recombination Ataxia Telangiectasia Nijmegen breakage syndrome Hereditary breast and ovarian cancer syndrome (BRCA1, BRCA2) AT-like disorder DNA sequence instability Bloom syndrome Werner syndrome Rothmund-Thomson syndrome Fanconi anemia DNA sequence instability and … Li-Fraumeni syndrome (TP53) Sengupta S., Harris C.C. p53: Traffic cop at the crossroads of DNA repair and recombination. Nat. Rev. Cancer 6: 44-55, 2005 Ø  p53 is associated with all types of DNA repair: NER, MMR, HR a NHEJ Ø  Model of p53 as a cellular rheostat: guarantees appropriate cellular response: 1.  Minor damage: p53 activates repair systems 2.  More extensive damage: stabilization of p53 leads to the DNA repair and cell cyle arrest 3.  Unrepairable DNA damage: p53 induces apoptosis or senescence 2. Chromosomal instability - CIN •  In comparison with NIN and MIN are chromosomal changes much more common in tumors – about 85 % of human CRCs are aneuploid. •  Common is loss of chromosome due to the LOH ⇒ Not always is the incorrect caryotype result of CIN! •  In CRCs and endometrial cancer there is an inverse correlation between MIN and CIN: tumors with defects in MMR are diploid and exhibit normal rate of chromosomal rearrangements, while tumors without defects in MMR are often aneuploid and have high frequency of chromosomal abnormalities. ⇒ At least in these two cancer types are MIN and CIN equal in respect of induction of genetic instability •  Both types of instability occur rather early during cancerogenesis and promote accumulation of genetic changes during later stages Relation between MIN and CIN Fusion of cells with CIN and MIN generates cells with CIN: Ø  Defects of MIN are complemented by MMR system of „CIN cells“ Ø  CIN phenotype is dominant: it indicates that to „achieve“ CIN only one mution may be sufficient Lengauer C et al, Nature 396 (1998) 643-649 Molecular mechanism of CIN •  In yeasts CIN may be caused by mutation of one of 100 genes: genes involved in chromatin condensation, sister chromatid cohesion, kinetochore assembly, centrosome and microtubule structure, etc •  During cell cycle there are several checkpoints that serve as surveillance mechanisms to monitor its proper progression: growth to the appropriate cell size, the replication and integrity of the chromosomes, and their accurate segregation at mitosis. Checkpoints during mitosis A.  Delays entry into mitosis until any damaged DNA is repaired B. Chromosomes are not condesened if microtubules are disturbed C. Prevents chromosome separation until the chromosomes are attached correctly to the spindle D. Separation of cells is delayed if spindle is incorrectly orientated Cortez D and Elledge SJ, Nature 406 (2000) 354-355 Restriction checkpoint vs. Other checkpoint Restriction checkpoint (G1 checkpoint, Start, Major checkpoint): - proliferation - quiscence, resting state - differentiation - senescence - cell death Restriction checpoint vs.other checkpoints -  In restriction checkpoint cell cycle may be stopped -  In other checkpoints cell cycle is delayed Spindle checkpoint •  metaphase checkpoint or spindle checkpoint or spindle assembly checkpoint or mitotic checkpoint ensures correct segregation of chromosomes •  it prevents anaphase movement of sister chromatids to the cell poles, unless: •  correct assembly of the mitotic spindle; •  attachment of all chromosomes to the mitotic spindle in a bipolar manner; •  congression of all chromosomes at the metaphase plate. •  Chromosomes are attached via kinetochores: protein structures that assemble on the centromeric DNA •  Kinetochores that are not linked to the microtubules of spindle form signaling complex transducing „wait anaphase signal“, that halts separation of chromatids until all kinetochores are attached. a.  Prophase: mitotic spindle is being formed from microtubule organising centre around cetrosomes at the cell poles b.  Metaphase: mitotic spindle is finished and chromosomes are attached to the spindle microtubules at kinetochores c.  Anaphase: chromatids are separated and pulled towards cell poles, kinetochores first Orr-Weaver TL and Weinberg RA, Nature 392 (1998) 223-224 Spindle checkpoint Ø  Kinetochores are large multiprotein complexes assembled on centromeres of chromosomes during mitosis or meiosis. They allow bipolar attachement to the microtubules of mitotic spindle and help with the movement to the opposite cell poles during anaphase. Kinetochores kinetochores red, microtubules green, chromosomes blue Spindle checkpoint Proteins Bub and Mad monitor the correct attachment of chromatids to spindle. They prevent activation of APC (APC/C, AnaphasePromoting Complex) if not all kinetochores are anchored to spindle. Orr-Weaver TL and Weinberg RA, Nature 392 (1998) 223-224 •  Key event in the spindle checkpoint is inhibion of ubiquitin ligase complex APC/C (anaphase-promoting complex/cyclosome): multiprotein complex that is active during the transition to anaphase. •  When all kinetochores are attached to the spindle, protein Cdc20 is released from inhibitory complex with checkpoint proteins BubR1, Bub3 (budding uninhibited by benomyl) and Mad2 (mitotic arrest deficient) •  Cdc20 binds and activates APC/C for interaction with cyclin B and securin •  Activated APC/C ubiquitinylates securin that functions as inhibitor of separase •  Separase then cleaves cohesin and sister chromatids are no longer connected – segregation Spindle checkpoint Spindle checkpoint After attachment of the last kinetochore, the Cdc20A P C / C b e c o m e s activated thus triggering degradation of securin, that results in release and activation of separase and proteolytic cleavage of its substrate cohesin. Musacchio A and Hardwick KG, Nat Rev Mol Cell Biol. 2002;3(10):731-41. Spindle checkpoint Separase, also known as separin, is a cysteine protease responsible for triggering anaphase by hydrolysing cohesin, which is the protein responsible for binding sister chromatids Jallepalli PV and Lengauer C, Nat Rev Cancer 1 (2001) 109-117 •  Securin prevents separation of sister chromatids by binding separin/separase – cystein protease that catalyzes cleavage of multiprotein complex cohesin. Cohesin bridges form immediately after DNA replication in S phase, bind sister chromatids and endure (at centromeric region) till anaphase. Securin functions as inhibitor of separase. •  Activation of APC/C at the onset of anaphase triggers degradation of securin and release of separase – degradation of cohesin –chromatid segregation Spindle checkpoint •  Securin have another function: Securin mutations cause chromosome nondisjunction! No separation of chromatids, if securin is inactive! Securin is required for correct localization and/or activation of separase ⇒ dual impact of interaction securin:separase – securin is necessary for full activation („priming“) of separase and also acts as separase inhibitor = double protection of correct chromatin segregation!! Spindle checkpoint Securin functions as a chaperone for separase – ensures that separase adopts its proper fold required for proteolytic activity (and promotes its nuclear accumulation) But: Inhibits proteolytic activity by disrupting the interaction between its Nand C-terminus and preventing interaction with its substrate (Scc1 is a subunit of cohesin). Model of interaction separase – securin Hornig N et al, Curr Biol (2002) In normal cell is segreģation of sister chromatids achieved by activation of APC/C – securin – separase axis. Jallepalli PV and Lengauer C, Nat Rev Cancer 1 (2001) 109-117 Spindle checkpoint In cell with only 1 functional allelle of Mad2 (heterozygot!!) there is not sufficient inhibition of APC/C, thus securin is prematurely degraded and chromatids separated without proper attachment to spindle, leading to chromosome losses. Jallepalli PV and Lengauer C, Nat Rev Cancer 1 (2001) 109-117 Spindle checkpoint Cell completely deficient in securin cannot „prime“ separase for full activation thus cohesin bridges are incompletely cleaved and sister chromatids are not separated (mitotic nondisjuction) that leads to aneuploidy. Jallepalli PV and Lengauer C, Nat Rev Cancer 1 (2001) 109-117 Spindle checkpoint Spindle checkpoint alterations in cancer •  Downregulation of hMad2 found in some breast cancers. Haploinsufficiency probably plays role Mad2 mutation phenotype •  Some CRCs have somatic mutations etiher in hBub1 or hBubR1. Mutations of hBub1 are dominant. •  hMad1 is targeted for degradation by protein Tax, product of Tlymphotropic virus type 1: this results in spindle checkpoint defects in adult T cell leukemias. •  Gene encoding securin was first described as PTTG (pituitary tumour-transforming gene) and it is highly expressed in some tumors (overproduction of securin probably cause missegregation of chromatids). •  Gene encoding components of mitotic checkpoint are rarely mutated in cancer, but frequently up/downregulated (↑ and ↓) •  Level of Mad2 must be tightly controlled, both ↑ and ↓ enhanced cancer development. In mice higher Mad2 levels result in wider range of tumor types and more agressive tumors. High levels of Mad2 cause not only changes in chromosome numbers (aneuploidy) but also chromosomal rearrangements (breaks, end-to-end fusions) •  Molecular mechanism (high Mad2 levels): (1) insufficient destruction of cyclin B and securin – reduced separese activity –separation of chromatids without cohesin desintegration ⇒ breaks (2) failure of cytokinesis - tetraploidy Spindle checkpoint alterations in cancer Van Deursen JM, Rb Loss causes cancer by driving mitosis Mad, Cancer Cell 11, pp. 1-2, 2007 •  Gene mad2 is transactivated by E2F1 and thus highly overexpressed in tumors with inactivated RB. APC and CIN Ø  Germ-line mutations of APC (adenomatous polyposis coli) cause FAP (familial adenomatous polyposis), somatic mutations are the most common (early) genetic changes in colorectal cancer. Ø  APC has 2 functions: 1. regulation of WNT signaling via binding to β-catenin – target genes of this pathway are Myc and cyclin D1 (⇒ increased proliferation) 2. APC is localized to the kinetochores of metaphasic chromosomes via binding of its C-teminus to the EB1 protein – it mediates binding of kinetochore to the microtubules; mutant APC proteins (Cterminally truncated forms) cannot bind to EB1 – the attachement of kinetochore to spindle is disrupted Dual function of APC in cell A. Regulation of levels of free β-catenin B. APC function in mitotic spindle A APC accumulates at the kinetochore, where it facilitates the binding of spindle microtubules to the kinetochore by interacting with the microtubuleassociated protein EB1. B In cells that express a truncated form of APC, the interaction between kinetochores and spindle microtubules is disrupted, leading to CIN. Fodde R et al, Nat Rev Cancer 1 (2001) 55-66 APC and CIN •  Mutation of APC gives cell dual „benefit“: increased proliferation and genetic instability. •  CRCs with mutated β-catenin and without mutated APC... do not progress that fast as APC mutated CRCs – they have enhanced cell division rate (thus advantage in respect to clonal expasion) (gatekeeper function), but slower progression to the more aggresive stages due to lack of genetic variability/instability (caretaker). Multiple centrosomes •  Presence of multiple centrosomes (more than 2) in one cell leads to the defects in mitotic spindle organisation (multipolar) – missegregation of chromosomes ⇒ results in increased genetic instability CIN Contribution of E6 and E7 proteins to transformation by papillomaviruses E6 - inactivates p53 (disrupted: blok G1, apoptosis, genetic stability) - interacts with p300/CBP (homeostasis pertrubation) - activates expression of hTERT (telomerase activation) - inactivates p16ink (distrubed cell cycle control) - interacts with Bak (inhibition of apoptosis) - interacts with E6BP/ERC-55 (inhibits diferentiation) - induces degradation of hDlg (and with other proteins with PDZ motif) (change of morphology, induction of motility) E7 – binds protein RB - inactivation of p21Cip and p27Kip (disconnected proliferation and differentiation) - prevent inhibitory effect of TGF-β on cell growth - cause formation of multiple centrosomes Centrosome •  Small organels consisting of two centriols (9 triplets of microtubules) and surrounding dense protein matrix = pericentriolar material (PCM). •  Functions as microtubules organization centre, determines polarity and orientation of microtubules during interphase, regulates mitotic spindle assembly Fukasawa K, Oncogene 21 (2002) 6140-6145 Centrosome duplication •  After mitosis each daughter cell has one centrosome, this is duplicated in late G1 (after restriction checkpoint) and early S phase •  Daugther centrioles form in the vicinity of pre-existing (mother) centrioles and grow during S an G2 phases Fukasawa K, Oncogene 21 (2002) 6140-6145 1. Deregulation of cell cycle checkpoint for duplication of centrosomes •  Check of initiation od centorosome duplication •  Suppression of centrosome re-duplication 2. Failure of cytokinesis 3. Uncontrolled splitting of centriol pair 4. Overexpression of certain PCM component and formation of acentriolar centrosome Ø  Multiple centrosomes as a cause of aneuploidy were observed in breast, lung, prostate, colorectal, brain tumors, Mechanisms leading to the multiple centrosomes Mechanisms leading to the multiple centrosomes Fukasawa K, Oncogene 21 (2002) 6140-6145 Regulation of centrosome duplication •  For regulation of centrosome duplication during cell cycle is critical function of Cdk2/cyclin E. •  Substrate of Cdk2/cyclin E is nucleophosmin, this protein is associated with unduplicated centrosomes and dissociates from them upon phosphorylation by Cdk2/cyclin E. •  In some breast, prostate and head and neck cancer mutation of TP53 correlates with presence of multiplicated centrosomes (possibly via target p21Waf-1 - inhibitor of Cdk2/cyclin E). •  Human homolog (BTAK/STK-15) of Droshophila gene aurora2/ STK-15 regulates structure of centrosomes and segregation of chromosomes and is overexpressed (amplified) in some cancers. •  Some cancers overexpress kinase PLK1 (Polo-like kinase) that regulates centrosome maturation. 3. Chromosomal translocation A. Simple type •  Specific rearrangement of chromosome segment in specific type of tumor. Common in leukemias and lymphomas, sometimes in some sarcomas. Typical for cancer type, used as diagnostic tool. •  Specific translocations may by caused by ionizing radiation, e.g. some thyroid cancer in children after Chernobyl accident have specific translcation of chromosome 10 resulting in fusion gene consisting of RET oncogene. •  This specific translocations are not associated with genetic instability, are likely the results of errors during VDJ recombination. 3. Chromosomal translocation B. complex type •  Common in solid tumors. Translocations include more that 2 chromosomes, are random, different in individual tumors of one histological type. Large parts of chromosomes are deleted, often also “marker chromosomes” that contains complex rearrangements of several chromosomes. •  They result in losses and gains of chromosomes similarly as during CIN, in addition new genes may be produced. •  Molecular mechanism is not completely known, presumably cells enter mitosis without previous repair of ds breaks. Candidate molecular players: ATM, ATR, BRCA1, BRCA2, TP53. 4. Gene amplifications •  Occur in some cancer types in later phases and sometimes underlies the acquisition of (chemotherapy-) resistance •  Most common genes undergoing amplification are N-myc, erbB (HER2) and ras, less frequently abl, myb, MET, GLI1, •  Generally amplifications are features of late tumors, associated with aggressive types and poor prognosis Gene amplifications •  Gene amplifications are more common in cells with inactivated p53. In cells with functional p53 is the presence of amplicon sensed as DNA damage and may induce apoptosis. Mutation of p53 may thus allow survival of cells with amplifications and promote their accumulation during subsequent cell divisions. This is a specific type of „amplification instability“ different from CIN. Level of genetic instability endogenous DNA damage Exogenous DNA damage smoking, alcohol, radiation… Mutationrate Repair systems Level of genetic instability DNArepaircapacity Level of genetic instability endogenous DNA damage Exogenous DNA damage smoking, alcohol, radiation… Mutationrate Repair systems Level of genetic instability DNArepaircapacity Proliferation rate: DNA replication stress Accumulation of somatic mutations during cancerogenesis 1. Mutator hypothesis Accumulation of mutations is enabled by enhanced genetic instability, that results from (germinal or somatic) defect in DNA repair systems and cell cycle checkpoints Significance of DNA repair and cell cycle checkpoints is confirmed by the fact, that congenital defects in these systems predispose to cancer development (hereditary cancer syndromes). 2. Model of DNA replication stress induced by oncogenes oncogenic pathways (cancer-driver mutations) have dual impact: 1.  Stimulate growth/ cell division 2.  Induce genetic instability, mostly CIN, by reducing mitotic fidelity Negrini S et al.: Nat Rev Mol Cell Biol 11 (2010) 220-228 Orr B., Compton D.A.: Frontiers in Oncology 3 (2013) 164 Orr B., Compton D.A.: Frontiers in Oncology 3 (2013) 164 Activated oncogenes (deregulated cell division) induce DNA replication stress Thank you for attention!